ASSESSMENT OF SURROGATE BIOMARKERS FOR OPHTHALMIC DISEASE IN COLINUS VIRGINIANUS INFECTED BY OXYSPIRURA PETROWI
by
JORDAN WILLIAM HUNTER, B.S.
A Dissertation
In
ENVIRONMENTAL TOXICOLOGY
Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of
DOCTOR OF PHILOSOPHY
Steven M. Presley Chair of Committee
C. Brad Dabbert
Joseph Fralick
Ernest Smith
Mark Sheridan
Dean of the Graduate School
December, 2016
Copyright © 2016, Jordan W. Hunter
Texas Tech University, Jordan Hunter, December 2016
ACKNOWLEDGEMENTS
My experience at Texas Tech University and The Institute of Environmental and Human
Health (TIEHH) has been invaluable for my growth as a scientist. I would like to thank my advisor Dr. Steven Presley for his wisdom, patience, and good-spirited encouragement through my graduate studies. Thank you to my committee members; Dr.
Ernest Smith, Dr. Brad Dabbert, and Dr. Joseph Fralick for providing their collective wisdom. I thank Dr. David Lewis for providing his assistance in ocular pathology, Mary
C. Hastert for her help in our histology preparations, Dr. Peter Keyel for his help and suggestions during the development of this study, and Dr. Gregory Mayer for his assistance with the instrumentation used for this project. The Rolling Plains Quail
Research Foundation and the participants of Operation Idiopathic Decline were vital in obtaining subjects and data for this project, and I thank them for their help and hospitality.
I would also like to thank Dr. Galen Austin for imparting his acumen during our field studies as well as my fellow VZL group members, all of whom played an integral part in my education and positive graduate experience, and have greatly contributed in the work necessary to complete this study.
Thanks to the faculty, staff and students at TIEHH I have been provided an exceptional environment to learn, work and have fun.
Thank you to my friends and family, my father Allen, and my mother Linda for all her support, sacrifice and encouragement through my journeys. Thank you to my partner
Patricia for helping me grow as a person during this process, filling me with confidence and making me whole.
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS ...... ii TABLE OF CONTENTS ...... iii ABSTRACT ...... vii LIST OF TABLES ...... ix LIST OF FIGURES ...... x CHAPTER I INTRODUCTION ...... 1 1.1 Eye parasite afflicting northern bobwhite quail ...... 1 1.2 The importance of understanding helminth associated diseases ...... 2 1.3 Other factors involved in bobwhite quail decline ...... 3 1.4 Host response to helminths ...... 4 1.5 Known effects of oxyspiruriasis in galliformes ...... 5 1.6 Immunopathology and oxidative stress caused by helminths ...... 5 1.7 Experimental design ...... 7 1.7.1 Histopathology ...... 8 1.7.2 Oxidative stress surrogate biomarkers ...... 8 1.7.3 Immune response surrogate biomarkers ...... 8 1.8 Conclusion ...... 9 CHAPTER II LITERATURE REVIEW ...... 11 2.1 Northern bobwhite quail (Colinus virginianus) ...... 11 2.1.1 Discovery and taxonomy ...... 11 2.1.2 History in the United States ...... 12 2.1.3 Habitat ...... 12 2.1.4 Diet ...... 13 2.2 Northern bobwhite quail behavior, life history and biology ...... 15 2.2.1 Behavior ...... 15 2.2.2 Breeding, fecundity and life cycle ...... 17 2.2.3 Anatomy and biology ...... 19 2.2.4 Toxicology ...... 20 2.2.5 Infectious disease and parasites ...... 22 2.3 Northern bobwhite quail population decline ...... 24 2.3.1 Population changes over time ...... 24
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2.3.2 Factors associated with bobwhite population decline ...... 25 2.3.3 Role of habitat loss...... 27 2.3.4 Potential role of pathogens and parasites in population changes ...... 28 2.3.5 Stress and reproduction ...... 29 2.4 Oxyspirura petrowi ...... 32 2.4.1 Taxonomy ...... 32 2.4.2 Background and history ...... 34 2.4.3 Habitat and environment ...... 35 2.4.4 Life cycle ...... 38 2.4.5 Non-pathological effects on host ...... 39 2.4.6 Pathology in host...... 39 2.5 Oxidative stress ...... 42 2.5.1 Physiological conditions and function ...... 42 2.5.2 In vivo consequences of free radical generation ...... 44 2.5.3 Role in disease pathogenesis ...... 46 2.5.4 Oxidative damage to neurological and ophthalmic tissue ...... 47 2.5.5 Host ocular antioxidant protection ...... 50 2.5.6 Oxidative stress induced by host immune response to pathogens ...... 53 2.6 Gene expression and immune response in the eye ...... 57 2.6.1 Innate and adaptive immune systems ...... 57 2.6.2 Immunology and immune privilege of the eye ...... 59 2.6.3 Th2 and Th1 host immune response ...... 61 2.6.4 Transcription factors and avian uncoupling protein ...... 64 2.7 ROS and immune response mediated disease in ocular tissue ...... 64 2.7.1 Cornea ...... 65 2.7.2 Aqueous humor ...... 65 2.7.3 Lens ...... 66 2.7.4 Retina ...... 67 2.8 Analysis of oxidative damage and immune response in ophthalmic tissue ...... 68 2.8.1 Hematoxylin and eosin staining of ocular tissue ...... 68 2.8.2 Antioxidant and lipid peroxidation surrogate biomarkers ...... 69 2.8.3 Immune response biomarkers associated with oxidative stress ...... 70
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2.9 Summary ...... 70 CHAPTER III HISTOPATHOLOGY OF OCULAR TISSUE ...... 74 3.1 Introduction ...... 74 3.2 Known areas of O. petrowi induced inflammation in ocular tissues ...... 76 3.3 Materials and methods ...... 77 3.3.1 Sample harvesting and tissue fixing ...... 77 3.3.2 Deparaffinization and hematoxylin and eosin staining ...... 77 3.3.3 Experimental design and analyses criteria ...... 78 3.4 Results ...... 79 3.4.1 Histopathological analyses of male uninfected tissue ...... 79 3.4.2 Histopathological analyses of female uninfected tissue ...... 80 3.4.3 Histopathological analyses of male infected tissue ...... 81 3.4.4 Histopathological analyses of female infected tissue ...... 82 3.4.5 General assessment off histology in uninfected vs infected tissue ...... 83 3.4.6 Comparing uninfected males to females ...... 84 3.4.7 Comparing O. petrowi infected males to females ...... 84 3.4.8 Potential ophthalmic disease and vision disorders in infected quail...... 85 3.5 Discussion ...... 88 CHAPTER IV OXIDATIVE STATUS OF OPHTHALMIC TISSUE ...... 95 4.1 Introduction ...... 95 4.2 Oxidative stress and eye disease ...... 97 4.3 Locations of antioxidants in ocular tissue ...... 99 4.4 Materials and methods ...... 102 4.4.1 Subject capture and sample harvesting ...... 102 4.4.2 Tissue Homogenization ...... 103 4.4.3 Glutathione (GSH) assay ...... 104 4.4.4 Thiobarbituric acid reactive substances (TBARS) assay ...... 104 4.4.5 Catalase (CAT) Assay...... 105 4.4.6 Superoxide dismutase (SOD) assay ...... 105 4.4.7 Glutathione peroxidase (GPx) assay ...... 106 4.4.8 Pierce BCA protein assay ...... 106 4.4.9 Experimental design and post-laboratory analysis criteria ...... 106
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4.5 Results ...... 108 4.5.1 GSH analysis of ocular tissue ...... 108 4.5.2 TBARS analysis of ocular tissue and vitreous ...... 109 4.5.3 CAT analysis of ocular tissue and vitreous...... 111 4.5.4 SOD analysis of ocular tissue ...... 113 4.5.5 GPx analysis of ocular tissue ...... 114 4.5.6 General assessment of antioxidant and lipid peroxidation status ...... 115 4.5.7 Comparing oxidative status between sexes...... 117 4.6 Discussion ...... 117 CHAPTER V IMMUNE RESPONSE IN QUAIL OCULAR TISSUE ...... 129 5.1 Introduction ...... 129 5.2 Eye Immunology ...... 131 5.3 Th2 host immune response to helminths—interleukins, transcription factors and avian uncoupling protein ...... 134 5.4 Oxidative stress and eye disease ...... 137 5.5 Materials and methods ...... 139 5.5.1 Subject capture and sample harvesting ...... 139 5.5.2 Tissue Homogenization ...... 140 5.5.3 RNA extraction and analysis ...... 141 5.5.4 Gene expression analysis ...... 141 5.5.5 Experimental design and post-laboratory analysis criteria ...... 144 5.6 Results ...... 145 5.6.1 Expression of all infected relative to uninfected...... 145 5.6.2 Expression of male infected relative to male uninfected ...... 146 5.6.3 Expression of female infected relative to female uninfected ...... 146 5.6.4 Expression of male infected relative to female infected ...... 147 5.6.5 Expression of male uninfected relative to female uninfected ...... 148 5.6.6 General assessment of immune response ...... 148 5.6.7 Comparing immune response between sexes ...... 149 5.7 Discussion ...... 150 REFERENCES ...... 161
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ABSTRACT
The decline of northern bobwhite quail (Colinus virginianus) populations across Texas over the last few decades is of significant ecological importance. Old World
(Phasiandiae) and New World (Odontophoridae) quail and related galliformes have been historically important birds for livestock, game, agriculture, and scientific study. The high incidence of the eye worm, Oxyspirura petrowi, and its infestation within the ocular tissue of northern bobwhites is a biotic factor with influence on C. virginianus physiology and behavior that has not been completely elucidated.
Conjunctivitis, intraorbital gland fibrosis, atrophy, and bilateral cataract are conditions resulting from oxyspiruriasis that have been observed in avian species. Helminthiasis of birds is also known to alter the oxidative status of the host’s systems, facilitating oxidative stress and damage to host tissues.
Oxidative stress (OS) is linked to etiology of many diseases including severe vision debilitating ophthalmic disorders, and OS can occur through persistent helminth induced dysregulation of host inflammatory immune responses. Under these conditions, host system immunopathology and oxidative stress induced by O. petrowi infestation can lead to the clinical manifestation of eye disorders within the intraocular microenvironment compromising the host’s vision.
We have utilized histopathology, enzymatic and non-enzymatic antioxidants, oxidative damage by-products, and gene expression studies to assess surrogate biomarkers associated with the formation of intraocular disease in O. petrowi infected bobwhite quail.
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We report O. petrowi infected northern bobwhite exhibited a consistent and higher degree of inflammation within the uveal tract, manifesting as choroiditis, as well as abnormalities of the lens relating to the formation posterior sub capsular cataract.
Variation in oxidative status of intraocular tissue was observed between bobwhites infected and those uninfected by O. petrowi. Antioxidant concentration and activity was observed to be either significantly reduced (glutathione and glutathione peroxidase), or increased (catalase and superoxide dismutase) among infected quail as compared to uninfected subjects. Lipid peroxidation markers were significantly elevated within infected quail intraocular tissue compared to uninfected bobwhites.
We observed increased gene expression of T-helper 2 (Th2) cell immune response cytokines, anti-inflammatory cytokines, and avian uncoupling protein, as well as lower expression of glutathione peroxidase among eye worm infected quail relative to uninfected quail.
These studies indicate that O. petrowi infected quail are mounting a Th2 immune response by signaling the recruitment of leukocytes and initiating respiratory bursts of cytotoxic reactive intermediates, altering the oxidative status of the host’s system and increasing intraocular tissue inflammation that could affect the host’s vision.
The surrogate biomarkers used in this study can act as indicators for clinical endpoints related to ophthalmic disease or symptoms of impaired vision, and can be useful in the study of animal vision and behavior as well as ophthalmic disease detection for human health.
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LIST OF TABLES
Table 5.1 Target gene primer sequences and annealing temperatures ...... 143
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LIST OF FIGURES
Figure 2.1 Hosts of Oxyspirura petrowi and Oxyspirura pusiallae ...... 37 Figure 3.1 Hematoxylin and eosin stain of male bobwhite intraocular tissues ...... 91 Figure 3.2 Hematoxylin and eosin stain of male uninfected bobwhite lens tissues ...... 91 Figure 3.3 Hematoxylin and eosin stain of female bobwhite intraocular tissues ...... 92 Figure 3.4 Hematoxylin and eosin stain of female bobwhite uveal tissue ...... 92 Figure 3.5 Hematoxylin and eosin stain of infected male bobwhite ocular tissues ...... 93 Figure 3.6 Hematoxylin and eosin stain of male infected bobwhite lens tissues ...... 93 Figure 3.7 Hematoxylin and eosin stain of female bobwhite extraocular tissues ...... 94 Figure 3.8 Hematoxylin and eosin stain of female bobwhite uveal tissue ...... 94 Figure 4.1 Glutathione concentration in intraocular tissues...... 122 Figure 4.2 Superoxide dismutase concentration in intraocular tissues ...... 123 Figure 4.3 Catalase concentration in intraocular tissues ...... 124 Figure 4.4 Catalase concentration in vitreous ...... 125 Figure 4.5 Glutathione peroxidase concentration in intraocular tissues ...... 126 Figure 4.6 Thiobarbituric acid reactive substances concentration in intraocular tissues ...... 127 Figure 4.7 Thiobarbituric acid reactive substances concentration in vitreous ...... 128 Figure 5.1 Relative gene expression for all quail ...... 156 Figure 5.2 Relative gene expression for male quail ...... 157 Figure 5.3 Relative gene expression for female quail ...... 158 Figure 5.4 Relative gene expression of infected quail (male relative to female) ...... 159 Figure 5.5 Relative gene expression of uninfected quail (male relative to female) ...... 160
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CHAPTER I
INTRODUCTION
The decrease of northern bobwhite quail (Colinus virginianus) populations across Texas over the last few decades is a phenomenon of considerable ecological importance
(Brennan, 1999). Old World (Phasiandiae) and New World quail (Odontophoridae) and related galliformes have been historically important birds as livestock, game, agriculture, and scientific study. Understanding both the environmental and biological factors that may have contributed to the decline of C. virginianus is vital in aiding the conservation efforts for this species and other wildlife sharing the same or similar habitats. Identifying the significant environmental factors or pathogenic agents that influence this avian species may also benefit our broader understanding of how these factors influence wildlife and human health on all trophic levels within ecoregions. In addition, species such as the northern bobwhite and Japanese quail (Corturnix japonica) have been used as effective animal models in the study of toxicological and the physiological responses of vertebrates to pharmaceuticals, pesticides and pathogens (Newsted et al., 2007; Romijn et al., 1995).
1.1 Eye parasite afflicting northern bobwhite quail
Oxyspirura petrowi (Spirurida: Thelaziidae) is a heteroxenous parasitic nematode often
found around the eyelids, nictitating membrane and orbital cavity of many ground
dwelling bird species, including the northern bobwhite quail (Skrjabin, 1929). Interest in
this helminth is considerable due to the frequency of its appearance in wild galliformes,
and its potentially debilitating disease burden among the declining quail populations in
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Texas and across the United States (Xiang et al., 2013).
Oxyspirura petrowi is one of seventy known species of oxyspirurids, including
Oxyspirura mansoni and Oxyspirura pusillae, which have been identified in wild and
domestic galliformes located in North America. Oxyspirura mansoni is known to
severely affect broiler chickens in the Americas and is of large concern among the
poultry industry (Sanders, 1928). Oxyspiruriasis in avian hosts is associated with clinical
signs of lacrimation, conjunctivitis, bilateral cataract, and inflammation of the nictitating
membrane among other maladies or be asymptomatic depending on the degree of
infestation in ocular or digestive organs (Santoyo-de-Estafano et al., 2014). However,
there is limited knowledge regarding intraocular pathology in adult galliformes with
chronic parasitic infestation and the impact of ophthalmic helminthiasis on the host’s
biology and behavior (Bruno et al., 2015). The northern bobwhite quail provides a useful
animal model for the study of the host and parasite physiological interactions, as well as
the clinical detection of ophthalmic disease in helminth-infected vertebrates. The
applications for identifying surrogate biomarkers for ocular disease not only contributes
to an ecological understanding of the role of eye associated pathogens in bobwhite quail
population decline, but may also assist in the development of methods to clinically detect
and treat eye disorders in wildlife and human populations.
1.2 The importance of understanding helminth associated diseases
Neglected tropical diseases (NTD) are estimated to cause twenty-six million disability adjusted life years (DALY) worldwide—a measure accounting for the total years of life lost from each infected person due to disability and premature mortality (Global Atlas of
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Helminth Infection, 2015). Helminthiasis, accounts for a significant amount of this disease burden, and affects close to one-third of the global population (Hotez et al.,
2008). The number of soil-transmitted helminths and filarial worm infections alone exceeds the cases of human immunodeficiency virus (HIV) and malaria combined
(Crompton & Savioli, 2007). Despite the magnitude of exposure and effect parasitic worms have on the quality of life globally, there are still many unanswered questions about the immunological and ecological relationship of these parasites for both human and animal hosts (McSorely & Maizels, 2012).
The dramatic population decreases of bobwhite quail can often be associated with changes in the environment that also affect humans and animals sharing the same ecosystem (Angelo et al., 2012). There are an estimated 100 worm species known to infect galliformes in the United States, and a significant number of nematode species are among these (Merck & Co. Inc., 2016). The lesser prairie chicken, and short-eared owl are among several different species of wildlife that rely on the same habitat as the quail and are also considered declining in populations (Angelo et al., 2012). In addition, identifying biomarkers characteristic of immunopathology in the eye of parasite- challenged animal models may be of great public health benefit for early clinical detection of eye related diseases, such as cataracts in human health.
1.3 Other factors involved in bobwhite quail decline
While alterations in habitat are believed to be important to the quail population decline,
other stressors such as environmental contaminants and pathogens could also play a
significant role (Stewart et al., 1997). The food sources available to quail and their
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exposure to infection through an arthropod intermediate host can be influenced by
anthropogenic or natural environmental changes (Githeko, Lindsay, Confalonieri, & Patz,
2000; Magori & Drake, 2013). This is similar in the epidemiology of outbreaks for many
vector-borne diseases of public health concern for humans, which are highly influenced
by environmental variations affecting population dynamics of arthropods and wild birds
(Magori & Drake, 2013).
1.4 Host response to helminths
The presence of micro and macro-parasites can alter the host’s immune response in various ways—and it is difficult to predict the type of host response to the pathogen depending on the parasite’s residence in the body and other biological factors. (Segura et al., 2007). Due to this ambiguity, it is suggested that helminths have both the ability to create either an asymptomatic environment in the host through suppression of the host’s immune defenses, or alternately trigger immunopathology in their host through severe inflammation accompanied by the increased generation of unreduced free radical intermediates (Sorci & Faivre, 2009). The existence of few reliable vaccines and pharmaceuticals that are effective against the entire spectrum of parasitic worm species is an indicator of our limited understanding of the vertebrate immune response (Hewitson &
Maizels, 2014; Hein & Harrison, 2005). Ocular immune privilege also makes helminth infections in proximity to these tissues a unique and challenging area of study, as the eyes are not prone to the same type of systemic immune response as other organs of the body and can offer more, or alternately less protection from xenobiotics and pathogens (Taylor,
2009).
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1.5 Known effects of oxyspiruriasis in galliformes
During the adult stage of its life cycle, O. petrowi is known to inhabit the area
surrounding the eye of its host including the nictitating membrane and conjunctival sacs
of avian vertebrates (Dunham et al., 2015). For most galliformes, symptoms of spiruroid
eye helminthiasis are typically inflammation, petechial hemorrhages, swelling of the
nictitating membrane, and conjunctivitis (Magalhaes et al., 2004).
Severe filariasis has also caused bilateral cataract, cloudiness in the cornea, partial
blindness, infection by secondary bacteria and eventually significant eye destruction
causing morbidity (Vellayan et al., 2012). As the clinical symptoms of eye worm
infection can have profound effects on visual acuity leading to fitness penalties for the
host—this can also influence the behavior and ecology of birds in the wild (Dunham et
al., 2014; Newborn & Foster, 2002).
The primary goal of this study was to identify surrogate biomarkers in the intraocular tissue of O. petrowi infected quail that could be used as indicators of more severe eye disorders compromising the host’s vision and causing morbidity. This was accomplished by examining the intraocular microenvironment of the eye for signs of immunopathology, an indirect consequence of helminthiasis in the host. The results and findings of this research provides useful information for the diagnosis and detection of early signs of eye related diseases, preempting harmful clinical manifestation.
1.6 Immunopathology and oxidative stress caused by helminths
Helminth infection is known to elicit a T-helper cell 2 (Th2) adaptive immune response in
mammals producing eosinophil granulocytes responsible for attacking macro parasites
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(Sorci & Faivre, 2009; Xiang et al., 2013). Eosinophils and the similar avian heterophil
granulocytes utilize respiratory bursts of reactive oxygen species (ROS) to breakdown
and degrade the cellular components of pathogens including the lipid bilayer, proteins,
and DNA (Barnes, 1990). The proliferation of reactive free radicals intended to destroy
the incoming pathogen may also have adverse effects on the host in cases of severe
infection and chronic parasitization (Sorci & Faivre, 2009).
Excessive ROS can have a biological cascading effect in the amplification of harmful free
radicals and the level of oxidation occurring in the host; depleting the body’s resources of
much needed antioxidants (Constantini & Moller, 2009). An imbalance of high cellular
ROS and low antioxidants creates an environment of oxidative stress in the host, a
condition that is associated with many illnesses including: cancer, cardiovascular disease,
neurodegenerative disease, and eye diseases among other maladies (Halliwell, 2007;
Ramalingam & Kim, 2012).
Glutathione (GSH) is an important antioxidant in vertebrates, and is responsible for normal maintenance of the eye lens (Slingsby & Miller, 1985). Studies have shown that depleting GSH levels in the eye under conditions of oxidative stress can lead to degradation of proteins in the lens tissue, causing lens opacification and the development of cataract (Giblin, 2000). Glutathione is one of the key biomarkers identified in this study, as previous research has shown that depletion of this antioxidant due to oxidative stress under disease conditions downregulates the T-helper cell or Type 1 (Th1) and upregulates the Type-2 (Th2) innate immune response in mammals, making the host more susceptible to other infectious agents such as viral pathogens and bacterial infection
(Kim et al., 2007). Chronic helminthiasis potentially creates a cycle in which the host
6 Texas Tech University, Jordan Hunter, December 2016 immune response could continuously express signaling proteins for eosinophil/heterophil activation and the perpetual release of ROS in response to eye worm antigens. The proliferation of free radicals that must be quenched by the host’s immunoregulatory processes could reduce the available antioxidants in the host and amplify the Th2 response further. This cycle of increasing immune response in conjunction with decreasing antioxidant availability might be induced by the chronic presence of the parasite. Any subsequent immunopathology in the quail ophthalmic tissue could be a by- product of constant stimulation of the host’s normal immune response to the helminth infection (Macdonald et al., 2002).
Glutathione class antioxidant levels in birds are also influenced by the pheomelanin
content of their plumages, with birds displaying brown to red phenotypes also having
lower physiological levels of GSH due to the sequestering of cysteine in feathers (Galvan
et al., 2012). This is believed to be associated with the development of cataracts among
certain bird species (Degen et al., 2005). Northern bobwhite quail have a sexual
dichromism in their plumage phenotype, as males exhibit more prominent white feathers
around their head, neck, throat and, eyes, which may grant them more resistance to
oxidative stress than females.
1.7 Experimental design
Samples consisted of eyes enucleated from euthanized wild northern bobwhite quail trapped as part of a collaborative field study of quail population decline across west
Texas. Birds infected with eye worms and those that have no apparent eye worm infection were separated into two cohorts to make a comparative study of host response
7 Texas Tech University, Jordan Hunter, December 2016 to O. petrowi. Adult males and females from both infected and non-infected cohorts were used to compare differences in physiological response associated with sex.
Tissue taken from sampling was studied in three phases:
1.7.1 Histopathology
Pathology induced by the helminth were determined through a qualitative assessment of
tissue inflammation. Signs of early or late stage ophthalmic disease were established
through hematoxylin and eosin staining of tissue sectioned across the eye (Wakamatsu et
al., 2010).
1.7.2 Oxidative Stress Surrogate Biomarkers
The vitreous humor, lens, and remaining intraocular tissue from the quail eye were
removed separately and each tissue sample were individually assayed for oxidative status
biomarkers including glutathione, superoxide dismutase, catalase, glutathione peroxidase,
and thiobarbituric acid reactive substances (TBARS) (Cohen et al., 2007). Commercially
available assay kits were used in conjunction with a fluorescence microplate reader to
compare concentration between O. petrowi infected and non-infected tissue.
1.7.3 Immune Response Surrogate Biomarkers
Tissue homogenates from the second phase were utilized in conjunction with real-time
polymerase chain reaction (PCR) to represent gene expression associated with
antioxidants, avian uncoupling protein, immune response linked cytokines and
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transcription factors linked to alterations of Th2 innate immune response in the quail
(Degen et al., 2005).
1.8 Conclusion
The impact of Oxyspirura petrowi helminthiasis on the immune response and physiology of the northern bobwhite quail is a complex eco-immunological study. We have assessed physiological characteristics from quail obtained from their natural ecosystems along with their respective habitat specific environmental and weather data collected over three years, encompassing several ecoregions across West Texas. This study has helped to further delineate aspects of quail immunology, expand our understanding of the bobwhite quail population decline, and our clinical knowledge of parasite burden and host relationships in vertebrates from an ecological perspective.
The SPECIFIC AIMS for this project were to:
Aim 1: Determine if pathology exists in the eyes of northern bobwhite quail infected by
Oxyspirura petrowi.
Aim 2: Determine if oxidative stress occurs in the ophthalmic tissue of infected quail via
antioxidant associated biomarker assays.
Aim 3: Evaluate the parasite induced immune response of infected northern bobwhite
quail using relative gene expression.
The HYPOTHESES were: 1) General antioxidant concentrations of parasite infected ophthalmic tissue are lesser than in tissue with no detectable eye worm infestation—a result of depletion under inflammatory immune responses. (Bertrand et al., 2006; Sorci &
Faivre, 2009) 2) Concentrations of thiobarbituric acid reactive substances corresponding
9 Texas Tech University, Jordan Hunter, December 2016 to oxidative damage of parasite infected tissue are greater than in non-infected tissue, per prior studies of helminthiasis in avian species. (Siwela, Motsi, & Dube, 2013). 3) There is an increase of Th2 immune response and alteration of oxidant mediating gene expression in parasite infected ophthalmic tissue.
4) Evidence of intraocular pathology in conjunction with increased expression of Th2 immune response and altered levels antioxidants in O. petrowi infected quail tissue can provide evidence for the pathogenesis of ophthalmic diseases compromising the host vision. Identifying these surrogate biomarkers corresponding to ocular disease in O. petrowi infected quail can provide information relevant to their fitness and population changes, as well as assist in the development clinical diagnoses of eye related disorders.
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2 CHAPTER II
LITERATURE REVIEW
2.1 Northern bobwhite quail (Colinus virginianus)
2.1.1 Discovery and taxonomy
The northern bobwhite quail (Colinus virginianus (Odontophoridae), in the order of
galliformes) has been reported throughout the United States, Mexico, and parts of the
Caribbean since the 1800’s (Brennan, 1999). Odontophoridae were first classified as
distant relatives to old world quail (Phasiandiae) in 1844 by the ornithologist John Gould,
who determined these small birds with similar appearance and habitat are generally found
in locations exclusive to the Americas. Within this family, the genus Colinus is separated
into four species in which a total 22 subspecies are known to exist among the following
groups; Eastern, Grayson’s, Black-breasted, and Masked. These taxonomic
classifications are separated largely by geographical location and morphology which is
highly variable among and within groups (Hernandez et al., 2006). This diverse
phenotypic variability has also made it difficult to distinguish taxonomic differences
between populations and species (Johnsgard, 1988). The main differences between
subspecies are typically more evident in males than in females, with majority of the
morphological variation between subspecies located around the head and underparts,
with plumages ranging in color from white to grey and brown (Del Hoyo et al., 1997).
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2.1.2 History in the United States
Due to its population and wide geographical distribution, the northern bobwhite is
considered one of North America’s most economically important game birds (Brennan,
1999). During the era of the first western settlers to North America, the bobwhite
inhabited a region encompassing the northeast of Maine down to Florida and west
towards the upper Mississippi Valley and Texas (Elliot, 1974). Westward expansion and
agricultural growth in the early history of the U.S. is believed to be the driving factor for
the expansion of the bobwhite’s habitat range and by the 1850s birds could be found in
southern Ontario and as far west as South Dakota (Klinger, 2011).
The quail farming industry in the U.S. before the 1980s was comprised primarily of stock
hunting plantations for the wealthy, however by 2007 the USDA estimates that nearly
forty million quail were produced for hunting and agriculture by U.S. farmers that year
(United States Department of Agriculture, 2009). The state of Georgia alone produces
over five million captive northern bobwhites for use at hunting preserves, and plantations
where flight-ready birds can be sold for $2.80-3.00 per quail (Dozier & Bramwell, 2002).
Per Texas quail unlimited, the total economic impact of quail hunting exceeded $26
million in 2004, but is roughly 50% lower than the total expenditures twenty years’ prior
(Angelo et al., 2012).
2.1.3 Habitat
Habitat requirements for northern bobwhites generally consist of early successional
vegetation and a mixture of different cover types near (Klinger, 2011). These
environments are typically characterized by the presence of grassland, woodlands,
bushland, and cultivated land (Leopold, 1933). The general features for bobwhite habitats
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across the U.S. are a combination of grassy areas for nesting, cover for dusting or resting,
screening overhead cover for feeding and traveling, mixed with brush used for woody
tangled cover during escapes, and protection and crops or native plants for food
(Edminster, 1954). Within Texas, northern bobwhite can be found from the Panhandle
region to as far south as the Rio Grande, with their principle range suggested to be from
the 101st meridian and eastward across North America (Jackson et al., 2011). Although
its habitat can differ in variations in soils and climate across the state, factors including
climate and abnormal weather conditions can also determine the range of bobwhites and
prolonged periods of drought, floods and other disruptive weather phenomenon can be
severely detrimental to these quail populations (Jackson et al., 2011). Roosting cover is
also required and found within the already ideal mixture of grasslands and woody cover
bobwhites prefer. Birds will roost on the ground and preferably in grassy or weedy
glades, old reverting fields, grassy hillsides and/or timbered areas with open spaces
(Jackson et al., 2011). Weather and availability of nearby food sources often dictates the
location of roosts (Jackson et al., 2011). The quality of habitat conditions, spread of
food, cover and nesting, roosting resources are significantly important for the long-term
survival of wild bobwhites, (Klinger, 2011).
2.1.4 Diet
Like most gallinaceous birds, the northern bobwhite is primarily a seed eater with
vegetative sources providing roughly 97-99% of their diet during most parts of the year,
excluding the summer season, when insects and arthropods provide close to one fourth of
its total food consumption (Edminster, 1954). The primary sources of food categories can
consist of: wild seeds from annual forbs, legume seeds, cultivated grains, mast, fruits,
13 Texas Tech University, Jordan Hunter, December 2016
grass seeds, greens, and insects depending on the habitat geography and season (Reid &
Goodrum, 1979). Wild and cultivated plant seeds can make up a significant amount of
the bobwhite’s diet including: beggarweeds, ragweeds, lespedezas, corn, partridge peas,
oaks, sumacs, pines, soybeans, and cowpeas (Kabat & Thompson, 1963).
During nesting, egg-laying and rearing, female birds require larger amounts of protein in
their diet supplied by increased feeding on invertebrates (Moreby, 2003; Wiens &
Rotenberry, 1979). During their first months after hatching bobwhite chicks require a
high protein diet to facilitate quick growth (Rosene, 1969). Insects present in low
growing herbaceous vegetation offering protection by wood brush plants often provide
young birds with an early nutrient rich food source (DeVos & Mueller, 1993). For many
young galliformes, insects and arthropods are a vital source of amino acids and proteins
used in growth, and maintenance and development of feathers (Savory, 1989).
A study by (Hurst, 1972) found that insects were the most important food source for
bobwhite chicks from 2-20 days old, and brood habitats that contain high amounts of
legumes and mixed forbs also contained higher insect abundance. Beetles (Coleopetra),
bugs (Hemiptera), and grasshoppers (Orthoptera) are known to provide as much as 94%
of the food eaten during the first 14 days of newly hatched bobwhite chicks (Eubanks &
Dimmick, 1974). Once the quail have reached seven to nine weeks of age the feeding
behavior of young birds begins to resemble that of adults. Conservatively, one bird can
eat approximately 50,000 and 75,000 insects per season depending on location and need
(Underwood, 2004).
Bobwhite existing in highly cultivated habitats, are also believed to fare better than those
who solely depend on native plants (Roseberry & Klimstra, 1984). The benefit of birds
foraging on agriculture is also suggested as the quail might complete the harvesting of
14 Texas Tech University, Jordan Hunter, December 2016
peanuts, corn, and grain, where crop has been lost in gathering by mechanical means
(Underwood, 2004).
The benefit of agricultural crops to supply food for quail for the winter is based upon to
the species of plant, harvesting method, weather, and availability of cover and
unharvested crops leftover during winter can be an important food source during this
season (Roseberry & Klimstra, 1984). However, agricultural pesticide usage can also be
detrimental to foraging quail, and the early spring season is difficult for birds due to the
reduced availability of seed and fruit crops (Reid & Goodrum, 1979).
In general, the most ideal food foraging habitats for bobwhite quail include bare ground,
low hanging vegetation and the presence of small-leaved leguminous plants and woody
protective cover nearby (Davis, 1964).
2.2 Northern bobwhite quail behavior, life history and biology
2.2.1 Behavior
The northern bobwhite quail is timid and elusive, and its behavior is like that of other
game birds (Rosene, 1969). Depending on its situational awareness, it will crouch and
freeze to camouflage itself when threatened, or it will rapidly launch into low flight to
escape (Bent, 1932). The vocalizations of northern bobwhite are an integral part of
communication between quail, and each specific vocal call is conditional upon the
situation. The characteristic clear, whistled "bobWHITE" is used primarily by unmated
males during the breeding season. In addition, there are different signaling calls used by
bobwhite to signal others in the covey for: gathering, scattering, establishing a decoy
ruse, distress, and alarm (Bent, 1932; Stoddard, 1931). Bobwhites can be found solitary
15 Texas Tech University, Jordan Hunter, December 2016
or paired in the late winter and spring seasons of the year, and transition to forming
family groups in the summer and then into larger roosts in the early winter which can
contain over two dozen birds in one covey (Klinger, 2011). Once a suitable habitat has
been located the bobwhite is typically a stationary bird with a sedentary lifestyle and is
not known to migrate far from it residence except for significant habitat changes brought
on through seasonal transitions. Coveys of birds formed during late summer and early fall
may migrate several kilometers to habitats where food and cover will be available during
the winter (Rosene, 1969). Bobwhites already residing in suitable habitats during the
winter will often rear their broods and form winter covey ranges up to a few hundred
yards of where they were hatched (Agee, 1957).
Home ranges can change depending on geographic location. According to Wiseman and
Lewis (1981), the average home range of coveys in Oklahoma was 4.4 hectares (ha) and
ranges were centered along stream channels with little variance in size of ranges between
fall and spring. During the winter season in Alabama and South Carolina ranges varied
from 1.6 to 31.2 ha (Rosene, 1969). Winter ranges in Tennessee were an average of 6.8
ha (Yoho & Dimmick, 1972) and southern Illinois ranges averaged 15 ha (Roseberry &
Klimstra, 1984). A study by Robinson (1957) determined that 4.9 ha is the minimum area
to maintain a covey of bobwhite quail.
During the summer season bobwhite feed in the early morning, rest during mid-morning,
loaf, and dust during the middle of the day, and feed again up to three hours before
sundown (Klinger, 2011). Bobwhites primarily travel by foot rather than flying to avoid
being detected by predators, this also allows them to conserve energy needed for quick
escapes (Klinger, 2011). Starting in the latter part of the summer the birds will begin to
participate in roosting patterns at night by forming tight circular groups on the ground
16 Texas Tech University, Jordan Hunter, December 2016
with their tails together and heads pointed outward—the function of this formation is
believed to be important for social behavior, escape, and conserving heat (Klinger, 2011).
By the early fall, birds begin to mix between broods and form coveys containing between
20 to 30 quail, which can be further dwindled down to groups of 10 to 15 birds
comprised of approximately 75-80% juvenile quail as the coveys begin to inhabit their
winter range (Klinger, 2011). This phase of behavior is referred to as the fall shuffle.
The lessened abundance of food in the fall also reduces the bird’s movements and this
period of the season is generally when bobwhite populations have reached their
maximum size for the year (Agee, 1957). By the following spring the maximum
mortality of the winter population can be from 50- 75%. Surviving quail coveys will
begin to breakup during the longer daylight and warmer weather and begin mating calls
in preparation for pairing in the next breeding season (Klinger, 2011).
2.2.2 Breeding, Fecundity and Life Cycle
Reports by (Klinger, 2011) describe early spring as a time of dispersal for bobwhites
preparing for reproduction, and during this time birds will transition from the shrubby
and woody habitats used in winter to more open and grassy environments. During the
breeding season, roughly 25% of the population will relocate to new areas approximately
2 kilometers from their winter range in search of mates and more ideal breeding habitats
(Suchy & Munkel, 1993). The increased volume of bobwhite mating calls that happens in
early spring is a sign that the reproductive season is beginning.
March and April is when mating pairs are formed and bobwhite pairs can break up and
reform for the duration of the breeding season taking place from late May through early
17 Texas Tech University, Jordan Hunter, December 2016
September (Curtis et al., 1993). The bobwhite’s high reproductive potential is attributed
to the long breeding season allowing for many attempts to establish suitable nests. The
length of time to setup a nest is approximately 35 to 48 days from assembling the nest to
chick hatching, which can happen between May and October (Burger et al., 1995). Nest
incubation is predominately done by females, but approximately 25% to 30% of males
take part as well during the middle of the season, and majority of the hatching occurs
around the middle of July (Klinger, 2011).
When nests are destroyed by weather or predation replacements can be readily assembled
in a day and females can potentially generate multiple broods in a season (Curtis et al.,
1993). Nest sites are chosen based on the proximity to native grasses with dense cover
and enough bare ground covered by forbs and shrubs for foraging chicks. Hens can lay
about one egg per day until a complete clutch averaging 12 eggs is produced between 15
to 20 days after the nest is assembled (DeVos & Mueller, 1993). After two to five days
following egg laying the pair will begin incubation. Approximately 55-70% of nests are
destroyed by predation, weather or agricultural processes and adult birds are killed in
roughly 25% of nest failures (DeVos & Mueller, 1993).
A clutch of bobwhite chicks will typically hatch within two hours after roughly 23 days
of incubation (DeVos & Mueller, 1993). The initial nesting for a pair of bobwhites
succeeds approximately 33% of the time, and hens can re-nest two to three times in a
season, which allows for three quarters of the surviving birds to hatch more than one
clutch (Burger et al., 1995).
Chicks weigh an average of 7 g after hatching, and bobwhite hens will begin encouraging
the young to forage on insects in safe and covered open areas near and away from the
nest (Stoddard, 1931). A brood can range between 2 to 100 acres during this early rearing
18 Texas Tech University, Jordan Hunter, December 2016
period, and the chicks are capable flight after two weeks (Klinger, 2011). Approximately
50% of new bobwhite chicks will not survive due to predators or severe weather
disturbances within these first two weeks (Klinger, 2011).
Females can be differentiated from males after eight weeks through the brown plumage
in the throat patch. Male bobwhites can be identified by their white throat patch and
black eye stripes and collar. Juvenile quail begin to appear more like adults within 12 to
16 weeks and will have their full plumage for the next early spring breeding season by 21
weeks of age (Burger et al., 1995). It takes about a year before juveniles are
indistinguishable from adults, as younger birds will still retain the more-pointed ninth
and tenth primary wing feathers and buff-colored tips of the greater primary coverts not
observed in adults.
2.2.3 Anatomy and biology
The average size of an adult northern bobwhite is 25.4 cm, with a wingspan between 23
and 28 cm, and a weigh 142 – 227 g (Wooding, 2009). Like most gallinaceous species,
the bobwhite anatomy is comprised of a light skeletal system along with strong muscular,
and respiratory systems to suit its flight style. Plumages of bobwhite have a sexual
dichromism in color particularly around the neck and eyes, and bird feathers will molt
once a year generally after breeding season (Butcher, 2016).
The alimentary tract of bobwhites is comprised of a pointed bill for picking up food, a
triangle-like tongue, esophagus, trachea, and a crop for storing grit, seeds and insect food
sources prior to digestion. The quail have two stomachs—the proventriculus or “true
stomach” for storing and secretory enzymatic breakdown of food. And the ventriculus,
where the physical grinding of food with digested grit takes place before being
19 Texas Tech University, Jordan Hunter, December 2016
transported back to the proventriculus to complete digestion. Movements of the small
intestine and rectum transport food and fluid into the cecal lumen which is generally
emptied once a day in the morning (Fenna & Boag, 1974). Bobwhites have a heart, liver
and pancreas with secretory ducts that open into the duodenum, these organs are
necessary for metabolism of blood for detoxification and systemic circulation. Bobwhite
anatomy includes kidneys for filtering waste, gall bladder and two bile ducts, a rectum,
and a cloaca that serves a dual purpose for waste excretion and reproduction. The quail
do not have sweat glands and cool themselves by rapid fluctuations of their air sacs.
Quail testicles can be white or yellow in color and enlarge during the breeding season
when fertile semen is produced. The birds reproduce using a posterior opening also used
for waste excretion called the cloaca that exists in males and females. Mating birds will
touch cloaca for enough time to suitably transfer sperm which can take as little as a few
seconds. Avian species also possess a bursa of Fabricius, located near the cloaca which
produces immune related antibodies in developing birds (Glick et al., 1956). Bobwhite
quail have an average life expectancy of eight and a half months and experience a high
rate of mortality within their first year either from succumbing to disease, predation, or
weather (Brennan, 1999).
2.2.4 Toxicology
Environmental toxicology studies of birds have played a significant role in understanding
the effects of toxic contaminants among all wildlife species dating back to publication of
Rachel Carson’s Silent Spring (Carson, 1962). Birds’ higher rates of food consumption
and metabolism, followed by periods of starvation that mobilize lipid reserves, hormone-
dependent behaviors, developmental strategies, and control of sexual differentiation,
20 Texas Tech University, Jordan Hunter, December 2016
influence the toxicological and toxicokinetic consequence of environmental contaminant
exposure (Touart, 2004). These physiological factors indicate that the biological fitness
of birds can be altered significantly by small changes in the function and balance of its
endocrine system (Scanes & McNabb, 2003).
The northern bobwhite has been utilized as an avian eco-toxicological model and in the
study of health status and disease among vertebrates (Gust et al., 2015; Williams et al.,
2000). Different quail species including Corturnix, and Colinus have been regularly used
in laboratory studies for the physiological and behavioral assessment of pesticides on
wildlife, and bobwhites were among the first wild animal species to have their life history
and habitat examined in a modern systematic study of environmental factors influencing
population ecology (Stoddard, 1931).
The attributes that make quail an asset in avian eco-toxicological modeling have helped
in studies to characterize its genomics and specific gene expression and tools have been
created in support of future bobwhite toxicological studies (Rawat et al., 2010).
Overall studies have shown that bobwhite quail are generally sensitive to exotoxins
and/or suitable avian models for toxicity tests (Brausch et al., 2010; Gust et al., 2015;
Kitulagodage et al., 2011; Ottinger et al., 2005; Quinn et al., 2012; Romijn et al., 1995).
The avian renal portal system provides increased support for metabolism and excretion of
xenobiotics (Pan & Fouts, 1979; Rennick, 1976) and has higher concentrations of P-45
enzymes compared to mammals (Walters et al., 1987). However, when compared to
mammals, the kidneys of birds are more susceptible to xenobiotic toxicity as venous
blood from avian digestive tracts is transported directly to kidneys instead of flowing
through the liver initially (Wolfe et al., 1998). In general, immature birds are less able to
detoxify xenobiotics compared to adults (Makri et al., 2004).
21 Texas Tech University, Jordan Hunter, December 2016
For nearly all toxic chemicals, the toxic action or stress exerted on an organism is
moderated by endocrine and/or immune processes that exist to maintain homeostasis
(Crisp et al., 1998). Thus, it is difficult to differentiate between a direct disruption of an
endocrine process or whether the disruption is a consequence of another systemic stress.
2.2.5 Infectious disease and parasites
The occurrence of parasitic and infectious diseases occurring in northern bobwhite quail
has been reported from many studies (Bruno et al., 2015; Kellogg & Calpin, 1971; Kocan
et al., 1979). Kellogg & Calpin (1971) catalogued various pathogens found in bobwhites
including: four viral, five bacterial, two mycotic, and twelve protozoans. Additionally,
other parasites detected in bobwhites included: one trematode, 13 cestodes, three
acanthocephalans, 30 nematodes, and 38 arthropods. Given the number of reported micro
and macro-parasites and the economic importance of bobwhite for food and game, the
prevalence of any infectious disease in wild and pen-raised birds is a significant concern.
Of the known biological threats to bobwhite, the three most common and damaging are
quail bronchitis, ulcerative enteritis, and quail pox (Dozier & Bramwell, 2002; Kellogg &
Calpin, 1971). In addition, more diseases such as mycoplasma, botulism, coccidiosis, and
capillaria worms are known to have also been problematic with quail (Dozier &
Bramwell, 2002).
The most common debilitating disease in bobwhites is believed to be ulcerative enteritis
(UE) which is caused by infection of a gram-positive bacterium known as Clostridium
colinum and can also be found in turkeys, grouse, pheasant and other gamebirds (Dozier
& Bramwell, 2002). Clinical signs of UE include a deterioration in body mass caused by
22 Texas Tech University, Jordan Hunter, December 2016
dehydration and lack of nutrition, changing the bird’s behavior and posture. The small
intestine of UE infected birds contains lesions in the lower small intestine, cecal pouches
and large intestine and ulcers can be seen through the intestinal wall.
Quail bronchitis (QB) is a naturally occurring and usually fatal disease caused by an
adenovirus (quail bronchitis virus). The virus can be transmitted vertically from parent to
embryo, and horizontally from one quail to another. The rates of morbidity and mortality
among infected birds are close 100% and 50%, respectively. Quail bronchitis spreads
quickly in quail populations and young and newly hatched birds four weeks of age and
less are at greater risk (Dozier & Bramwell, 2002). Signs of sub-clinical infection may
also appear in quail exceeding eight weeks of age. There is no treatment for QB and the
virus can still be shed by sub-clinical birds and quail recovering from infection. Reduced
appetite, and severe respiratory issues as well as white mucous developing throughout the
body are clinical signs of QB infection. Quail pox, or fowl pox, is a disease caused by
the avipoxvirus (poxviridae) and is generally transmitted by mosquitoes, but can enter the
body through skin abrasions, ingestion with minor abrasions to the upper digestive tract,
and potentially by swallowing infected tears. The incidence of avian pox transmission
increases during the fall and winter months. Clinical signs of fowl pox are raised,
blanched nodule lesions found on the skin and dipthertic membrane of the respiratory
tract. Wart-like growths can appear around the eyes in cutaneous pox, and in wet pox
raised yellow blemishes can appear on mucous membranes around the mouth, esophagus,
trachea, and lungs causing difficulties in breathing and swallowing. Reduced egg
production, growth, blindness, and increased mortality as well as elevated predation
secondary infections, trauma, reduced male mating success, and death are associated with
avipoxvirus (Dozier & Bramwell, 2002).
23 Texas Tech University, Jordan Hunter, December 2016
External parasites such as lice, ticks, mites, and fleas, are common among wild quail, as
are internal helminths found in the intestines and other organs. The primary parasites of
interest in our study, the Spiruroid class nematodes represented by Oxyspirura petrowi
and Oxyspirura mansoni, both commonly found within the eye orbit, between the globe
and the nictitating membrane of bobwhite quail and poultry respectively.
2.3 Northern bobwhite quail population decline
2.3.1 Population changes over time
It is believed that northern bobwhite populations throughout the United States have been
declining since the latter part of the 20th century, and that broad scale declines likely
started sometime between 1875 to 1905 (Halley et al., 2014; Klinger, 2011). In addition
to these lower numbers, reduction in the habitat range of quail within the last 30-40
years, reflected in the disappearance of bobwhite populations near the northern edge of
its historical range (Klinger, 2011).
The population fluctuations of quail have been known to change significantly and the birds are characterized as having “boom or bust” short-term population changes depending on the season or year. This species has undergone a large and statistically significant decrease over the last 40 years in North America, which has been approximated to be a 35.2% decline per decade (Butcher and Niven, 2007).
While short-term population reductions for bobwhites can be expected due to seasonal
variations, the evidence of consistent population decline over the last four decades is
concerning. In Texas alone, bobwhite quail populations have declined around 5.6% per
year since 1980, with scaled quail populations declining at a rate of about 2.9% per year,
24 Texas Tech University, Jordan Hunter, December 2016
accounting for a 75% loss in bobwhites and a 66% loss in scaled quail (Angelo, Elliot, &
Brennan, 2012).
Data from the North American Breeding Bird Survey (BBS, 2007) indicate that quail
have declined 4.2% annually between 1966 and 2007 and during the past 15 years the
The Institute of Environmental and Human Health at Texas Tech University has
conducted two separate surveys to monitor quail population trends. The quail call count
survey suggests populations have declined 4.0% annually and the rural mail carrier
survey indicates populations are declining at a 2.9% annual rate. The decline in quail is
not unique to one specific location or region, as the range-wide population has declined
66% since 1980 (Dimmick et al., 2002). Populations of other bird species that are
dependent on grasslands and shrub habitats have declined at similar rates (Norman &
Puckett, 2010).
2.3.2 Factors associated with bobwhite population decline
The downward trends in bobwhite population declines and range reduction have been
associated with several different environmental and biological factors. The range of
potential influences reported regarding the quail decline include: variations in annual
rainfall (Bridges et al., 2001; Hernandez et al., 2005; Lusk et al. 2002); the effect of
higher climate temperature on quail embryos (Guthery et al., 2000; Reyna & Burggren,
2012); changes in agricultural land use and size decreasing available habitat (Lusk et al.,
2002; Williams et al. 2004; Hernandez & Peterson, 2007; Brady et al., 1998; Peterson et
al. 2002); quail egg predation by red fire ants (Solenopsis invicta) (Allen et al., 2000;
25 Texas Tech University, Jordan Hunter, December 2016
Mueller et al., 1999); quail sensitivity to exotoxins (Kitulagodage et al. 2011; Ottinger et
al., 2005); hunting and harvesting by humans during drought periods
(Peterson & Perez, 2000; Peterson, 2001; Williams et al., 2004; Hernandez et al. 2005).
Investigations involving in the role of environmental contamination have also been
conducted, as ground foraging birds in the wild can potentially consume harmful
chemicals or byproducts from polluted soil or grit to facilitate food digestion (Gionfriddo
& Best, 1996).
When the toxicological effects of munitions constituents were examined in bobwhite
quail it was reported that 2-amino-4,6-dinitrotoluene (2A-DNT) was acutely toxic at high
doses (LD50 = 1167 mg/kg), and sub chronic 60d exposures of consistent oral doses of
2A-DNT caused mortality at a much lower dose (14 mg/kg-d) (Gust et al., 2015; Quinn
Jr. et al., 2010). The effects of perfluorooctane sulfonate (PFOS)—a by-product of many
commercial products such as pesticides, surfactants and adhesives-on northern bobwhite
quail were also examined (Newsted et al., 2005; Newsted et al., 2007). Acute studies
showed no noticeable signs of toxicity or mortality in birds orally dosed with 70.3 mg/kg
feed or, equivalent to 24 mg/kg bw/day in quail. Chronic dosing of quail showed the
lowest observable effect level was 10mg PFOS/kg food and overall liver weights were
larger in birds treated with PFOS and the male testes showed higher incidences of
shrinkage compared to controls.
Studies involving environmentally prevalent polycyclic aromatic hydrocarbons (PAH)
showed that sub chronic exposure of benz[a]anthracene (BAA) ranging from 0.1 to 10
mg/kg induced higher levels of metabolic enzymes ethoxyresorufin O-deeththylase
(EROD) and pentoxyresorufin O-deethylase (PROD) in both kidney and liver tissue
compared to controls, but no acute toxicity (Brausch et al., 2010).
26 Texas Tech University, Jordan Hunter, December 2016
Instances where bobwhites have been relocated to historically inhabited environments
with presently lower populations have had varying outcomes (DeVos & Speake, 1995;
Terhune et al., 2010; Scott et al., 2012). A study by Scott and colleagues (2012) found
that relocated bobwhites were unsuccessful in re-establishing new suitable habitats in
now fragmented environments. The release of pen-reared bobwhites to restock sparsely
populated environments has also not been successful with pen-reared birds having low
survival rates (Baumgartner, 1944; Bucchner, 1950; DeVos & Speake; Evans et al., 2006
1995). It has also been suggested that surviving pen-reared quail that successfully mate
once released into the wild may reduce local genetic adaptations (Evans et al., 2006;
Halley et al., 2014).
2.3.3 Role of habitat loss
Significant weather fluctuations as well as predation by raccoons, hawks, skunks, and fire
ants among other wildlife have been consistent sources of interest in the study of
bobwhite quail decline, however, habitat has been regarded as one of the most important
sources of population changes among bobwhites (Angelo et al., 2012). Significant
alterations in habitat are often indicators of the health condition and hospitality of ranges
and ecosystems, and shrinking bobwhite rangelands can increase rates of predation as
these previously suitable regions are reduced and spread farther apart (Angelo et al.,
2012; Cook, 2004; Rollins, 1999; Rollins & Carroll, 2001).
The role of human alterations in habitat must also be considered as human population
growth as well as endeavors to suppress natural fires, utilize new ranching and grazing
techniques, and timber/forest industrial usage can make it difficult for quail populations
27 Texas Tech University, Jordan Hunter, December 2016
to rebound when coupled with severe weather habitat altering events including flooding,
snow, ice, and drought.
Sharecropping that started in east Texas around the 1940s is believed to have played a
significant part in the record numbers of bobwhite in this region by creating an ideal
quail habitat. The disruption to these habitats over time through changes in farm
machinery usage and agricultural practices reducing the cover and protective plants
required by quail are believed to have made it difficult for the birds to continue to thrive
(Angelo et al., 2012).
The dramatic changes in farmland habitats due to increased land use and development in
Pennsylvania over the last 40 years are also believed to have had a harmful impact on
many grassland bird populations including bobwhites and pheasants along the northeast
United States (Staback & Klinger, 1998).
2.3.4 Potential role of pathogens and parasites in population changes
The disease susceptibility of galliformes has been well documented and potential
pathogens include many known viral, bacterial, mycoplasma, parasitic, chlamydial,
rickettsia, and fungal biological agents (Butcher, 2016). In a study of pathogens in
wild northern bobwhite quail in Oklahoma, Kocan and colleagues (1979) found that
37% of quail contained one or more species of helminths, and 68% contained intestinal
protozoa spread across four genera and five species.
Seasonal weather variations and colder temperatures, social stress, and reduced food,
among other conditions can cause gallinaceous birds to adapt physiological conditions of
increased resistance to bacterial infection (Gross & Siegel, 1965; Juszkiewicz, 1967;
Siemensen & Olson, 1981; Mutalib et al., 1983). As quail populations during cold winter
28 Texas Tech University, Jordan Hunter, December 2016
months have historically experienced large die-offs (Roseberry & Klimstra, 1984). The
role of acute thermal stress on the immune system of bobwhites was studied by Dabbert
and collaborators (1997). Similarly, Houstek and Holub (1994) observed that bobwhites
challenged with Pasteurella multocida also attained increased bacterial resistance under
cold stress (3.6 to -20 C) conditions, likely through an increase in phagocytic leukocyte
activity. A study of intestinal helminth parasitism in wild bobwhites done by Moore et
al. (1988) observed that the social interaction provided by quail coveys can potentially
increase the likelihood of disease transmissions in accordance with previous research that
found a similar correlation with other species (Brown & Brown, 1986; Freeland, 1979;
Hoogland, 1979). Host-parasite associations where parasite transmission happens mostly
within stable social groups are where the relationship between group size, prevalence and
intensity are most likely to occur (Moore et al., 1988).
Moore and colleagues (1998) have suggested that parasites with longer life cycles that
also require more than one host to complete its cycle (heteroxenous) have a weaker
relationship with the group size, the percentage of hosts infected (prevalence), and the
number of parasites found in a host (intensity) (Margolis et al., 1982) than single-host or
monoxenous parasites with rapid life cycles as time and other conditions associated with
the intermediate host play a role in overall transmission.
2.3.5 Stress and Reproduction
Stress factors and quail population flux have been explored through statistical modeling
of bobwhite population demographics through several years of data obtained from
multiple locations (Sandercock et al., 2008). The northern bobwhite’s short life
expectancy coupled with its rate of population growth make it susceptible to variations in
29 Texas Tech University, Jordan Hunter, December 2016
fecundity caused by environmental and biological stressors (Lebreton & Clobert, 1991).
Lipid reserves, diet, and stress are additional factors that can affect the initiation of
breeding for bobwhites, possibly adversely affecting reproductive success as well as
shortening the duration of the reproductive season (Mueller & Dabbert, 2002). Sensitivity
to disruptions in population growth can be compensated by the bird’s species
characteristic abilities such as high rates of reproduction, egg hatchability as well as large
clutch sizes (Klinger, 2011). The quail can also intensify seasonal reproduction by re-
nesting after an unsuccessful clutch, participate in secondary brooding and enable males
for egg incubation when needed. However, variances in nest survival (0.19–0.70) and
chick survival (0.14–0.72) reduce the rates of reproductive output. These figures along
with the short life span of bobwhites offer the quail lower survival prospects during the
six-month summer (0.01–0.92) and winter periods (0.00–0.73) (Klinger, 2011).
Reproduction and immune system processes require significant biological resources from
the quail beyond routine physiological maintenance (Monteith et al. 2014; Wobeser,
2007). Wild animals can shift metabolic priorities to make survival a primary condition
over reproduction (Morano et al., 2013; Stearns, 1992)
Weather related stressors such as severe winter affecting temperature and availability of
food sources have been known to impact the reproductive output of bobwhite quail for
two seasons after the stress event (Lathame & Studholme, 1952). This is believed to be
due to depletion of vitamin A, fat loss and muscle protein resulting in a delayed and
shortened nesting season and a reduction in clutch output and overall fertility of eggs
(Lathame & Studholme, 1952). Low body protein and macronutrients can cause
immunosuppression (Downs & Stewart, 2014). Reserves of protein are important to
30 Texas Tech University, Jordan Hunter, December 2016
sustain effective levels of immunocompetence in juvenile bobwhites (Brunner et al.,
2014; Klasing, 1998; Klasing & Leshchinsky, 1999; Lochmiller & Deerenberg, 2000).
Body fat content for use during winter fasts, thermoregulation, migration and incubation
is important for many animals and is closely correlated with the reproduction and
survival of wildlife (Barboza et al., 2009). The effect of body mass on egg laying makes
female reproduction and important factor and females are particularly sensitive to
decreased body fat during breeding, however, suitable body fat is also significantly
important for productivity in both adult male and female species of certain birds (Babbitt
& Frederick, 2008; Gates & Woehler, 1968).
Dependence on stored fat and protein sequesters energy reserves that can be also useful
for flight and storage, therefore it may be more efficient for birds breeding in
unpredictable seasonal conditions to rely on local resources when they are more plentiful
(Babbitt & Frederick, 2008; Jonsson, 1997).
The breeding season is linked with major energy and physiological requirements, and
avian immune processes might be less effective during this period as energy stored and
used for protection from disease is primary purposed for reproduction (Svensson et al.,
1998, Vleck et al., 2000; Mallory et al., 2007). Parasites of many avian species are
capable of being highly influential in the dispersal and abundance of their host (Grenfell
& Dobson, 1995). Parasite infections convey varying fitness penalties and consequences
on various bird hosts (Allander & Bennett, 1995; Dawson & Bortolotti, 2000; Grillo et
al., 2012; Marzal et al., 2005; Siikamaki et al., 1997; Sol et al., 2003). These penalties
may be based upon the epizootiology of the parasite species and the length of the
relationship established between the parasite and its intermediate and/or final host
species, the biology of the host, and the type of environment both organisms inhabit
31 Texas Tech University, Jordan Hunter, December 2016
(Atkinson & Van Riper, 1991; Cornelius et al., 2014). Seasonal changes, climate and
temperature fluctuations also affect arthropod intermediate and/or vector life cycles and
the transmission of parasites between hosts can be affected by altering these conditions
(Worms, 1972).
2.4 Oxyspirura petrowi
2.4.1 Taxonomy
Oxyspirura petrowi (Spirurida: Thelaziidae) is a heteroxenous nematode that is known to
infect both arthropod intermediate hosts and different species of galliformes and
passerine host (Bruno et al., 2015; Skrjabin, 1928).
Research by Pence (1972) has extensively reported the taxonomy of O. petrowi and it is
described as slender, with an appearance that ranges from yellow to cream in color, an
anterior that is bluntly rounded, and a posterior that is sharply attenuated. These
helminths have cervical alae, a transversely striated cuticle with amphids, a mouth with
four sub-median pairs and three circumoral pairs of cephalic papillae, and an undivided
cuticularized buccal capsule. The esophagus is divided into muscular and glandular
portions. The nerve ring has deirids present anteriorly and an excretory pore located
posteriorly. The uterus when gravid can vary in its position and extend into the region of
the esophagus. Both the anus and vulva are in the posterior section of the body. Spicules
within the body can vary in size and similarity. Both sexes have phasmids, but neither
male or female O. petrowi have gubemaculum or cauda alae. Males do have ventral
caudal papillae. Male O. petrowi vary in length between 6.27-8.63 µ (7.40), with a
maximum width between 0.185-0.330 µ (0.237). The length and width of the buccal
32 Texas Tech University, Jordan Hunter, December 2016
capsule is between 14-29 M (21); and 13-23 p (18) respectively. The length of the
esophagus is between 0.530-0.680 µ (0.590) long, the nerve ring is 0.110-0.165 µ (0.137)
and excretory pore is 0.219-0.330 µ (0.255) from the anterior extremity. The right spicule
0.121-0.320 µ (0.183) is described as long and boat-shaped. The left spicule is slim with
a sharp tip and is approximately 0.264-0.517 µ (0.381) long. The tail is sharply pointed
and roughly 0.181-0.330 µ (0.255) long. There are between 4-6 asymmetrically arranged
papillae located before and after the anus and phasmids lateral to the posterior extremity,
between 0.055-0.154 µ (0.111) in size.
Female O. petrowi are on average greater in size than males, and range in length between
7.70-12.35 µ (10.17), with a maximum width between 0.200-0.455 µ (0.308). The buccal
capsule is 18-28 µ (22) long and 15-26 µ (21) wide and the esophagus is between
0.5500.730 µ (0.620) long. The female nerve ring is between 0.110-0.15 µ 8 (0.137) and
its excretory pore measures 0.136-0.308 µ (0.260) from the anterior extremity. The vulva
is 0.500-0.700 µ (0.591) from posterior extremity. The anus length is 0.242-0.400 (0.328)
from posterior extremity. Phasmids are lateral from the posterior extremity and range in
length between 0.100-0.176 (0.136). Embryonated eggs are 35-44 µ (39) long, and 15-31
µ (26) wide. (Addison & Anderson, 1969; Barus, 1965; Cram, 1937). Juvenile worms of
this species are typically more transparent as well as smaller in length and size than their
adult counterparts which are usually significantly larger in length and thickness and have
more opacity in color (Dunham, 2014).
This oxyspirurid is associated with the eyelid and nictitating membrane (Cram, 1937;
McClure, 1949; Saunders, 1935), conjunctiva of the eyelids and nasolacrimal ducts
(Hunter & Quay, 1953; Robel et al., 2003), orbital cavity (Addison & Anderson, 1969;
33 Texas Tech University, Jordan Hunter, December 2016
Al-Moussawi & Mohammad, 2013; Barus, 1965;), and nasal sinuses (Dunham et al.,
2014) of its host.
The predominant species of oxyspirurids found in North American galliformes are,
Oxyspirura petrowi and Oxyspirura mansoni (Pence, 1972). O. mansoni is most
commonly found in poultry and O. petrowi is known to occur mostly in wild birds. O.
mansoni’s impact on the financial stability of the poultry industry has stimulated
significant research efforts to understand the life cycle and pathogenicity of this helminth
within its hosts (Kobayashi, 1927; Ransom, 1904; Sanders, 1929; Schwabe, 1950). The
pathogenicity and life history of O. petrowi is not as well established as O. mansoni, but
the two parasites are believed to share similar physiology and behavior (Bruno et al.,
2015; Wehr, 1972).
2.4.2 Background and history
There are believed to be 84 species of the subgenus Oxyspirura throughout the world,
and among these only three have been reported in North America; Oxyspirura mansoni,
Oxyspirura petrowi, and Oxyspirura pusillae (Addison & Anderson, 1969; Anderson,
2000). Infections of Oxyspirura or similar nematodes of this genus of parasites have been
found in different regions including Asia, Europe, Australia, and the Americas, and
reported in species including poultry, primates and other zoo animals dating back to 1904
where Ransom, reported O. mansoni in Louisiana and Puerto Rico (Ransom, 1904).
Proceeding these first documented observations many authors have since reported similar
eye-related helminth infections in animals described as ocular Oxyspiruriasis or
oxyspirurosis (Addison & Prestwood, 1978; Ali, 1960; Ivanova et al., 2007; Jairapuri &
Siddiqi, 1967; Schwabe, 1950; Vellayan et al., 2012; Xiang et al., 2013).
34 Texas Tech University, Jordan Hunter, December 2016
O. mansoni in Florida domestic fowl was reported by Sanders (1928) who experimented
in infecting pigeons, bobolinks, shrikes, and blue jays. Oxyspirura petrowi was first
described by Skrjabin (1929), and Cram (1937) first reported a species identified as O.
petrowi in ruffed grouse, prairie chicken, sharp-tailed grouse, eastern robin, and the
meadow lark in Michigan. Pence (1972) and Jackson (1969) were among the first reports
“The eye worm Oxyspirura sygmoides…under the nictitating membrane of the eyes in
approximately half (49.5 percent) of the [northern bobwhite] quail.” in the Rolling Plains
of Texas.
2.4.3 Habitat and environment
The natural history and ecology of Oxyspirura petrowi infections suggest that these
helminths have a wide geographic range, can be found in the America’s, Europe, and
Asia, exhibit considerable metrical variation, and occur mostly in avian hosts inhabiting
open fields and sub-marginal grass and marsh land-type vegetation ecosystems. The
nematode has been observed in many different bird species which share similar
ecological habitats, suggesting that the parasite’s host specificity may not necessarily be
dependent on the host’s physiology, but based on the occurrence of intermediate
arthropod hosts and their contact with final avian hosts located in these habitats
(Junquera, 2015; Pence, 1972).
O. petrowi is a spirurida order nematode, and oxyspirurids are known to naturally infect
the eyes of at least 28 species of bird host’s in North America, and studies by Pence
(1972) suggest that this helminth species demonstrates little host specificity.
35 Texas Tech University, Jordan Hunter, December 2016
Infection by spirurida is estimated to be present in between 47–56% of northern bobwhite
and scaled quail (Calilipepla squamata) found in Texas. (Jackson, 1969; Landgrebe et
al., 2007; Villareal, 2012).
In a study of northern bobwhite quail within the Rolling Plains region of West Texas
reported by Dunham et al. (2016) during the months of August and October between
2011-2013, 348 quail were sampled for O. petrowi. From this pool of samples, a total of
1,018 eye worms were taken from 144 (41.4%) infected birds. The abundance and
intensity of eye worms was reported to be was (mean±SE) 2.9±0.4 (range 0-64), and
(mean±SE) of 7.1±0.6, respectively. The prevalence of O. petrowi was also much higher
in adult bobwhites (58.7%) than what was observed in juveniles (35.4%).
36 Texas Tech University, Jordan Hunter, December 2016
Figure 2.1
Source: (Pence, 1972)
37 Texas Tech University, Jordan Hunter, December 2016
2.4.4 Life Cycle
Of the 84 species of known oxyspirurids, the life cycle of the poultry infecting eye worm
O. mansoni is the only species of this subgenus to have received significant investigation
(Anderson, 2000; Villareal et al., 2012). O. petrowi shares a similar life history with O. mansoni in that both helminths are heteroxenous parasites requiring at least two different hosts to complete their life cycles. The nematode will undergo three larval stages while in the intermediate host and a fourth and final larvae stage in the definitive host. The known intermediate hosts of Oxyspirura are arthropods including; cockroaches (Pycnocelus surinamensis) (Fielding, 1926; Sanders, 1928) and grasshoppers (Melanoplus spp.)
(Cram et al., 1931). Insects feeding on feces containing helminth eggs will then carry encapsulated first stage parasite larvae in their gut, and body cavity. Sometime after three weeks the larvae reach their second stage which is believed to be the period in which the major part of larval growth occurs, where molting has mostly been completed and the larvae begin to resemble miniature adults. After fifty days, the larvae reach their third stage where their morphology is more clearly defined. O. mansoni infected arthropods are consumed by birds and stored in the quail host’s crop prior to digestion. The third stage nematode larvae will leave the intermediate arthropod host likely through conditions induced by temperature changes between insect and bird hosts while in the crop (Fielding, 1926). In studies performed by Fielding (1926;1927) O. mansoni larvae will then travel through the esophagus and pharynx towards the mouth and either through the infra-ocular sinus cavity or nasolacrimal ducts into the eyes within a few minutes where they will permanently reside and develop during their fourth larval stage in which sexes can be distinguished (Ruff, 1984).
38 Texas Tech University, Jordan Hunter, December 2016
Adult eye worms will mate and embryonated eggs in females will pass down from the
eyes through the nasolacrimal tear ducts, where they are swallowed by the host, digested
and passed out in the feces within 32 days. Eggs in the feces can then be consumed by
another arthropod intermediate host continuing the life cycle of the parasite (Permin &
Hansen, 1998)
2.4.5 Non-pathological effects on host
The impact of Oxyspirura petrowi on northern bobwhite quail has been the subject of
increased interest for this avian species and other declining game bird populations such
as the prairie chicken (Dunham et al., 2014; Villareal et al., 2012). In observations by
Jackson (1969) the author reported “…erratic and peculiar behavior…” in O. petrowi
infected quail in one of the earliest reported observations of eye worms in bobwhites, and
deceased infected birds have been found near buildings suggesting that death was caused
by direct flight and trauma from impact due to visual obstruction of the eyes (Dunham et
al., 2014). Visual acuity and comfort disruptions caused by nematodes located around
the nictitating membrane and sclera create non-pathological problems that make these
infected hosts less aware of their surroundings and more susceptible to predators and
other hazards in their environment (Villareal et al., 2012).
2.4.6 Pathology in host
There are few studies investigating the pathological effect of Oxyspirura petrowi on host
organisms, however there has been considerable research focusing on the pathological
implications of other helminths in birds, as well as the related heteroxenous parasite
Oxyspirura mansoni due to its veterinary importance (Vellayan et al., 2012). Former
39 Texas Tech University, Jordan Hunter, December 2016
studies found evidence of conjunctivitis and petechial hemorrhages in the nictitating
membranes of infected hosts, as well as blindness and complete destruction of the eye-
ball (Vattandorn, 1984; Vellayan et al., 2012; Zahedi et al., 1982). Clinical signs of
bilateral cataract, inflammation of the eye tissues, mucopurulent discharge through the
nasal passage, cloudiness in the cornea and partial blindness were also symptoms seen in
oxyspiruriasis in zoo birds (Vellayan et al., 2012). In studies of domestic poultry
infected by O. mansoni Sanders (1928) and Schwabe (1950), found evidence of grave
pathology in hosts, observing heightened inflammatory responses, nasal discharge,
corneal opacity, and other eye related damage as well as blindness. However, in a study
of jungle fowl infected with eye-worms found no distinct clinical sign of pathology in 7
of 14 subjects (Mustaffa-Babjee, 1968).
There is limited and conflicting information regarding the pathological effects of O.
petrowi oxyspirurids on northern bobwhites on an individual and population basis.
Studies investigating O. petrowi and O. pusillae by have found up to 30 worms of these
species per eye in heavy infections and reported no gross pathogenicity or histopathology
in these infected hosts (Pence, 1972). In a recent study, Dunham and colleagues (2014)
noted O. petrowi infections in bobwhite quail and found helminths attached to the nasal-
lacrimal-orbital tissues, ingesting blood, with significant numbers of eye worms around
this location inducing inflammation and localized petechial hemorrhaging around the
lacrimal duct. Histopathological analysis and comparison of 10 samples of infected and
non-infected northern bobwhite found that eyes infected with O. petrowi under the
eyelid, nictitating membrane, and conjunctival sac can potentially cause mechanical
abrasion and mucosal discharge, as well as inflammatory responses causing corneal
scarring, conjunctivitis, and keratitis (Bruno et al., 2015). Eye worms located in the
40 Texas Tech University, Jordan Hunter, December 2016
intraorbital glands can also cause inflammatory responses, alter the structure of glands
and reduce secretory cells.
Additional consequences of eye worm infection in bobwhite quail can extend beyond
ocular pathology, and can affect the function other organs in the host. The transmission
cycle of O. petrowi consists of parasite migration between the host’s crop, larynx,
nasolacrimal ducts, orbit, esophagus, and digestive tract, allowing these organs to
encounter the helminth at some stage of its development. Other possible problems caused
by O. petrowi infection could include: increased susceptibility to impaired respiratory
function, and a loss in visual acuity reducing food foraging capability (Dunham et al.,
2014).
Other helminth species are also capable of secreting several enzymatically active
products that believed to be significant in facilitating infection through degradation of
soluble anti-parasite molecules or weakening of host’s immune cells (Everts et al. 2010).
Oxidative stress in the cells and tissue of helminth infected hosts is another possible
outcome of eye-worm infection—leading to many potential pathological states induced
by this parasite. An oxidative stress imbalance of antioxidants and reactive radical
species could proceed through mechanisms associated with physical and mechanical
abrasive tissue damage in proximity to the parasite and/or by heightened host immune
response and inflammation (Halliwell, 1997; Mougeot et al., 2010). Helminths are known
to elicit a Type-2 (Th2) signaling process involving many additional cytokine directed
and phagocytosis mechanisms releasing reactive species in hosts as part of immune
defense (Cliffe et al., 2005; Herbert et al., 2009; Jang & Nair, 2013). Multiple studies
have shown that parasitic helminths including, Codiostomum struthionis, Libyostongylus
41 Texas Tech University, Jordan Hunter, December 2016
douglassii, and Trichostrongylus tenuis can reduce antioxidant capability and increase
markers of oxidative damage in different avian species such as the red grouse (Lagopus
lagopus), and ostrich (Struthio camelus) (Mougeot et al., 2010; Sorci & Faivre, 2009;
Siwela et al., 2013).
2.5 Oxidative stress
2.5.1 Physiological conditions and function
Oxidative stress is the biological tipping point of the imbalance between reactive oxygen
species/reactive nitrogen species (ROS)/(RNS) and the depletion in concentration and/or
the capability and effectiveness of antioxidants (AOX) to reduce or prevent harmful
proliferation of ROS metabolites (Halliwell & Gutteridge, 2007; Pinazo-Duran et al.,
2014; Sies, 1991). The resulting physiological imbalance of antioxidant and free radicals
can create conditions causing oxidative damage on cells and tissue which in turn lead to
organ dysfunction and eventually disease. Oxidative stress is believed to be important in
affecting reproductive performance, cell senescence and aging (Beckman & Ames, 1998;
Constantini & Moller, 2009; Hulbert et al., 2007), and play a significant role in the
etiology of many diseases (Droge, 2002; Kadiiska et al., 2015). Research suggests an
ecological and evolutionary connection between the concentration of free radicals and
antioxidants and functional interactions among history traits (Constantini, 2008;
Constantini & Moller, 2009; Monaghan et al., 2009; von Schantz et al., 1999). An
abundance of ROS without the necessary antioxidants to compensate for their
proliferation can be harmful, however free radical molecules are also an integral part of
biological evolution and life (McCord, 2000; Uttara et al., 2009). Radical oxygen species
42 Texas Tech University, Jordan Hunter, December 2016
are necessary for normal biochemical function of cell processes, including, signal
transduction, gene transcription, and regulating soluble guanylate cylase activity. Other
free radical signaling molecules including nitric oxide (NO) are involved in the induction
of leukocyte proliferation and relaxation of vascular smooth muscle cells, platelet
aggregation, leukocyte adhesion, angiogenesis, vascular tone, hemodynamics, and
thrombosis (Uttara et al., 2009; Zheng & Storz, 2000).
The presence of free radicals in the environment are ubiquitous and organisms can be
consistently exposed via different pathways including, cosmic radiation, natural
resources, and electromagnetic radiation from anthropogenic pollution. Normal cellular
metabolic functions within the organism such as immune response mediated respiratory
bursts of ROS, and enzymatic reactions are also sources of naturally occurring free
radicals (Uttara et al., 2009).
Animals and even aerobic bacteria are capable of producing oxygen free radicals and
ROS metabolites through many different biochemical and physiological functions
necessary for cell signaling, maintenance, and homeostasis (Lushchak, 2011). Hydoxyl
radicals, superoxide, hydrogen peroxide (H2O2), nitric oxide, peroxynitrile, and
hypochlorous acid can all be generated within the organism predominately through
aerobic metabolism (Halliwell, 1994; Poulson et al., 1998).
To maintain efficient regulation of these molecules, small molecule antioxidants such as,
glutathione, arginine, citrulline, taurine, creatine, selenium, zinc, vitamin E, vitamin C,
vitamin A are utilized. These molecules are reinforced by various antioxidant enzymes
that include, superoxide dismutase, glutathione reductase, catalase, and glutathione
peroxidase (Poulson et al., 1998; Uday et al., 1990; Uttara et al., 2009; Yun-Zhong et al.,
2002).
43 Texas Tech University, Jordan Hunter, December 2016
Examining oxidative stress and the many conditions associated with this phenomenon is
a field of research that has gained increased interest as it can be applied to many
different areas of study including medicine, environmental science and toxicology
(Lushchak, 2011).
2.5.2 In vivo consequences of free radical generation
The established consequences of free radical generation in vivo are lipid peroxidation,
DNA strand scission, nucleic acid base modification, and protein oxidation
(Bandyopadhyay et al., 1999). Lipid peroxidation occurs as oxygen radicals catalyze the
oxidative modification of lipids (Gardener, 1989). Alkoxy (OH) and peroxy (ROO)
radicals typically act as both reaction initiators and byproducts of lipid peroxidation.
Reaction between lipid peroxy radicals and other lipids, proteins, and nucleic acids
produces more free radical products and this propagation enhances electron transfer and
substrate oxidation. The high polyunsaturated fat content of cell membranes makes them
more susceptible to oxidative reactions, altering membrane fluidity, permeability, and
changing the metabolic functions of the cell (Turrens & Boveris, 1980).
Oxidative damage to nuclear and mitochondrial DNA by ROS is also possible.
Deoxyribose oxidation, DNA-protein cross-links, and strand breakage are outcomes of
damage to nucleic acids. Hydoxyl radicals can react with DNA bases to produce different
by products including, C-8 hydroxylation of guanine to form 8-oxo-7,8 dehydro-2′-
deoxyguanosine, a ring-opened product; 2,6-diamino-4-hydroxy-5-
formamimodipyrimidine, 8-OH-adenine, 2-OH-adenine, thymine glycol, cytosine glycol
(Bandyopadhyay et al., 1999; Wiseman & Halliwell, 1996). Mutagenic alterations can
occur from ROS stimulated DNA damage, and selective modification of G:C sites are
44 Texas Tech University, Jordan Hunter, December 2016
indicators of oxidative damage. The promutagenic behavior of 8-oxo-deoxy-guanosine
and disruption of methylase binding subsequently preventing cytosine methylation has
been linked to cancer development (Weitzman et al., 1994). Cell signaling interference
by ROS can also potentially alter gene expression by redox regulation of transcription
factors and/or activators or by oxidative changes to proteins kinase cascades
(Bandyopadhyay et al., 1999).
Stress response genes such as c-fos, c-jun, jun-B, jun-D, c-myc, erg-1, and heme
oxygenase-1 can also be stimulated and activated through ROS interactions affecting
signal transduction, and cell proliferation and transformation (Watts et al., 1995).
Mitochondrial DNA that has undergone oxidative damage experiences modification of
bases and strand breaks, creating irregular parts in the electron transport chain, and
causing dysfunction leaking electrons and proliferating additional ROS and cell damage
(Bandyopadhyay et al., 1999; Richter, 1988).
Degradation of proteins can occur from reactive oxygen species and/or free radicals
generated during the mitochondrial electron transport chain. Metabolic processes that
degrade damaged proteins used to stimulate the synthesis of new proteins can initiate
oxidative damage. Modification of proteins through oxidation is a significant factor in the
development of cataractogenesis and the subsequent cross-linking of crystalline lens
protein, formation of high molecular weight aggregates, solubility loss and lens
opafication (Bandyopadhyay et al., 1999; Guptasarma et al., 1992). Aged cells,
Alzheimer’s disease neuronal cells, and iron overloaded hepatocytes can accumulate the
lipofuscin aggregates consisting of peroxidized lipid and proteins in lysosomes (Wolf et
al., 1986). Thermolabile, catalytically inactive or less active forms of enzyme in
accumulate in aging cells which show a significant increase in protein carbonyl content,
45 Texas Tech University, Jordan Hunter, December 2016
can be used as a measure of metal-catalyzed oxidation of proteins (Oliver et al., 1984).
Erythrocytes in humans show declining levels of glyceraldehyde-3-P-dehydrogenase,
aspartate aminotransferase, and phosphoglycerate kinase associated with age and an
increase of protein carbonyl content (Oliver et al., 1987). An in vitro study of the
mechanisms involved in protein oxidative damage generated reactive free radical species
in solution global and within protein, and showed that protein damage can be global
(non-specific) or localized (site specific) (Fisher & Statdtman, 1992). Non-specific
damage can potentially cause aggregation and fragmentation of protein and modification
of nearly all amino acid residues (Davies, 1987). Site specific oxidative damage occurs
during the formation of hydroxyl radicals or other reactive species at presumed metal
binding sites occupied by iron or copper within proteins. The resulting ROS then reacts
with certain amino acids in proximity to the binding site, causing site specific damage
that is not seen in any other parts of protein (Bandyopadhyay, Das, & Banerjee, 1999;
Stadtman, 1990; Levine, 1983). Site specific, metal-catalyzed damage has been reported
in many studies and is important in understanding the difficulty of treating oxidative
stress as the capability for AOX, or reactive species scavengers are reduced due to their
inability to access the microenvironment of metal binding catalytic ion sites
(Bandyopadhyay et al. 1999; Fisher & Statdtman, 1992; Halliwell & Gutteridge, 1986;
Stadtman, 1986).
2.5.3 Role in disease pathogenesis
There are many endogenous defense mechanisms available to protect against reactive
oxygen species (ROS), however when there is low availability of cell antioxidants and/or
significantly high amounts of ROS, oxidative cell damage is eventual creating an
46 Texas Tech University, Jordan Hunter, December 2016
environment for many pathological conditions (Bandyopadhyay et al., 1999; Halliwell,
1997). Approximately 100 disorders have been linked to ROS mediated processes (Das et
al. 1997; Halliwell & Gutteridge, 1990).
Oxidative stress conditions allowing for an abundance of ROS and reactive nitrogen
species in an organism causes free radical toxicity as these species interact with cell
lipids (polyunsaturated fatty acids), protein, and DNA causing inflammation,
degradation, tissue damage, and eventually cell apoptosis (Uttara et al. 2009). The
resulting oxidative damage to these components can cause severe health problems and
eventually cause diseases like, chronic inflammation, stroke, diabetes, cardiovascular
diseases, atherosclerosis, post-ischemic perfusion myocardial-infarction, Alzheimer’s
disease, gastroduodenal pathogenesis, metabolic dysfunction of vital organs,
degenerative diseases, septic shock, and cancer (Freidovich, 1999; Uttara et al., 2009;
Yun-Zhong et al. 2002).
External injury to tissues by ischemia reperfusion, heat, freezing, trauma, toxin, radiation
and infection can also generate reactive oxygen species and lead to oxidative stress and
disease development (Bandyopadhyay et al., 1999; Halliwell, 1997).
2.5.4 Oxidative damage to neurological and ophthalmic tissue
ROS activity is prevalent in the brain and neuronal tissue through sources of oxidative
stress like the metabolism of excitatory amino acids and neurotransmitters unique to this
organ. Glial cells and neurons are post-mitotic cells and are generally sensitive to free
radicals and ROs causing neuronal damage (Gilgun-Sherki et al., 2001; Uttara et al.,
2009).
47 Texas Tech University, Jordan Hunter, December 2016
The eye is often exposed to oxidative stress conditions from exogenous and/or
endogenous sources, subsequently altering the redox balance in these tissues and
potentially causing a variety of ophthalmologic disorders such as, cataract of the lens
(Varna et al., 1984;Varna et al., 2011), diabetic retinopathy (Lopes de Faria et al., 2011;
Yang et al., 2010; Yamagishi & Matsui 2011), age related macular degeneration (AMD)
(Ding et al., 2009 Kaarniranta et al., 2011; Liutkeviciene et al., 2010), and experimental
retinal detachment (Mantopoulos et al., 2011; Roh et al., 2011).
The intraocular anterior and posterior portions of the eye and associated tissues are also
particularly susceptible to ROS attack and degradation. Oxidative stress is believed to be
a primary factor in the formation of cataractous changes in the crystalline lens of the eye
(Babizhayev et al., 2004; Babizhayev & Costa, 1994; Babizhayev, 2012; Babizhayev et
al., 1988; Spector, 1995). The structure and integrity of the lens epithelial cells can be
damaged by hydrogen peroxide, dehydrostatic acid, and lipid peroxides. This damage
relates to the induction of protein disulfide crosslinks, reduced protein solubility and
lower cellular adhesions (Colitz et al., 2004; Spector, 2000; Babizhayev & Yegorov,
2014).
Research has shown that oxidants and ROS can induce premature senescence in human
lens epithelial cells, accelerating telomere shortening and reducing telomerase activity
(Huang et al., 2005; Babizhayev et al. 2011; Babizhavey & Yegorov, 2010). Data from
studies by (Babizhayev & Yegorov, 2014) have demonstrated that human
cataractogenesis involves senescence of lens cells, induced by oxidative stress linked to
DNA damage, inhibition of telomerase and marked telomere shortening. The
ribonucleic protein telomerase in mammal lens epithelial cells has been suggested to
assist damage telomeres during oxidative stress and preventing acceleration of cell
48 Texas Tech University, Jordan Hunter, December 2016 senescence. The epithelial cell layer is the initial location of oxidative stress incidence, followed by attack on the lens fibers causing cortical cataract (Spector et al., 1995).
Glutathione (GSH) is important for normal maintenance of lens transparency and is the predominant reducing antioxidant molecule found in large concentration of the lens.
GSH in collaboration with the glutathione redox cycle in the lens epithelium and superficial cortex detoxifies many harmful reactive oxidants including H2O2 and dehydroascorbic acid (Babizhayev & Yegorov, 2014).
The importance of GSH and the build-up of lipid peroxidation products through oxidative stress in the lens membranes during cataract formation makes the progression of this eye disorder highly correlated with GSH deficiency and disruption of the lens redox balance
(Babizhayev & Yegorov, 2014).
The mitochondrial respiratory chain of cellular mitochondria is a potent generator of reactive species including superoxide and hydrogen peroxide, and it contributes to a large degree of oxidative damage in vital cells. Oxidative damage from mitochondria is of large clinical importance, however direct treatment of mitochondria with antioxidants has not been entirely successful likely due to their wide range of dispersal throughout the body, ophthalmic tissues and fluids (Babizhayev, 2016).
Cataracts are one of the most significant ophthalmologic diseases in veterinary medicine and ophthalmology (Babizhayev & Yegorov, 2014). The correlation between age related cataract and oxidative damage of lens epithelial cells and the internal lens in suggests that this could also potentially be used as general biomarker for assessing systemic damage done by reactive oxygen species and life expectancy in animals (Babizhayev & Yegorov,
2014; Urfer et al., 2011). Antioxidant and lipid peroxidation markers in the blood and tissue are generally viewed as the factors most indicative of redox state in an organism,
49 Texas Tech University, Jordan Hunter, December 2016
however, there is currently no universally acknowledged method among researchers
regarding the most consistent and accurate measure of oxidative stress (Kadiiska et al.,
2015).
2.5.5 Host ocular antioxidant protection
Both the definitive host avian species (Paskova et al., 2008) and nematode parasites
(Selkirk et al., 1998) are known to utilize free radical scavenging endogenous and/or
exogenous antioxidants to prevent oxidative stress and damage. Helminths are also
known to express numerous enzymes, including; cystatins, that disrupt the hosts capacity
for antigen presenting by dendritic cells (Dainichi et al., 2001), and cysteine proteases
(cathespins) that are able to mediate or suppress hosts Th1 immunity (Everts et al., 2010;
O'Neill et al., 2001) O. petrowi’s presence in the eye cavity of quail and it’s
undetermined pathological implications suggest that the protection mechanisms of the
host’s ophthalmic tissue require further investigation.
The oxidative damage defenses of ocular tissue can be categorized into enzymatic and
non-enzymatic antioxidant classes (Varna, 1991). Enzymatic antioxidants catalyze
electron transfer from a reducing agent or substrate to a reactive oxygen species (ROS).
In this process the substrate can be recycled for additional usage by nicotinamide adenine
dinucleotide phosphate oxidase (NADPH) generated from different metabolic pathways
(Cabrera & Chihuailaf, 2011; Chihuailaf et al., 2002). Protecting enzymes catalyze the
reduction of specific ROS and the primary antioxidant enzymes in the eyes of most
animals are superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase
(GPx) (Cabrera & Chihuailaf, 2011; Cejkova et al., 2000).
50 Texas Tech University, Jordan Hunter, December 2016
Superoxide dismutase is metalloprotein with three isoforms; Cu-SOD and Zn-SOD which
are found in the cytosol and extracellular fluid, and Mn-SOD which is located within the
mitochondrial matrix (Benitez, 2006). This enzyme catalyzes the dismutation of O2- and
H2O2 and O2. All isoforms of SOD have been reported in the cornea epithelium and
endothelium (Cejkova et al., 2004), the lens epithelium, aqueous humor (Satici et al.,
2002), iris, ciliary body (Phylactos & Unger, 1998) and inner segment layer of
photoreceptor cells and pigment epithelium of the retina (Frank et al., 1999).
Glutathione peroxidase (GPx) is a selenoprotein that reduces H2O2 and hydroperoxides
into H2O and alcohol utilizing glutathione for electron donation. GPx is an important
antioxidant in the defense of ROS damage to lipid membranes and polyunsaturated fatty
acids susceptible to oxidation (Chihuailaf et al., 2002). This enzyme has four isoforms:
cellular, extracellular and plasmatic class isoforms, as well as phospholipid
hydroperoxide, and gastrointestinal forms. The cornea epithelium and endothelium
(Cejkova et al., 2004a; Cejkova et al., 2004b), lens epithelium, aqueous humor (Ozmen et
al., 2002), ciliary body, choroid, and retina contain this enzyme (Agardh et al., 2006;
Bilgihan et al., 2003; Penn et al., 1992).
Catalase (CAT) is a hemoprotein that catalyzed the transformation of H2O2 into H2O and
O2 and contains four heme groups and is in peroxisomes mitochondria and cytoplasm.
The presence of CAT has been reported in the cornea epithelium and endothelium
(Cejkova et al., 2004a; Cejkova, Vejrazka, Platenik, & Stipek, 2004b), lens epithelium,
aqueous humor (Satici et al., 2002), ciliary body, iris (Phylactos & Unger, 1998) and
retina (Agardh et al., 2006; Frank et al., 1999).
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Non-enzymatic antioxidants are a diverse category of electron donating molecules that
reduce and stabilizes free radical species to create less harmful molecules (Chihuailaf et
al., 2002). Ascorbic acid, vitamin E, vitamin A, and GSH are the predominant
antioxidants found within the eye globe tissue (Williams, 2006).
Ascorbic acid is an antioxidant that is generally found in its soluble form ascorbate anion,
and is present in this form in most tissues at physiological pH. It reduces O2 and OH
radicals into more stable forms (Yu, 1994) and is also involved in the recycling of alpha-
tocopheryl radical, a form of vitamin E, to its stable state. Ascorbate also performs as a
pro-oxidant during increased concentrations of Fe3+ and Cu2+ ions (Chihuailaf et al.,
2002; Halliwell et al., 1992), and has been reported in the cornea (Brubaker et al., 2000),
aqueous humor (Barros et al., 2003; Ringvold et al., 2000), lens (Ohta et al., 2004),
vitreous humor (Koide et al., 2006), and retina (Penn et al., 1992).
Vitamin E is the general description for eight similar compounds, four tocopherols, and
four tocotrienols, with alpha-tocopherol being the predominant and most active
liposoluble antioxidant in membranes. Alpha-tocopherol transforms ROS including O2,
OH and LOO radicals into less reactive forms, thus this reducing agent is converted to a
stable non-reactive alpha-tocopheryl radical that can be recycled by vitamin C, GSH, and
lipolic acid, and it capability is dependent on the availability of these compounds
(Chihuailaf et al., 2002). The lens (Yeum et al., 1995), aqueous humor, and retina (Zhang
et al., 2008), have all been reported to contain this molecule, however, the tissue
concentration of vitamin E is potentially reduced with increased proliferation of ROS
(Powers & Lennon, 1999).
52 Texas Tech University, Jordan Hunter, December 2016
Vitamin A is a group of compounds sharing the precursor B-carotene, a known
carotenoid neutralizer of O2. The long chains and conjugated double bonds of these
molecules allow them to convert O2 and LOO into less reactive substances (Yu, 1994).
Carotenoids are not dispersed equally among ocular tissues, the predominate forms being
lutein and zeaxanthin which are found in the macula, retina, and lens in high
concentration (Bernstein et al., 2001; Yeum et al., 1995).
Glutathione (GSH) is a tripeptide (gamma glutamyl-cysteinyl-glycine) containing a thiol
group in its active site. This agent acts by transferring electrons to reactive oxygen
species including hydroxyl radical and carbonyls, and is subsequently converted into an
oxidized form (GSSG) (Benitez, 2006; Yu, 1994). This antioxidant is vital for lenticular
protein maintenance (Zhang et al., 2008) and as a co-substrate of GPx. GSH has been
reported in the lens (Saaki et al., 1995), cornea and retina (Kannan et al., 1993), and
along with ascorbic acid, is essential for oxidative damage defense in these tissues
(Cabrera & Chihuailaf, 2011; Taylor et al., 1995).
2.5.6 Oxidative stress induced by host immune response to pathogens
There has been significant research into the correlation between immune response and
oxidative stress (Rahal et al., 2014). ROS and RNS oxidants are used by the body’s
immune defense mechanisms to break down and destroy invading pathogens
(Splettstoesser & Schuff-Werner, 2002). When immune challenge occurs a cascade of
signaling processes works to generate ROS using nicotinamide adenine dinucleotide
phosphate oxidase (NADPH) enzymes found in several phagocytic cells that include
macrophages, eosinophils, and heterophils, and in B and T lymphocytes (Babior, 1999;
53 Texas Tech University, Jordan Hunter, December 2016
Hampton et al., 1998). Phagocytes are known to boost their oxygen uptake by 10 to 20
folds during a process called respiratory burst, and the O- produced initiates the
production of reactive oxygen species (Clark, 1990). These cells are known to produce
many prooxidants including; hydrogen peroxide (H2O2), hypochlorous acid (HOCl),
peroxynitrite (ONOO–), hydroxyl (OH) and ozone (O3). These reactive metabolites are
non-specific in their reactive targeting capability, allowing the phagocytes to mount an
effective cytotoxic immune response on any antigenic components, however the
consequence of this indiscriminate process can cause chronic inflammation and harm to
the host’s tissues as well via immunopathology (Rice-Evans & Gopinathan, 1995; Rahal
et al., 2014; von Schantz et al., 1999).
Nitric oxide (NO) is an additional oxidant species that is believed to be produced and
utilized in phagocytic defense mechanisms (Nussler & Billiar, 1993). Studies have
reported that NO is produced by activated murine macrophages as an effective reactive
species capable of eliminating cancer cells, parasites, and intracellular pathogens,
(Anggard, 1994), and inducible NO synthase activity has been reported in human
monocytes as well (Splettstoesser & Schuff-Werner, 2002; Weinberg et al., 1995). The
close relationship between oxidative stress, the immune system and inflammation have
been examined (Sorci & Faivre, 2009). Monocyte cultures have shown increased NO
expression in response to viral infections, and increased lipid peroxidation and altered
enzymatic and non-enzymatic ant oxidative responses have been correlated with
increased NO production (Valero et al., 2013).
All classes of pathogens with weakened antioxidant defenses including bacteria, viruses
and parasites, are more vulnerable to phagocytic degradation and microbial ROS
production in host tissues (Halliwell & Gutteridge, 2007; Rahal et al., 2014). In a study
54 Texas Tech University, Jordan Hunter, December 2016
by Nathan & Shiloh (2000) subjects that had lower antioxidant mechanism capability
were more vulnerable too dangerous bacterial and fungal infections. The catalysis of
superoxide anion radical O2 by NADPH oxidase and the increased production of ROS
can induce inflammatory processes can also modify immune response to pathogens
changes in IFN-y secretion, CD14 expression, acute-phase protein production and
induction of TNF-alpha can alter innate and adaptive immune responses (Puertollano et
al., 2011).
The pathology and protective immunity to most pathogens is generally T-cell, B-cell, and
macrophage dependent or cytokine mediated. Helminth infection, however is associated
with immune effector responses which feature IgE antibody production, tissue and
peripheral blood eosinophilia and inflammatory mediator mast cell activity like many
allergic disease states. These immune responses are integral for the host to protect against
helminth parasitism, but they are also involved in pathologic reactions (Damonneville et
al., 1986; Gleich et al., 1979; Klion & Nutman, 2004; Ridel et al., 1988).
Eosinophils are one of the main phagocytes associated with ROS generating immune
response and can cause damage in tissues through various means such as, direct cellular
cytotoxicity, occupying physical space by tissue infiltration, thromboembolic occurrences
caused by eosinophil hypercoagubility, and toxic cationic proteins (Costa et al., 1997;
Gleich et al., 1979; Gleich et al., 1984). Additional potential toxic inflammatory
mediators from eosinophils include; leukotrienes (Shaw et al., 1985), platelet-activating
factor (Cromwell et al., 1990), reactive oxygen species (McCormick et al., 1996), and
lysosomal hydrolases (Costa et al., 1997). Tissues in proximity to these mediators can be
damaged (Thomas et al., 1997; Yazdanbakash et al., 1987) and instigate activation and
expression of the local immune response (Klion & Nutman, 2004).
55 Texas Tech University, Jordan Hunter, December 2016
It is believed that an integral function of eosinophils is host defense against infection by
larger organisms like parasitic helminths. In vivo and in vitro studies have shown that
eosinophils will degranulate in proximity and onto the surface of helminths to destroy
them in the presence of antibodies or complements (Butterworth, 1984; Costa, Weller, &
Galli, 1997; Hitoshi et. al, 1991). They can migrate from the blood and aggregate in
locations of helminths in vivo (Teixeira et al., 1995). Large amounts of eosinophils have
been observed near intact and damaged helminths (Behm & Ovington, 2000;
Butterworth, 1984; Weller, 1991). The cytokine IL-5 can generally be found at increased
levels in hosts with helminth infections during Th2 cytokine immune signaling response,
and is integral part of mucosal immune responses causing helminth induced eosinophilia
(Behm & Ovington, 2000).
Animal and human examples have shown that eosinophils can support the pathogenesis
of helminth infections in the host ocular tissues. Eyes infected with the intraocular
parasite Onchocerca volvulus have demonstrated eosinophilic infiltration of the cornea
and conjunctival tissues during onchochercal sclerosing keratitis (Cooper et al., 1999,
Pearlman et al., 1995).
Similarly, the nematode parasite Trichostrongylus tenuis was reported to cause an
increase in oxidative damage and decrease in circulating antioxidants based upon the
number of worms observed within red grouselikely through helminth immune response
and release of ROS/RNS in the host (Mougeot et al., 2010).
Experimental manipulations of immunology and redox state using lipopolysaccharide
(LPS) injection to activate innate immune and inflammatory responses in Zebra finches
(Taeniopygia guttata) demonstrated decreased antioxidant status within 24 hours
(Bertrand et al., 2006).
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Phagocytes including macrophages eosinophils, neutrophils, and heterophils that are
exclusive to avian species, are integral in the innate inflammatory response to pathogens
and parasites (Sorci & Faivre, 2009). The exact mechanisms of bobwhite quail host
immune response to O. petrowi in the intraocular tissues and their ability to initiate
pathology are not well established, however, given that both the initiation and reduction
of cellular damage is controlled by the redox balance of oxidation and antioxidation that
occurs in aerobic and immune based scenarios observed in different animal and human
models, the immune system defense mechanism of quail might also interact in some
capacity with some components involved in oxidative stress (Rahal et al., 2014). These
potentially significant alterations in antioxidant status could have a substantial impact on
the prediction of short-term survival prospects in organisms (Alonso-Alvarez et al.,
2006).
2.6 Gene Expression and immune response in the eye
2.6.1 Innate and adaptive immune systems
The innate immune response is the initial method of host defense against
microorganisms and infection (Alberts et al., 2002). Innate immunity of the ophthalmic
tissues can be characterized by physical barriers of the bony orbit and eyelids, as well as
cellular and molecular components that protect the ocular surfaces from microorganisms
such as, tears, corneal nerves, the epithelium, keratocytes, polymorphonuclear cells, and
cytokines (Akpek & Gottsch, 2003). Epithelial cells of the cornea and stromal
keratocytes can secrete cytokines to induce immune defense including IL-1α
(Niederkorn et al., 1989). Keratocytes can produce IL-6 and defensins which provide
57 Texas Tech University, Jordan Hunter, December 2016
antimicrobial activity against bacteria, fungi and viruses and increase epithelial healing
(Cubitt et al., 1995, Ganz et al., 1985; Gottsch et al., 1998) and can also be found in
neutrophils within the conjunctiva (Haynes et al., 1999). Complement are innate
response mediating molecules and refers to several effector and regulatory proteins that
activate biologically active enzymes, opsonins, anaphylotoxins, and chemotaxins (Law
& Reid, 1995). Interferons (IFN) are proteins synthesized by leukocytes, natural killer
cells, and T-cells to respond to viral infections and induce production of major
histocompatibility complex (MHC) which enable viral infected cells to present antigens
to T-cells (Akpek & Gottsch, 2003; Su et al., 1990, Thacore et al., 1982). Other cells of
innate immunity include neutrophils that are important effectors in microbial
elimination and phagocytosis (Burg & Pillinger, 2001), and eosinophils that contain
surface receptors for IgE antibodies and complement components. Activation of
eosinophils by IL-3, IL-5, and granulocyte colony stimulating factor allows these
phagocytes to utilize granule proteins to initiate toxicity against parasites (Trocme &
Aldave, 1994). Macrophages can mediate T-cell immune response by presenting
antigens secreting inflammatory cytokines as well as perform as phagocytes (Underhill
& Ozinsky, 2002). Natural killer cells are granular lymphocytic cells that identify MHC
class 1 molecules and can degrade target cells infected by viruses, are covered by
antibodies, or tumor cells (Moretta et al., 2002).
As the innate immunity often induces a non-specific inflammatory response the ROS and
RNS produce do distinguish between host and pathogen DNA, lipids, or proteins (Sorci
& Faivre, 2009). Acquired immunity of the eye is utilized when innate immunity is
ineffective and foreign organisms and/or antigens are chronically exposed to tissues, cell
58 Texas Tech University, Jordan Hunter, December 2016
mediated processes then to reduce the threat of microorganisms (Akpek & Gottsch,
2003).
2.6.2 Immunology and immune privilege of the eye
Intraorbital glands comprised of the lacrimal and harderian glands are responsible for
maintaining normal hydration, nutrition and defense of the ocular surface tissues (Knop
& Knop, 2005). Aqueous secretions are provided by the lacrimal gland while the
harderian gland plays a vital role in ocular immunity (Dimitrov & Genchev, 2011;
Kozlu & Altunay, 2011) through secretion of immunoglobulins IgA, IgG, and IgM. This
gland also provides vascular distribution of immune cells including macrophages,
lymphocytes and granulocytes for the eye orbit (Baba et al., 1990; Boydak & Mehmet,
2009; Bruno et al., 2015).
Ophthalmic tissues and the eye globe can induce innate and acquired immune responses
to mitigate the effects of microorganisms (Akpek & Gottsch, 2003). However, the eye is
an organ with immune privilege as it does not elicit the same systemic responses to
foreign bodies or from pathogen challenge as other organs in the body do (Benhar,
London, & Schwartz, 2012; Taylor, 2009).
The exact reason for this organ’s immunological responses have not been completely
determined, however research suggests that the difficulty required for immune-
mediating cells to pass the blood ocular barrier tissues and the absence of direct
lymphatic drainage required for antigens to pass onto lymphatic nodes and induce
immune response play important part in immune cell regulation out of the eye (Barker
& Billingham, 1968; Medawar, 1948; Taylor, 2009).
59 Texas Tech University, Jordan Hunter, December 2016
The intraocular microenvironment also contains large amounts of immunosuppressive
molecules and processes which can mediate immune cell activity. Macrophages in the
aqueous humor can present antigens to promote immune suppression (Wilbanks &
Streilein, 1992). Aqueous humor can suppress IFN-γ generation and promote TGF-β
production by CD4 T-cells and contains many neuropeptides including; α-MSH, CGRP,
VIP, and SOM to suppress activation of inflammatory activity of macrophages (Taylor et
al., 1992; Taylor, 2005), cytokine TGF-β2 and the molecules, IDO and FasL to suppress
the activation of Th1 cells (Griffith et al., 1995; Taylor et al., 1994; Taylor et al., 1998;
Ryu & Kim, 2007). These features among others indicate that there is a high threshold
and many conditions that must be met before the start of inflammation and immunity
within the normal ocular tissue microenvironment (Taylor, 2009).
Experimental studies have demonstrated that ocular immunosuppressive capability can
be altered when antigens such lipopolysaccharide (LPS) are introduced into the ocular
microenvironment as in the cases of endotoxin-induced uveitis (EIU) and endotoxin
autoimmune uveitis (EAU) (Kitaichi et al., 2005; Luger et al., 2008; Ohta et al., 2000).
These studies have shown that before detection of retinal infiltration, the
immunosuppressive capabilities of the aqueous humor are inactive and large amounts of
pro-inflammatory cytokines in and T-cell subsets could be detected (Kitaichi et al.,
2005; Luger et al., 2008; Ohta et al., 2000). During these conditions the anterior
chamber-associated immune deviation (ACAID) that induces immunosuppression when
foreign antigens are present in the ocular microenvironment was inhibited at the onset of
retinal inflammationthe microenvironment did not resume its ability to induce ACAID
until after uveitis was resolved. These experiments indicate that a direct attack on ocular
60 Texas Tech University, Jordan Hunter, December 2016
tissue causes an accelerated inflammatory response, and diminished immunosuppression
for some time, and the resulting uveitis could be a way in which the ocular
microenvironment attempts to recover its immune privilege (Taylor, 2009). This creates
a potential pathway for intraocular pathogenesis through increased inflammatory
response exacerbated under chronic antigen exposure from pathogens or parasites.
For regular maintenance (moistening, nutrition, and defense), the eye surface depends
primarily on the conjunctiva and secretions from the lacrimal and Harderian glands,
collectively called intraorbital glands (Knop and Knop, 2005). While the lacrimal gland
is responsible for more aqueous secretions, the avian Harderian gland plays a vital role
in controlling local orbital immunity (Dimitrov & Genchev, 2011; Kozlu & Altunay,
2011) via immunoglobulins IgA, IgG, and IgM, as well as vascular released immune
cells (macrophages, lymphocytes, granulocytes) onto the eye orbit (Baba et al., 1990;
Boydak and Mehmet, 2009; Bruno, Fedynich, Smith-Herron, & Rollins, 2015).
2.6.3 Th2 and Th1 host immune response
Leukocytes consist of many subsets including: granulocytes, monocytes, and
lymphocytes, that are known to travel to the areas in proximity of infected tissue and
secrete metabolites. Injury to these tissues may occur as the immune response is either
misdirected or overexpressed, which can then cause immunopathology in the host.
Organisms have mechanisms to mediate this process and prevent inflammation and tissue
damage using certain cytokines like IL-10, TGF-β to mitigate and resolve this
inflammation as the pathogen is eliminated (Belkaid, 2007). Research has suggested that
cases of chronic infection by pathogen or parasite an over-stimulated inflammatory
61 Texas Tech University, Jordan Hunter, December 2016
response may cause more harm than the direct effects of the pathogen (Mackintosh et al.,
2004; Graham et al., 2005; Raberg et al., 1998)
There are two sub-groups of T-helper (Th) cells that encompass different types of
cytokine production cascades with different purposes (Cherwinski et al., 1987; Mosman
et al., 1986). Th1 cell types are associated with cells infected by viruses and other
intracellular microbes and have features that include: cytolytic activity, synthesis of IL-2
and IFN-γ, as well as help B-cells with IgG, IgM, IgA synthesis. Th2 cells are not
cytolytic but are known to generate IL-4 and IL-5 cytokines, assist in IgE helminth
specific antibody synthesis, as well as IgG, IgM and IgA antibody production. The
immune system chooses what CD4 Th1 or CD4 Th2 cells are induced based upon the
type of infection or autoimmune disease. Inflammatory states with delayed type
hypersensitivity reactions and low antibody generation leads to Th1 cell selection.
Inflammatory reactions associated with constant antibody production such as allergic
reactions or larger pathogenic organisms initiating significant amounts of IgE generation
are typically associated with Th2 cells. The cytokine profiles associated with Th cells
dictate the intensity, duration and type of immune or inflammatory response the host
produces and are important in the resulting impact of infectious diseases. Studies have
shown that the Th1 response of hosts with schistosomiasis is linked to purging this
parasite, and that Th2 responses will generally result in granuloma formation and disease
development (Infante-Durante & Kamradt, 1999).
In most cases helminth infection, will initiate a Th2 immune response in the host that
results in eosinophilia, goblet and musocal mast cell hyperplasia and synthesis of
noncomplement fixing antibodies (Gause et al., 2003)
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The human eye worm onchocerca volvulus for example has been reported to cause
increased CD4 cells and IL-4 production eventually leading to inflammatory response in
the ocular tissue, a primary characteristic Th2 response (Akpek & Gottsch, 2003; Chan et
al., 1993, Chan et al., 1989). IL-5 is another cytokine associated with Th2 response and is
known to induce phagocytic eosinophils to purge parasites (Spencer & Weller, 2010). IL-
13 is a cytokine secreted by CD4 Th2 cells that is known to activate IgE in human B
cells, and initiate physiological changes in tissue that enable parasite clearance (Wynn,
2003). IL-8 is a chemokine referred to as a neutrophil chemotactic factor, it is a key
mediator of inflammation and induces movement of phagocytes to areas of tissue in
proximity to infection and signals phagocytosis around these areas (Modi et al., 1990).
During an oxidative stress state IL-8 secretion increases, leading to more recruitment of
inflammatory cells and oxidant stress mediators (Vlahopoulos et al., 1999). Studies have
shown that there is a close relationship between Th1 and Th2 immune responses in
mammals and birds, with factors upregulating or downregulating the activity of these
cells in inverse proportion (Degen et al., 2005). Depletion of glutathione, an outcome of
oxidative stress, has been shown to downregulate Th1 immunity and affect the polarity of
immune response as oxidative stress skews the immune response towards Th2 cells and
suppresses Th1 cell differentiation (Kim et al., 2007).
IL-10 is known to have many different effects on the hosts immunoregulation and
inflammation including downregulation of Th1 cytokine expression, MHC class II
antigens, and co-stimulatory molecules on macrophages (de Waal Malefyt et al., 1991).
The other purposes of this cytokine are to inhibit bacterial antigen product initiation of
pro-inflammatory cytokines, facilitate B cell survival, and antibody production (Varma et
al., 2001).
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2.6.4 Transcription factors and avian uncoupling protein
Other regulators of immune response and oxidative stress include GATA-3 and avian
uncoupling protein. GATA-3 is a transcription factor that is known to induce IL-4, IL-5,
and IL-13 secretion in Th2 cells and suppress the differentiation of naïve T-cells towards
the Th1 cell type while facilitating differentiation of Th0 cells to Th2 cell subtypes (Yagi
et al., 2011). The capabilities of GATA-3 are thought to be related to tissue type (Wilson,
2008), and its transcriptional level expression closely linked to IL-4 receptor cytokines
(Cook & Miller, 2010).
Uncoupling proteins are part of a group of mitochondrial proteins involved in uncoupling
the oxidation of reduced substrates from the phosphorylation of ADP to ATP through a
leak of protons within the cellular inner membrane (Ricquier & Bouillaud, 2000). The
role of avian uncoupling proteins (avUCP) has not been completely characterized, but it
is believed to be associated with facultative thermogenesis, and lipid metabolism (Colin
et al., 2003).
Studies by (Criscuolo et al., 2005) have reported that avUCP overexpression in yeast
instigates mediation of ROS, and one of primary purposes of this protein in birds is to
stop mitochondrial ROS proliferation and oxidative stress during periods of high
metabolic activity (Rey et al., 2010).
2.7 ROS and immune response mediated disease in ocular tissue
There is high metabolic activity in the eye and it requires a high consumption of ATP to
function. Components of the eye such as the cornea, lens, vitreous and retina interact in
some way with external light and therefore photochemical ROS generation is also a
64 Texas Tech University, Jordan Hunter, December 2016
factor (Barros et al., 2003; Varna, 1991). These physical and metabolic characteristics
make this organ particularly susceptible to oxidative stress (Cabrera & Chihuailaf, 2011)
2.7.1 Cornea
A decrease in corneal antioxidants including CAT, GPx, and SOD, and decreasing
Na+/K+ ATPase in the corneal epithelium and endothelium are factors that contribute to
corneal hydration, changing its transparency and releasing proinflammatory cytokines
(Cejkova & Lodja, 1996; Cejkova et al., 2000; Lofgren & Soderberg, 2001; Newkirk et
al., 2007). Physiological components of glycosaminoglycans in the stroma can be altered
by Ultaviolet (UV) radiation, making this tissue more susceptible to deterioration from
phagocytic cell enzymes (Carubelli et al., 1990; Cejkova et al., 2004). Ascorbic acid is
highly concentrated in the corneal center and is one of the primary antioxidants in the
tissue along with SOD, and during oxidative stress CAT, GPx and SOD are reported to
decrease, heightening the levels of ROS that can injure the cornea (Cejkova et al., 2000).
2.7.2 Aqueous humor
Inflammatory responses in proximate tissues and UV radiation are the primary sources
of ROS proliferation, the aqueous humor (Cejkova et al., 2004; De Biaggi et al., 2006).
UV radiation absorbed in the humor has been reported to increases ROS activity and
decrease GSH metabolismascorbic acid is highly concentrated in the environment,
however and able to act as a primary UV filter (Varna, 1991). During idiopathic acute
uveitis, the total antioxidant capacity of the aqueous humor has been reported to decrease
by 40% (Barros et al., 2003; Cheng et al., 1999; De Biaggi et al., 2006). Subsequent
increases of H2O2 concentrations in aqueous humor can lead to injury of the corneal
65 Texas Tech University, Jordan Hunter, December 2016
endothelium, lens, and ciliary body trabecular meshwork. Increasing ROS concentration
has also showed a decrease in aqueous humor outflow and has been considered a factor
in the formation of eye diseases like glaucoma (Andley, 1987).
2.7.3 Lens
The avascular structure of the lens and lenticular protein components are factors in its
susceptibility to oxidative damage. Its high exposure to UV radiation causes a reduction
in antioxidant concentrations in the lenticular nucleus. Alterations of the aqueous and
vitreous humor near the lens can also cause inflammation of adjacent eye intraocular
components or make the lens susceptible to defects from systemic metabolic disorders
like diabetes (Andley, 1987; Williams, 2006). The lens GSH is kept in a reduced state by
NADPH produced through the pentose phosphate pathway using glucose-6-phosphate
dehydrogenase (G-6-PD) (Saaki et al., 1995); and oxidation of crystalline proteins (α, β,
and γ-) is believed to be the mechanism of lenticular opacity (Andley, 1987; Truscott,
2005). Oxidation of crystalline protein thiol groups generates disulfide adducts and by
products that cause protein aggregation leading to development of cataract (Williams,
2006). Oxidative damage based protein deterioration, disulfide group increases, and
sulfhydryl group decreases, are linked to cases of cataract compared to transparent lenses
(Boscia et al., 2000; Kulshrestha et al., 1983). High levels of GSH and ascorbic acid are
the primary antioxidant defenses of the lens (Taylor et al., 1995). MDA levels have been
reported to increase as antioxidant levels decreased in animals with cataracts (Barros et
al., 1999).
Ninety-five percent of the high glutathione content in the lens is in the reduced form
(GSH), to detoxify reactive oxygen species that might damage the tissue (Giblin, 2000,
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Paterson & Delamare, 1992). A redox cycle in the lens epithelium cells and superficial
cortex uses glutathione reductase and NADPH to reduced oxidize glutathione (GSSG).
This cycle is also associated with the detoxification of H2O2 and phospholipid
hydroperoxides that are present in the aqueous humor (Babizhayev, 2012; Colitz et al.,
2004). The purposes of lens GSH are believed to be associated with keeping -SH proteins
thiols in their reduced state preventing high molecular weight protein aggregates from
affecting lens opacity, and protecting these thiol groups that are used in cation transport
and permeability (Babizhayev & Yegorov, 2014; Reddy, 1990).
2.7.4 Retina
The retina part of the neurosensorial intraocular tissue of the eye and contains a high
number of polyunsaturated lipids making it susceptible to reactive oxygen species
(Miranda et al., 2006). ROS is generated in the retina in large part due to cells with high
oxygen consumption (Wang et al., 1997), UV exposure and diseases of the eye tissue like
glaucoma that affect vascular irrigation (Agardh et al., 2006). Pigments within the lens
including melanin, lipofuscin and lutein are factors in ROS production from
photosensitizing processes (Andley, 1987). It is believed that oxidative stress is a primary
factor in glaucoma which is associated with decreased function of retinal ganglion cells
and cell death from lipid peroxidation (Levin et al., 1996), optic nerve enlargement,
visual field reduction and blindness (Kang et al., 2003). High ROS imbalance has also
been linked to ischemia and reperfusion in the retina increasing NO proliferation in the
vitreous and aqueous humor (Kallberg et al., 2007). The main sources of antioxidant
protection in the retina are vitamin C, vitamin E, carotenoids, GPx, SOD and CAT
enzymes, and GSH (Andley, 1987; Nakajima et al., 2008; Penn et al., 1992).
67 Texas Tech University, Jordan Hunter, December 2016
2.8 Analysis of oxidative damage and immune response in ophthalmic tissue
2.8.1 Hematoxylin and eosin staining of ocular tissue
Hematoxylin-Eosin staining of ocular tissue can be used to determine pathology in the
eye related to oxidative damage (Saenz-de-Viteri et al., 2014). The hematein and
aluminum ion complex produces basic/positive hemalum which stains the basophilic
nuclei containing acidic negatively charged DNA/RNA as well as granules within the
cells blue. A counter stain of acidic and negative eosin Y colors eosinophilic positively
charged amino acids, proteins and other molecules of tissues red or pink. Using this
method, signs of tissue inflammation from inflammatory eosinophils and cell related
damage can be observed.
In a study of phototoxicity in the intraocular tissues of animal models a greater decrease
in neurosensory retina and the outer nuclear layer thickness in rabbits exposed to intense
light as evidenced by an increase in vacuolation inside the outer segments of
photoreceptors, apoptotic cell death and inflammatory processes (Saenz-de-Viteri et al.,
2014).
Studies of O. petrowi infected bobwhite quail by (Bruno, Fedynich, Smith-Herron, &
Rollins, 2015) used H&E stain of ophthalmic tissue to show inflammatory reactions.
These were evidenced by the appearance of immune cells within corneal epithelium,
Descemet’s membrane, and the connective tissue of the stroma. This histopathology also
revealed a decreased amount of antibody presenting plasma cells and increases in other
lymphocytes in eye worm infected birds. The lacrimal and harderian gland tissues of
infected birds also showed more acinar atrophy which reduces the amount secretory
molecules and immune cells that can be utilized to protect the eye’s surface.
68 Texas Tech University, Jordan Hunter, December 2016
2.8.2 Antioxidant and lipid peroxidation surrogate biomarkers
There is no definitive biological component, technique or assay that represents the most
complete measure of antioxidants and oxidative stress in an organism (Cohen et al.,
2007; Frankel & Meyer, 2000; Prior & Cao, 1999). The antioxidant protection in tissues
is comprised of mostly enzymatic scavengers and micromolecular components to a
lesser extent (Cohen et al., 2007), and the enzymatic antioxidants such as superoxide
dismutase, and catalase contained in mitochondria are considered the initial forms of
ROS defense (Godin and Garnett, 1992).
The two main factors most associated with oxidative stress in tissues that can be
measured are the (1) ability of the available enzymes and micro-molecules in the sample
to quench free radicals, and (2) oxidative damage, generally measured as lipid
peroxidation levels. Glutathione (GSH), glutathione peroxidase (GPx), superoxide
dismutase, and catalase (CAT) assays have been generally used as common markers of
antioxidant and oxidant status in tissue samples (Agardh et al., 2006; Barros et al., 1999,
Taylor et al., 1995; Cohen et al., 2007; Siwela et al., 2013), and lipid peroxidation has
been typically measured through thiobarbituric acid-reactive substances assay (TBARS)
(Cohen et al., 2007; Gaal et al., 1997).
Observation of the decrease in endogenous antioxidants in response to redox imbalance is
the most common method of oxidative stress assay assessment (Kadiiska et al., 2015).
Ferreira et al. (2004) suggest that reduced superoxide dismutase, GPx activities in the
aqueous humor could provide reasonable surrogate biomarkers in the aqueous humor of
glaucoma patients as one example. Malondialdehyde in TBARS assays is the measured
by-product of lipid peroxidation from oxidative damage. This analyte is inversely
correlated to antioxidant levels as it has been reported to increase in ocular tissues
69 Texas Tech University, Jordan Hunter, December 2016
(Mancino et al., 2011) under oxidative stress, and in cases of helminth infection (Siwela
et al., 2013).
2.8.3 Immune response biomarkers associated with oxidative stress
Immune response studies involving oxidative stress induced by pathogens and parasites
in tissues have mostly focused on the factors influencing the T-helper cell immune
response and the associated cytokines and transcription factors signaling and/or
expressing inflammatory responses and leukocytes (Moghaddam et al., 2011; Sorci &
Faivre, 2009). Research examining these relationships with endogenous antioxidants
(Kim et al., 2007), and other studies have suggested that oxidative stress promotes
polarization of human naïve T-cells toward Th2 type effectors (King et al., 2006), and
that gene expression of IL-4, IL-13, IL-8, and IL-10 cytokines, as well as the
transcription factor GATA-3, are all closely related to conditions characteristic of
oxidative stress in helminth and pathogen challenged tissues (Frossi et al., 2008; Hong et
al., 2006).
2.9 Summary
The general trend of declining northern bobwhite quail populations across West Texas is
of significant ecological and economic concern (Rolling Plains Quail Research Ranch,
2010). Bobwhite quail share habitats with other wildlife and human populations, and
alterations in these environments associated with quail survivability could signal larger
ramifications for these ecosystems (Angelo et al., 2012). Broadly examining bobwhite
population dynamics and the ecoregions they inhabit merits further research and more
70 Texas Tech University, Jordan Hunter, December 2016
specifically studying the role of pathogens and parasites on the host quail’s fitness,
behavior and fecundity (Moore et al., 1988).
The parasitic eye worm Oxyspirura petrowi is a helminth frequently found in the eye
orbit, and around the ocular surface, lacrimal tissues and nictitating membrane of ground
dwelling birds including the northern bobwhite quail (Skrjabin, 1929). Existing
knowledge of O. petrowi induced pathology on host quail and the impact of this parasite
on overall visual acuity and fitness in the host is limited (Bruno et al., 2015). Saunders
(1935) noted that some infected quail are reported to exhibit signs of inflammation
around sites of worm infestation. Studies have shown that pathogens and parasitic
helminths can induce harmful oxidative stress in animal and bird host’s tissues which
could be linked to these signs of inflammation (Mougeot et al., 2010; Siwela et al., 2013;
Sorci & Faivre, 2009).
Oxidative stress is a phenomenon caused by significant proliferation of free
radical/reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) and the
impaired function or availability of antioxidants such as, glutathione, superoxide
dismutase, and catalase, as well as their processes (Rahal et al., 2014). The imbalance of
high ROS and low antioxidants can cause unmediated to reactive species attack on
DNA/RNA, proteins, and lipids leading to apoptosis and tissue damage (Bandyopadhyay
et al., 1999). Oxidative damage is believed to be the etiologic agent in the formation of
many neurodegenerative and ophthalmic diseases and is often associated with vertebrate
immune response (Rahal et al., 2014; Sorci & Faivre, 2009).
Helminths are known to elicit a T-helper 2 (Th2) immune response in many vertebrate
host’s by signaling cytokines including; IL-4which induces naïve T-cells to Th2 type,
71 Texas Tech University, Jordan Hunter, December 2016
the inflammatory mediating IL-13which induces B-cell secretion of IgE helminth
related antibodies, anti-inflammatory IL-10, and eosinophil activating IL-5 in tissues
(Degen et al., 2005; Frossi et al., 2008; Hong et al., 2006). The vertebrate host immune
response also signals polymorphonuclear leukocytes, such as neutrophils, basophils,
eosinophils, and avian heterophils which utilize respiratory bursts of ROS to degrade and
eliminate extracellular parasites from the body (Behm & Ovington, 2000, Spencer &
Weller, 2010; Splettstoesser & Schuff-Werner, 2002). ROS are non-specific attackers of
cellular proteins and lipids and can damage parasite as well as hosts tissues (Rahma et al.,
2012). Under these circumstances chronic helminthiasis coupled with an active immune
response and depletion of endogenous antioxidants could potentially cause oxidative
stress in host tissues leading to oxidative tissue damage and disease (Sorci & Faivre,
2009). The high metabolic activity and constant external environment exposure of the
eye and ophthalmic tissues makes it susceptible to oxidative stress (Cabrera &
Chihuailaf, 2011). Increased proliferation of ROS in the ocular environment has been
linked to diseases such as conjunctivitis, uveitis, macular degeneration, retinopathy,
cataract, and glaucoma (Babizhayev et al., 1988; Rahma et al., 2012;).
The correlation between O. petrowi infections and their potential to cause
immunopathology induced diseases of the eye through oxidative stress mechanisms in
northern bobwhite quail has not been examined. The purpose of this study is to identify
signs of pathology within the ocular tissues of O. petrowi infected bobwhite quail using
histopathological techniques, assess oxidative stress in infected tissues from antioxidant
and oxidative damage marks, and determine if a Th2 immune response occurs in these
tissues through gene expression. The combined information gained from these studies
72 Texas Tech University, Jordan Hunter, December 2016 could provide information useful in the study of northern bobwhite quail fitness and behavior and the selection of surrogate biomarkers for the early detection of pre-clinical manifested ophthalmic disease.
73 Texas Tech University, Jordan Hunter, December 2016
3 CHAPTER III
HISTOPATHOLOGY OF OCULAR TISSUE
3.1 Introduction
The decline of northern bobwhite quail (Colinus virginianus) populations across Texas
over the last few decades is a phenomenon of considerable ecological importance
(Brennan 1999; Williams et al., 2004). Old world (Phasiandiae) and new world
(Odontophoridae) quail and related galliformes have been historically important birds as livestock, game, agriculture, and scientific study (Gust et al., 2015; Stoddard, 1931;
Williams et al. 2000). Understanding both the environmental and biological factors that may have contributed to the decline of C. virginianus is vital in aiding the conservation efforts for this species and other wildlife sharing the same or similar habitats (Brausch et al., 2010; Gust et al., 2015; Kitulagodage et al. 2011; Romijn et al., 1995; Ottinger et al.
2005; Quinn et al. 2012). Identifying the significant environmental factors or pathogenic agents that influence this avian population may also benefit our broader understanding of how these factors influence wildlife and human populations on all trophic levels within ecoregions (Angelo et al., 2012).
Species such as the northern bobwhite and Japanese quail (Corturnix japonica) have been used as effective animal models in the study of toxicology and the physiological responses of vertebrates to pharmaceuticals, pesticides and pathogens (Lusk et al., 2002;
Newsted et al., 2007; Quinn et al., 2009; Quinn et al., 2012; Romijn et al., 1995).
The occurrence of parasitic and infectious diseases affecting northern bobwhite quail
have been examine din numerous studies (Bruno et al., 2015; Dunham et al., 2014;
74 Texas Tech University, Jordan Hunter, December 2016
Kellogg & Calpin, 1971; Kocan et al., 1979). The parasitic eye worm Oxyspirura petrowi is a helminth frequently found in the eye orbit, and around the ocular surface, lacrimal tissues and nictitating membrane of ground dwelling birds including the northern bobwhite quail (Skrjabin, 1929). Existing knowledge of O. petrowi induced pathology on host quail and the impact of this parasite on overall visual acuity and fitness in the host is limited (Dunham et al., 2014). Some infected quail have been reported to exhibit signs of inflammation around sites of worm infestation including the conjunctiva, and intraorbital lacrimal and harderian gland tissues (Bruno et al., 2015; Saunders, 1935).
Studies have shown that pathogens and parasitic helminths can also induce harmful oxidative stress in animal and bird host’s tissues which could be linked to these signs of inflammation (Mougeot et al., 2010; Siwela et al., 2013; Sorci & Faivre, 2009).
The correlation between O. petrowi infections and their potential to cause intraocular pathology and diseases of the eye through oxidative stress mechanisms in northern bobwhite quail has not been examined. The purpose of this study was to identify signs of pathology within intraocular environment of O. petrowi infected birds using a comparative histopathological analysis of ophthalmic tissue taken from eye worm infected and uninfected wild northern bobwhite quail captured in West Texas and
Oklahoma. H&E stained sections of quail eye globe were histopathologically examined and infected quail were observed to have a higher incidence and degree of inflammation in the intraocular tissues than those with no detectable eye worm infection. The symptoms of inflammation within ophthalmic eyes of infected birds could potentially create more severe visual acuity problems in these hosts.
75 Texas Tech University, Jordan Hunter, December 2016
3.2 Known areas of O. petrowi induced inflammation in ocular tissues
Bruno and colleagues (2015) examined O. petrowi infested eye globe tissue from wild northern bobwhite quail to assess signs of pathology. These studies predominately have focused on the areas of ophthalmic tissue in the host which the eye worm is known to physically contact including, the intraorbital lacrimal gland, harderian gland, conjunctiva, and nictitating membrane. The authors reported “relatively healthy” tissue in the cornea with a layer of neutrophils aggregated beneath the endothelium in bobwhite eyes with no noticeable O. petrowi infection. This was contrasted by evidence of connective tissue infiltration associated with the corneal stroma and epithelium by inflammatory cells as well as vascularization and epithelial distress seen in O. petrowi infected birds. Stromal keratitis was also observed as indicated by aggregation of inflammatory cells, lymphocytes and macrophages in all eye worm infected quail eyes. The authors also identified signs of pathology in the intraorbital lacrimal and harderian glands of eye worm infested bobwhites as well. They reported indicators such as increased number of aggregated lymphocytes in the harderian gland, and the level of interlobular tissue fibrosis and acinar cell atrophy. Displacement of the central duct of the harderian gland was also observed and which suggested to be a result of size differences between the nematode and the hosts tissue glands. The extensive work of the authors in examining the extraocular host tissues in response to eye worm infection raises questions about the O. petrowi’s capability to cause damage in the intraocular microenvironments of the eyes as well. This injury could occur because of direct physical/mechanical disturbances or through immunopathology induced oxidative stress which is the focus of our present study.
76 Texas Tech University, Jordan Hunter, December 2016
3.3 Materials and methods
3.3.1 Sample harvesting and tissue fixing
Northern bobwhite quail were captured in grain sorghum baited wire mesh traps placed around locations within West Texas and Oklahoma between 2011 and 2013. Captured quail were randomly selected for euthanization via cervical dislocation and immediately frozen. Birds were transported from each field location to the hub laboratory where necropsied and organs relevant to this study were harvested.
The remaining carcass and ocular tissue were stored at -80 °C until use. Eight
Oxyspirura petrowi infected quail and nine quail with no detectable O. petrowi infestation were selected for histological analysis. The selected samples consisted of nine female birds (including five O. petrowi infected, and four uninfected) and eight male birds (including three O. petrowi infected, and five uninfected) for a total of (n=17) samples. Whole eyes were enucleated after thawing and fixed in a formalin solution
(Sigma-Aldrich, 10% neutral buffered formalin) for 48 hours then transferred to a solution of 90% ethanol (Fisher Scientific) for 24 hours. After ethanol dehydration, each eye was embedded in paraffin wax from which 5 µm thick sections of tissue were cut via microtome and subsequently. Samples were then attached on superfrost plus
(Fisherbrand) microscope slides and stored at room temperature until further analysis.
3.3.2 Deparaffinization and hematoxylin and eosin staining
Tissue attached slides were deparaffinized by submerging them in three changes of histology grade Xylene (Sigma-Aldrich). Following paraffin removal, the slides were
77 Texas Tech University, Jordan Hunter, December 2016
submerged in a sequence of 100%, 95%, and 75% ethanol for thirty seconds each and
rinsed with deionized water for 1 minute.
Rinsed slides were stained using a commercial H&E staining kit (Scytek Industries).
Slides were incubated in hematoxylin for five minutes, and rinsed with a sequence of
water, bluing reagent and absolute ethanol. The slides were incubated in eosin Y for
three minutes, and dehydrated in three changes of absolute ethanol (Fisher Scientific)
before mounting with organo-limonene solution (Sigma-Aldrich) for analysis.
3.3.3 Experimental design and analyses criteria
Studies have suggested that the proportion of pheomelanic plumage in different bird species may be correlated with their susceptibility to oxidative stress (Galvan et al.,
2012), (Galvan & Alonso-Alvarez, 2009). Galvan and colleagues (2012) reported that avian species with a larger proportion of plumage containing pheomelanin had higher prevalence of cataracts likely due to their reduced capability to protect the crystalline lens of the eye from ROS attack and oxidative stress, leading to oxidative stress and damage.
This may be due to the processes of pheomelanogenesis requiring the same cysteine amino acids for which glutathione (GSH) is the main physiological reservoir (Meyskens et al., 1999). Along with ascorbic acid, GSH is one of the primary antioxidants found in the eye, and is vital in maintaining redox balance in the intraocular tissues and crystalline lens--keeping protein thiols in their reduced state and preventing harmful protein aggregation which causes lens opafication associated with cataractous eyes (Reddy,
1990). A physiological competition between the processes of pheomelanogenesis and endogenous antioxidant capability in the eye may exist (Galvan et al., 2012).
78 Texas Tech University, Jordan Hunter, December 2016
Based the studies of (Galvan et al., 2012), oxidative stress induced by the parasitic eye worm O. petrowi could also play a factor in depleting glutathione levels in birds. These authors have determined that GSH concentration can be influenced by the synthesis of pheomelanin in avian plumages—as birds displaying brown to red phenotypes may have lower physiological levels of GSH due to the sequestering of cysteine in feathers.
Northern bobwhite quail have a sexual dichromism in their plumage phenotype, as females exhibit more brown and auburn coloration in the plumages around their body and eyes while males have more prominent white feathers around their eyes and throat areas which may grant them more resistance to oxidative stress than females.
Eye disorders and cataractous changes in the eye lens are also known to come with age
(Taylor et al., 1995). We have attempted to account for both sex and age factors that could influence oxidative stress and eye disease progression by limiting the subjects of our study to adult males and females, which were sexed per coloration on the head and neck and aged based on molting patterns at the primary wing coverts (Koerth et al. 2016).
3.4 Results
3.4.1 Histopathological analyses of male uninfected tissue
H&E stain histological analysis of two eye globes sectioned from two different
uninfected adult male bobwhites exhibited no overall gross pathology among all subjects
examined. Pockets of inflammatory cells could be seen around the choroid in one sample
and were the only signs of tissue abnormality.
A cross section of one globe sample showed calcified material as well as inflammatory
cells present in and around the choroid and the retina pigment epithelium (RPE). The
79 Texas Tech University, Jordan Hunter, December 2016 presence of inflammatory cells were indicated by purple/blue basophilic spots aggregated in sections of the choroid and below the retinal tissue layer (Figure. 3.1). Mast cells were also potentially present in the corneal stroma.
Examination of another male bobwhite eye with no detectable O. petrowi infestation showed the presence of parent lens epithelial cells (LEC) responsible for normal growth and development of protein lens fibers that are necessary for lens transparency. These were indicated by dark blue spots lining the lens epithelium (Figure. 3.2) LEC were only present in and around the lens epithelium in the anterior capsule region and not seen in the posterior regions of the lens tissue.
3.4.2 Histopathological analyses of female uninfected tissue
H&E stain histological analysis of five eye globes sectioned from five different uninfected adult female bobwhites exhibited no overall gross pathology among all subjects examined. Pockets of inflammatory cells could be seen around the choroid, and large inflammation was present in the lacrimal gland tissue of another sample that also displayed evidence of a potential former O. petrowi infection.
One subject exhibited pockets of inflammatory cells inside and outside the eye and within sections of the choroid and RPE tissues. Mast cells or eosinophils were also potentially identified in locations below the choroid and above the inner limiting membrane of the retina (Figure. 3.3). The presence of mast cells is generally associated with infection by pathogenic parasites including parasites through IgE signaling. Any disease associated with this pathway is less likely to be the result of an allergic reaction and more likely to be associated with a chronic condition.
80 Texas Tech University, Jordan Hunter, December 2016
Examination of the eye in another subject revealed inflammation around the intraorbital lacrimal gland as well a cross section of a potential O. petrowi foreign body in this area and possible mast cells around this location (Figure. 3.4). Pockets of inflammation could also be seen in this sample. Inflammation in and around the intraorbital glands could be a sign of keratoconjunctivitis sicca or chronic dry eye syndrome, a disorder that is also associated with conjunctivitis.
Calcium deposits in the location of the optic nerve opening could be evidence of cellular turnover in two samples sample that otherwise showed no inflammation or abnormalities around the ocular tissue. Mild inflammation around the optic nerve was also seen in another globe sample section that did not display any additional signs of pathology. Our last sample examined in this category did not show any signs of abnormality.
3.4.3 Histopathological analyses of male infected tissue
H&E stain histological analysis of three eye globes sectioned from three different infected male bobwhites exhibited an overall trend of significant inflammation within the intraocular and extraocular areas of the eye tissues, signs of hemorrhage, posterior sub capsular cataract, and the appearance of unidentified foreign bodies.
One sample exhibited significant inflammation around the muscles, choroid, conjunctiva, and lacrimal gland, as indicated by blue/purple spots in these locations (Figure. 3.5). The second subject also displayed a higher than normal level of inflammation inside the area between the choroid and the RPE area in cross sections of the globe. This inflammation was largely across the perimeter of the choroid in the sample.
Another eye sample in this category displayed the most obvious signs of abnormality, particularly around the lens tissue. Proliferation of nuclei were apparent around the
81 Texas Tech University, Jordan Hunter, December 2016 posterior lens capsule (Figure. 3.6), and below the sub epithelial fibers of the lens.
Epithelial cells with retained nuclei could also be seen farther into the lens capsule and closer to the lens cortex which would be another unusual occurrence in healthy lens tissue. Light pink regions of the stained lens could represent non-artifact swollen vacuoles or swollen lens fibers.
The degree of non-artifact abnormalities in this tissue suggests that this sample has potential signs for posterior sub capsular cataract. This could cause severe opafication of the rear of portion of the eye lens, with an initial clinical manifestation of farsightedness.
The progression of posterior sub capsular cataract is believed in a state flux, implying the intervention of natural repair mechanisms in the eye (Adrien Shun-Shin et al., 1989).
3.4.4 Histopathological analyses of female infected tissue
H&E stain histological analysis of five eye globes sectioned from five different infected female bobwhites exhibited an overall trend characterized by abnormal degree of inflammation within the intraocular and extraocular areas of the eye tissues including; the choroid, eyelid, intraorbital lacrimal gland, as well as signs of hemorrhage.
Pockets of inflammation could be seen around the lacrimal gland and muscle tissue as well as locations around the choroid in one subject (Figure. 3.7). This could cause potential keratoconjunctivitis in the host characterized by dry eyes. Signs of a potentially older hemorrhage could also be seen in this tissue.
Another subject displayed potential epiretinal membrane a condition that could occur in inflammatory diseases associated with the eye (Figure. 3.8). All ophthalmic sections from the remaining three samples in this category displayed pockets of inflammation around the choroid and RPE. Among these three, one sample featured inflammation in
82 Texas Tech University, Jordan Hunter, December 2016 the muscle of the eye lid as well as outside of the eye. A small amount of hemorrhage could also be seen in this tissue.
3.4.5 General assessment off histology in uninfected vs infected tissue
Histopathological examination of six globe cross sections of presumed O. petrowi uninfected quail exhibited no gross pathology in either male or female quail, however one sample appeared to have more abnormalities than the others. Uninfected quail eyes were enucleated based upon the infection status reported after careful examination of the orbit during necropsy. However, one sample from this group had a cross-section of a foreign body with morphology that resembled O. petrowi as reported by Bruno et al.,
(2015). Due to the likelihood of this tissue’s association with eye worm infestation, any histopathology observed in this sample could likely be a result of infection.
Some mild inflammation could be seen in the uninfected bobwhite eye tissue examined.
Pockets of inflammatory cells were observed in 3 out of the 6-remaining quail which showed no visible trace of O. petrowi infestation. Inflammatory cells were generally seen in locations around the choroid and retinal pigment epithelium space in these samples.
One sample displayed some mild inflammation around the optic nerve. Potential mast cells were seen in the intraocular environment of two of the female subjects examined, one that was previously mentioned to have evidence of eye worm infection and in another subject, that displayed aggregated inflammatory cells both in and outside of the globe. Histopathological examination of eight globe cross sections taken from O. petrowi infested bobwhite quail exhibited a general trend of pathological signs for both male and female birds. The intraocular tissue of all subjects examined in this category had noticeable signs of ocular inflammation that ranged from mild to significant. A
83 Texas Tech University, Jordan Hunter, December 2016 higher and more consistent degree of inflammatory cells were detected around the cornea, lacrimal gland, conjunctiva and choroid in the infected quail examined. All samples had pockets of inflammatory cells in the choroid tissue which could be seen in locations all around the perimeter of the intraocular tissue. Signs of hemorrhage were also seen in two infected samples as well
A comparison of mostly intact structures of the eye lenses from one uninfected male and one uninfected male showed the most distinct differences seen between tissues in this area. Normal lens epithelial cell growth lining the anterior perimeter of the eye from the uninfected quail was contrasted by the presence of retained nuclei epithelial cells in the posterior and sub capsular region of the lens in the infected quail subject.
3.4.6 Comparing uninfected males to females
There were no distinct differences in inflammation or other physiological states between male and female bobwhites with no detectable O. petrowi infestation. In our comparison of 2 male subjects, and 4 verified uninfected females only 1 male and 2 females exhibited mild inflammation around areas of the choroid and RPE of intraocular tissue, and one female showed some inflammation around the optic nerve. These assessments were characterized by the presence of inflammatory cell aggregates in some locations of the eye. One of these females also had mast cells present around this tissue and more inflammation that is more consistent with potential signs of infection.
3.4.7 Comparing O. petrowi infected males to females
There were no distinct differences in inflammation or other physiological states between male and female bobwhite quail in our qualitative visual assessment of O. petrowi
84 Texas Tech University, Jordan Hunter, December 2016 infected ocular tissue. In a comparison between three eye worm infected male and five infected female quail all subjects displayed significant inflammation around the area of the choroid and retina pigment epithelium. One male and two female subjects both exhibited inflammation around the lacrimal gland and muscle tissue as well. Signs of epiretinal membrane were also noticeable in in one female subject, an inflammatory induced condition that was not seen in all other subjects of both sexes.
3.4.8 Potential ophthalmic disease and vision disorders in infected quail
Ocular helminthiasis in humans and animals is known to cause various types of injury to ophthalmic tissues depending on the type of parasite species, and location of infestation
(Otranto & Eberhard, 2011). In our histopathological examination of ocular tissue taken from O. petrowi infested northern bobwhite quail, moderate to significant inflammation was identified in the tissue of every subject in our study. Signs of pathology were generally seen in the areas around the conjunctiva, intraorbital glands and orbital eye muscle in the extraocular environment.
The conjunctiva is a highly-vascularized tissue that covers the eyelids and the sclera around the ocular surface. Its function is to assist in lubricating the eye by producing mucous and aqueous solution, as well as assist in immune surveillance (Azari & Barney,
2013). Signs of inflammation in this tissue could be associated with conjunctivitis, a condition characterized by a reddish or pink color around the inner surface of the eye, with symptoms that include burning, itchiness and swelling of the tissues surrounding the eye (Azari & Barney, 2013). Eye worm infection by Oxyspirura mansoni has been reported to cause severe forms of conjunctivitis in broiler chickens (Santoyo-de-Estafano et al., 2014).
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The lacrimal gland is the primary source of the aqueous component of tear film through its secretion of proteins, electrolytes and water, providing nutrients to protect and maintain the ocular surface (Zoukhri, 2006). The pathology of lacrimal tissue inflammation includes the presence of focal lymphocytic infiltrates, and an upregulation of proinflammatory cytokines (Fox et al., 1994). Severe inflammation of this gland can lead to the inhibition of its ability to secrete nutrients, and create symptoms than clinically manifest themselves as aqueous tear deficient dry eye syndrome (DES) or keratoconjunctivitis sicca (KCS) (Pflugfelder, 2004). The symptoms that results from this are often dryness in the cornea and conjunctiva, irritation, discharge, eye fatigue, blurred vision and potential damage and scarring to the cornea (National Eye Institute, 2013).
The harderian gland acts in conjunction with the lacrimal gland, secreting fluid to assist movement of the quail’s nictitating membrane. It also functions as a source of immune response, growth factors, pheromones, thermoregulatory lipids, and osmoregulation around the eye (Payne, 1994). Inflammation of the harderian tissue can alter the normal maintenance of the eye and its ocular surface, making it difficult for the host to manipulate the nictitating membrane, and protect itself from harmful pathogens by mounting an immune response (Payne, 1994). Diseases associated with pathology of the lacrimal system and associated intraorbital glands can be a result of infection, as could potentially be the case for the O. petrowi infected bobwhites seen in this study (Klotz et al., 2000).
Histological assessment inside the globe of infected birds revealed aggregates of inflammatory cells within the intraocular environment of the eye as well. All subjects had more inflammation when compared to their uninfected counterparts. Much of the intraocular inflammation observed was located around the choroid and retina pigmented
86 Texas Tech University, Jordan Hunter, December 2016 epithelial tissue. The choroid is the vascular layer of the eye located between the retina and sclera, and supplies oxygen and nutrients to the components of the retina necessary to receive light and produce images. Inflammation of this tissue is commonly referred to as choroiditis, and has symptoms that include, blurred vision, floating black spots, pain and redness in the eye, sensitivity to light and excessive tearing (Berman, 2016). The presence of epithelial cells with retained nuclei in the posterior and sub capsular region of the lens and potential vacuoles in the sub capsular area seen during histological assessment of one infected male quail is also evidence of pathology in this intraocular tissue. The eye lens functions by focusing light onto the retina making it a critical component of healthy vision. The type of pathology seen in our histological analysis is characteristic of posterior sub capsular cataract, a condition resulting from the opafication the crystalline lens which obscures the passage of light within the eye. The pathogenesis of cataract is believed to be the result of the oxidation of lens proteins that make-up lens fibers. The degradation of these proteins forms high molecular weight adducts that aggregate and precipitate within areas of the lens creating opacity in the tissue (Taylor et al., 1995).
Inflammation observed around the optic nerve is another potential sign of pathology associated with the eye. Optic neuritis is a condition characterized by inflammation of the eye nerve tissue, with symptoms that include, blurred vision, reduced color vision, dimness of vision, and partial or full vision loss (Cleveland Clinic, 2014). Some zoonotic helminths including Taenia, Trichinella, and Ochocherca are known to cause damage to optic nerve which manifest as papilledema or optic atrophy (Otranto & Eberhard, 2011).
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3.5 Discussion
Histopathological ophthalmic analysis of six (2 males and 4 females) northern bobwhite quail with no detectable O. petrowi infestation during necropsy or H&E stain assessment, and eight O. petrowi infested quail (3 males and 5 females) resulted in a notable difference in tissue inflammation between these groups based upon their infection status.
All subjects examined from the infected quail group showed some degree of inflammatory cell presence in the choroid and around the uveal tissues. Some infected subjects also exhibited inflammation around the conjunctiva, intraorbital glands and optic nerve.
Histological examination of three subjects from the uninfected group showed evidence of inflammatory cells present primarily in the choroid and retina pigment epithelium of the intraocular environment. One sample from this group exhibited some evidence of inflammation around the lacrimal gland and mast cells.
There were no significant pathological differences observed between male and female birds in either infected or uninfected groups. Any potential plumage differences in pheomelanin content between the two sexes did not appear to have any effect on the overall degree of inflammation or pathology seen. However, potential mast cells were only seen in tissue sections taken from two females during this study.
In general, both the instance and degree of inflammation was higher in O. petrowi infested bird eyes than those did not have detectable eye worm infection. The presence of potential mast cells in two uninfected female tissue sections suggests some immune response was mounted either against a parasite or another pathogen during the lifetime of the host. Uveitis in birds is known to occur in cases of trauma, infections and immune- mediated inflammation and neoplasia (Schmidt et al., 1986; Tsai et al., 1993), but this
88 Texas Tech University, Jordan Hunter, December 2016 has been generally associated with microorganisms. Anterior uveitis can also develop in mammals as result of corneal ulcers (Bayon et al., 2007). However, it remains unclear why a more consistent and higher degree of inflammatory cell aggregates were seen around some of the uveal tissues in all the O. petrowi infected birds. O. petrowi is not known to penetrate the intraocular microenvironment of the eye during its residence in the hosts lacrimal system and extraocular eye orbit, therefore the possibility of immune response induced by direct or physical damage to these components in the internal environment is reduced.
One explanation for this trend could involve immunopathology induced by the eye worm through oxidative stress. O. petrowi’s proximity to the globe and the typical inflammation seen in the eye muscle tissues, intraorbital glands and conjunctiva could signal more inflammatory signaling processes throughout the ophthalmic space. The generation of reactive oxygen species from phagocytic processes, and/or the increased rate of metabolic activity and mitochondrial respiration could cause the proliferation of harmful oxidants that cascade and chain into and around the highly-vascularized areas of bird intraocular tissue such as, the choroid and pectin oculi. ROS attack on proteins, lipids, DNA and RNA could cause interference in many different host signaling pathways, leading to a higher expression of inflammatory cells around vulnerable areas of the eye.
Glaucoma in birds is reported to be secondary to uveitis in some reports and could potentially cause progressive visual field loss, and optic nerve damage (Bayon et al.,
2007).
One infected male subject in this study exhibited pathological signs of posterior sub capsular cataract which could potentially result in severe loss of visual acuity.
89 Texas Tech University, Jordan Hunter, December 2016
Cataractous changes are often associated with age in most vertebrates, however they can also be associated with; trauma, UV photooxidation, nutritional deficiencies, chronic uveitis, and encephalomyelitis in birds (Kern, 1989; Williams, 1994). The overwhelming majority of obvious pathology we observed in this study are signs relating to choroiditis.
Given the close choroid’s proximity to the retina within the eye it is also possible that many of our infected subjects could potentially have signs relating to choroiretinis as well. Severe inflammation of the choroid as in the case serpiginous choroiditis can manifest in symptoms that include a painless decrease in vision, blind gaps in visual field
(scotomata), and a sensation of flashes of light (photopsia) (WebMD, 2015).
In summary, our results suggest that Oxyspirura petrowi infected northern bobwhite quail exhibit a more consistent and higher degree of inflammation in both the extraocular and intraocular tissues. The predominant form of pathology observed was choroiditis, a condition of the highly vascular intraocular layer of the host’s eye with the potential to cause more severe visual acuity problems during the lifetime of infected birds. The exact mechanism that links eye worm infestation with higher intraocular inflammation requires further study, but parasite induced immunopathological pathways leading oxidative stress in the host could be a potential factor in the etiology of this disease.
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Figure 3.1 H&E staining of a male northern bobwhite quail intraocular tissue with no detectable
Oxyspirura petrowi infection. Inflammatory cells groupings can be seen in locations around the choroid (right and left).
Figure 3.2 H&E staining of a male northern bobwhite quail lens with no detectable Oxyspirura petrowi infection. Nuclei of lens epithelium cells (LEC) present in anterior
(right). LEC are absent in posterior lens region (left).
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Figure 3.3 H&E staining of a female northern bobwhite quail intraocular tissue with no detectable
Oxyspirura petrowi infection. Potential mast cells present around the uveal tissues (left).
Inflammatory cells present around choroidal tissues (right).
Figure 3.4 H&E staining of a female northern bobwhite quail intraocular tissue with no detectable eye worm infection at time of necropsy. Pockets of inflammatory cells around choroid and a cross section of possible O. petrowi on the bottom left along with potential mast cells.
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Figure 3.5
H&E staining of male northern bobwhite quail intraocular tissue with O. petrowi infection. Aggregates of inflammatory cells in a section of the choroid (left) and around extraocular muscle tissue (right).
Figure 3.6 H&E staining of a male northern bobwhite quail lens with O. petrowi infection.
Aggregates of inflammatory cells in a section of the choroid (left) and around extraocular muscle tissue (right).
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Figure 3.7
H&E staining of female northern bobwhite quail intraorbital gland tissue with
O. petrowi infection. Significant aggregation of inflammatory cells in lacrimal gland
(left) and around extraocular muscle tissue (right). Pockets of heightened inflammation in choroid areas (right).
Figure 3.8
H&E staining of female northern bobwhite quail intraocular tissue with O. petrowi infection. Aggregation of inflammatory cells in the choroid and possible epiretinal membrane.
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4 CHAPTER IV
OXIDATIVE STATUS OF OPHTHALMIC TISSUE
4.1 Introduction
The decline of northern bobwhite quail (Colinus virginianus) populations across Texas
over the last few decades is a phenomenon of considerable ecological importance
(Brennan, 1999; Williams et al., 2004). Old world (Phasiandiae) and new world
(Odontophoridae) quail and related galliformes have been historically important birds as livestock, game, agriculture, and scientific study (Gust et al., 2015; Williams et al.,
2000; Stoddard, 1931). Understanding both the environmental and biological factors that may have contributed to the decline of C. virginianus is important to assist the conservation efforts for this species and other wildlife sharing the same or similar habitats (Brausch et al., 2010; Gust et al., 2015; Kitulagodage et al., 2011; Ottinger et al., 2005; Quinn et al., 2012; Romijn et al., 1995). Identifying the significant environmental factors or pathogenic agents that influence this avian population may also benefit our broader understanding of how these factors influence wildlife and human populations on all trophic levels within ecoregions (Angelo et al., 2012).
Species such as the northern bobwhite and Japanese quail (Corturnix japonica) have been used as effective animal models in the study of toxicology and the physiological responses of vertebrates to pharmaceuticals, pesticides and pathogens (Lusk et al., 2002;
Newsted et al., 2007; Quinn et al., 2009; Quinn et al., 2012, Romijn et al. 1995).
The occurrence of parasitic and infectious diseases affecting northern bobwhite quail has
involved many studies (Dunham et al., 2014; Kellogg & Calpin, 1971; Kocan et al.,
95 Texas Tech University, Jordan Hunter, December 2016
1979; Bruno et al., 2015). The parasitic eye worm Oxyspirura petrowi is a helminth frequently found in the eye orbit, and around the ocular surface, lacrimal tissues and nictitating membrane of ground dwelling birds including the northern bobwhite quail
(Skrjabin, 1929). Existing knowledge of O. petrowi induced pathology on host quail and the impact of this parasite on overall visual acuity and fitness in the host is limited
(Dunham et al., 2014). Some infected quail have been reported to exhibit signs of inflammation around sites of worm infestation including the conjunctiva, and intraorbital lacrimal and harderian gland tissues (Bruno et al., 2015; Saunders, 1935). Studies have shown that pathogens and parasitic helminths can also induce harmful oxidative stress in animal and bird host’s tissues which could be linked to these signs of inflammation
(Siwela et al., 2013; Mougeot et al., 2010; Sorci & Faivre, 2009).
The correlation between O. petrowi infections and their potential to cause intraocular pathology and diseases of the eye through oxidative stress mechanisms in northern bobwhite quail has not been examined. The purpose of this study is to identify physiological signs of oxidative stress within the ophthalmic tissues and vitreous of O. petrowi infected quail.
A comparative assessment between eye worm infected and uninfected wild northern bobwhite quail was performed using vital non-enzymatic and enzymatic antioxidants as well as lipid peroxidation by-products. The average concentration of antioxidants measured in O. petrowi infected bobwhite ophthalmic tissue was significantly lower than that observed in the tissues of uninfected subjects. There were statistically significant differences in thiobarbituric reactive substances (TBARS) as well as antioxidant concentrations or activity between uninfected and infected quail. Some significant differences could be seen even when factoring the sex of bobwhite quail as well. The
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levels of antioxidants observed in this study indicate that there is potential for an
oxidative stress state in O. petrowi infected tissue given the general status conditions of
reduced levels of available antioxidants and increased levels of oxidative damage
markers.
The use of these biomarkers correlating to the general redox state of ocular tissue
provide valuable insight into the host’s physiological response to eye worm
helminthiasis. The correlation between oxidative stress and disease progression in many
organs including the eye (Babizhayev & Costa, 1994) has been explored by many
researchers (Rahal et al., 2014; Rahma et al., 2012), and ocular oxidative stress in
infected birds has the potential create more severe visual acuity and fitness related
problems in these hosts (Galvan et al., 2012).
4.2 Oxidative stress and eye disease
Oxidative stress conditions are imposed on the eye from exposure to exogenous and/or endogenous sources, subsequently altering the redox balance in these tissues and potentially causing a variety of ophthalmologic disorders such as; cataract of the lens
(Varna et al., 1984; Varna et al., 2011), diabetic retinopathy (Yang et al., 2010;
Yamagishi & Matsui, 2011; Lopes de Faria et al., 2011), age related macular degeneration (AMD) (Liutkeviciene et al., 2010; Kaarniranta et al., 2011; Ding et al.,
2009), and experimental retinal detachment (Roh et al., 2011; Mantopoulos et al.,
2011).
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The intraocular anterior and posterior portions of the eye and associated tissues are also particularly susceptible to ROS attack and degradation. Oxidative stress is believed to be a primary factor in the formation of cataractous changes in the crystalline lens of the eye
(Babizhayev et al., 2004; Babizhayev & Costa, 1994; Babizhayev, 2012, Babizhayev et al. 1988; Spector, 1995). The structure and integrity of the lens epithelial cells can be damaged by hydrogen peroxide, dehydrostatic acid, and lipid peroxides. This damage relates to the induction of protein disulfide crosslinks, reduced protein solubility and lower cellular adhesions (Babizhayev & Yegorov, 2014; Colitz et al. 2004; Spector,
2000).
Research has shown that oxidants and ROS can induce premature senescence in human lens epithelial cells, accelerating telomere shortening and reducing telomerase activity
(Babizhayev et al., 2011; Babizhavey & Yegorov, 2010; Huang et al., 2005). Data from studies by (Babizhayev & Yegorov, 2014) have demonstrated that human cataractogenesis involves senescence of lens cells, induced by oxidative stress linked to DNA damage, inhibition of telomerase and marked telomere shortening. The ribonucleic protein telomerase in mammal lens epithelial cells has been suggested to assist damage telomeres during oxidative stress and preventing acceleration of cell senescence. The epithelial cell layer is the initial location of oxidative stress incidence, followed by attack on the lens fibers causing cortical cataract (Spector et al. 1995).
Glutathione (GSH) is important for normal maintenance of lens transparency and is the predominant reducing antioxidant molecule found in large concentration of the lens.
GSH in collaboration with the glutathione redox cycle in the lens epithelium and superficial cortex detoxifies many harmful reactive oxidants including H2O2 and dehydroascorbic acid (Babizhayev & Yegorov, 2014).
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The mitochondrial respiratory chain of cellular mitochondria is a potent generator of reactive species including superoxide and hydrogen peroxide, and it contributes to a large degree of oxidative damage in vital cells. Oxidative damage from mitochondria is of large clinical importance, however direct treatment of mitochondria with antioxidants has not been entirely successful likely due to their wide range of dispersal throughout the body, ophthalmic tissues and fluids (Babizhayev, 2016).
Cataracts are one of the most significant ophthalmologic diseases in veterinary medicine and ophthalmology (Babizhayev & Yegorov, 2014). The correlation between age related cataract and oxidative damage of lens epithelial cells and the internal lens in suggests that this could also potentially be used as general biomarker for assessing systemic damage done by reactive oxygen species and life expectancy in animals (Babizhayev & Yegorov,
2014; Urfer et al., 2011). Antioxidant and lipid peroxidation markers in the blood and tissue are generally viewed as the factors most indicative of redox state in an organism, however, there is currently no universally acknowledged method among researchers regarding the most consistent and accurate measure of oxidative stress
(Kadiiska et al., 2015).
A large amount of work has been done in different harmful microorganisms and pathology of the eye as well (Mircheff, 2011, Klotz et al., 2000). However, there is limited research in ocular helminthiasis of the type O. petrowi is known to impose on its host, and any subsequent oxidative stress induced by this infestation.
4.3 Locations of antioxidants in ocular tissue
The oxidative damage defenses of ocular tissue can be categorized into enzymatic and non-enzymatic antioxidant classes (Varna, 1991). Enzymatic antioxidants catalyze
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electron transfer from a reducing agent or substrate to a reactive oxygen species (ROS).
In this process the substrate can be recycled for additional usage by nicotinamide
adenine dinucleotide phosphate oxidase (NADPH) generated from different metabolic
pathways (Cabrera & Chihuailaf, 2011; Chihuailaf et al., 2002). Protecting enzymes
catalyze the reduction of specific ROS and the primary antioxidant enzymes in the eyes
of most animals are superoxide dismutase (SOD), catalase (CAT), and glutathione
peroxidase (GPx) (Cabrera & Chihuailaf, 2011; Cejkova et al., 2000).
Superoxide dismutase is metalloprotein with three isoforms; Cu-SOD and Zn-SOD
which are found in the cytosol and extracellular fluid, and Mn-SOD which is located
within the mitochondrial matrix (Benitez, 2006). This enzyme catalyzes the dismutation
of O2- and H2O2 and O2. All isoforms of SOD have been reported in the cornea
epithelium and endothelium (Cejkova et al., 2004), the lens epithelium, aqueous humor
(Satici et al. 2002), iris, ciliary body (Phylactos & Unger, 1998) and inner segment layer
of photoreceptor cells and pigment epithelium of the retina (Frank et al., 1999).
Glutathione peroxidase (GPx) is a selenoprotein that reduces H2O2 and hydroperoxides
into H2O and alcohol utilizing glutathione for electron donation. GPx is an important
antioxidant in the defense of ROS damage to lipid membranes and polyunsaturated fatty
acids susceptible to oxidation (Chihuailaf et al., 2002). This enzyme has four isoforms;
cellular, extracellular and plasmatic, phospholipid hydroperoxide, and gastrointestinal.
The cornea epithelium and endothelium (Cejkova et al., 2004a; Cejkova et al., 2004b), lens epithelium, aqueous humor (Ozmen et al., 2002), ciliary body, choroid, and retina contain this enzyme (Agardh et al., 2006; Bilgihan et al., 2003; Penn et al., 1992).
Catalase (CAT) is a hemoprotein that catalyzed the transformation of H2O2 into H2O and
O2 and contains four heme groups and is in peroxisomes mitochondria and cytoplasm.
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The presence of CAT has been reported in the cornea epithelium and endothelium
(Cejkova et al., 2004a; Cejkova et al., 2004b), lens epithelium, aqueous humor (Satici et al., 2002), ciliary body, iris (Phylactos & Unger, 1998) and retina (Agardh et al., 2006;
Frank et al., 1999).
Non-enzymatic antioxidants are a diverse category of electron donating molecules that reduce and stabilizes free radical species to create less harmful molecules (Chihuailaf et al., 2002). Ascorbic acid, vitamin E, vitamin A, and GSH are the predominant non- enzymatic antioxidants found within the eye globe tissue (Williams, 2006). Ascorbic acid is antioxidant that is generally found in its soluble form, ascorbate anion in most tissues at physiological pH. It reduces O2 and OH radicals into more stable forms (Yu,
1994) and is also involved in the recycling of alpha-tocopheryl radical, a form of vitamin E. to its stable state. Ascorbate also performs as a pro-oxidant during increased concentrations of Fe3+ and Cu2+ ions (Chihuailaf et al., 2002; Halliwell et al., 1992), and has been reported in the cornea (Brubaker et al., 2000), aqueous humor (Barros et al.,
2003, Ringvold et al., 2000), lens (Ohta et al., 2004), vitreous humor (Koide et al.,
2006), and retina (Penn et al., 1992).
Vitamin E is the general description for eight similar compounds, four tocopherols, and four tocotrienols, with alpha-tocopherol being the predominant and most active liposoluble antioxidant in membranes. Alpha-tocopherol transforms ROS including O2,
OH and LOO radicals into less reactive forms, thus this reducing agent is converted to a stable non-reactive alpha-tocopheryl radical that can be recycled by vitamin C, GSH, and lipolic acid, and it capability is dependent on the availability of these compounds
(Chihuailaf et al., 2002). The lens (Yeum et al., 1995), aqueous humor, and retina (Zhang
101 Texas Tech University, Jordan Hunter, December 2016 et al., 2008), have all been reported to contain this molecule, however, the tissue concentration of vitamin E is potentially reduced with increased proliferation of ROS
(Powers & Lennon, 1999).
Vitamin A is a group of compounds sharing the precursor B-carotene, a known carotenoid neutralizer of O2. The long chains and conjugated double bonds of these molecules allow them to convert O2 and LOO (peroxyl radical) into less reactive substances (Yu, 1994). Carotenoids are not dispersed equally among ocular tissues, the predominate forms being lutein and zeaxanthin which are found in the macula, retina, and lens in high concentration (Bernstein et al., 2001; Yeum et al., 1995).
Glutathione (GSH) is a tripeptide (gamma glutamyl-cysteinyl-glycine) containing a thiol group in its active site. This agent acts by transferring electrons to reactive oxygen species including hydroxyl radical and carbonyls, and is subsequently converted into an oxidized form (GSSG) (Benitez, 2006; Yu, 1994). This antioxidant is vital for lenticular protein maintenance (Zhang et al., 2008) and as a co-substrate of GPx. GSH has been reported in the lens (Saaki et al., 1995), cornea and retina (Kannan et al., 1993), and along with ascorbic acid is significantly important as one of the primary mechanisms of oxidative damage defense in these tissues (Cabrera & Chihuailaf, 2011; Taylor et al.,
1995).
4.4 Materials and methods
4.4.1 Subject capture and sample harvesting
Northern bobwhite quail were captured in grain sorghum baited wire mesh traps placed around locations within west Texas and Oklahoma between 2011 and 2013. Several
102 Texas Tech University, Jordan Hunter, December 2016 captured quail were randomly selected for euthanization via cervical dislocation and immediately stored under freezing conditions. Birds were transported from each field location to our hub laboratory where necropsy was performed and study relevant organs were harvested. The remaining carcass and ocular tissue were stored at -80 °C until use.
Twenty-Four Oxyspirura petrowi infected quail and twenty-two quail with no detectable
O. petrowi infestation were selected for antioxidant analysis. The selected samples consisted of twenty-five female birds including twelve O. petrowi infected, and thirteen uninfected as well as twenty-one male birds that consisted of twelve O. petrowi infected, and nine uninfected for a total of (n=46) samples.
4.4.2 Tissue Homogenization
Whole pairs of eyes were enucleated after thawing and each pair of ocular tissue, lens, and vitreous from one quail subject were consolidated into their individual working samples. Eye vitreous was aspirated using a small syringe and transferred to a clean micro centrifuge tube for storage at -80 °C. A small incision was made on the perimeter of the globe and forceps were used to remove the lens for separate storage and homogenization. Eye pair of lens’ and the remaining globe tissues were homogenized separately via a glass dounce homogenizer in pH 7.0 50 mM potassium phosphate
(Fisher Chemical P288-100), 1mM EDTA (Ambion AM92605) buffer prepared in nuclease free water (Fisher Scientific # W5-1) to obtain an approximately 10% weight by volume homogenate (Saxena et al., 1992). Homogenates were centrifuged at 28,000 x g for 30 minutes at 4 °C, and the supernatants were transferred to a clean micro centrifuge tube for storage at -80 °C for stable use within one month or when ready for assay.
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Colorimetric assays using tissue samples in duplicate were performed for all assays using
a Biotek Synergy 4 Hybrid 96-well microplate reader and Gen 5 software. All assays
were performed per their manufacturers specifications. Globe and lens tissue
concentrations of antioxidants and TBARS from assay were normalized to their wet
weight, and vitreous concentrations were normalized from their total protein
concentration.
4.4.3 Glutathione (GSH) assay
Glutathione analysis in tissue and lens samples was performed using a kit and reagents manufactured by Cayman Chemical (Cayman Chemical, Glutathione Assay Kit Manual
No. 703002, 2015). This method determines the concentration of glutathione (GSH) using an enzymatic recycling method in which glutathione present in the sample reacts with 5,5’-dithio-bis-2-(nitro benzoic acid) (DTNB) to produce a yellow 5-thio-
2nitrobenzoic acid (TNB) product. The mixed disulfide, GS-TNB, that is produced is reduced by glutathione reductase to recycle the GSH and produce additional TNB. The rate of TNB production as measured by absorbance at 405 nm is directly proportional to the recycling reaction and the concentration of GSH in the sample.
4.4.4 Thiobarbituric acid reactive substances (TBARS) assay
TBARS analysis in tissue, lens, and vitreous samples was performed using a kit and reagents manufactured by Cayman Chemical (Cayman Chemical, TBARS (TCA
Method) Assay Kit Manual No. 7000870, 2015). This method determines the concentration of lipid peroxidation by products such as malondialdehyde (MDA) using
104 Texas Tech University, Jordan Hunter, December 2016 high temperature to create an adduct between MDA in the tissue sample and thiobarbituric acid. The resulting MDA-TBA adduct can be measured colorimetrically at
530 nm and is proportional to the amount of MDA or thiobarbituric acid reactive substances in the sample.
4.4.5 Catalase (CAT) Assay
Catalase analysis in tissue and vitreous samples was performed using a kit and reagents manufactured by Cayman Chemical (Cayman Chemical, Catalase Assay Kit Manual No.
707002, 2015). This method determines the activity of catalase through a reaction of the enzyme with methanol in the presence of optimal concentration of H2O2. The formaldehyde produced is measured colorimetrically at 540 nm, with 4-amino-
3hydrazino-5-mercapto-1,2,4 triazole (purpald) as the chromogen.
4.4.6 Superoxide dismutase (SOD) assay
Superoxide dismutase analysis in tissue and lens samples was performed using a kit and reagents manufactured by Cayman Chemical (Cayman Chemical, Superoxide Dismutase
Assay Kit Manual No. 706002, 2015). This method determines the activity of SOD via tetrazolium salt detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. One unit of SOD is defined as the amount of enzyme needed to exhibit
50% dismutation of the superoxide radical. This assay measures all three types of SOD
(Cu/Zn, Mn, and FeSOD) at 440 nm.
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4.4.7 Glutathione peroxidase (GPx) assay
Glutathione peroxidase analysis in tissue and lens samples was performed using a kit and reagents manufactured by Cayman Chemical (Cayman Chemical, Glutathione
Peroxidase Assay Kit Manual No. 703102, 2015). This method determines the activity of GPx indirectly by a coupled reaction with glutathione reductase (GR). Oxidized glutathione (GSSG), produced upon reaction of hydroperoxide by GPx, is recycled to is reduced state by GR and NADPH. The oxidation of NADPH to NADP+ is accompanied by a decrease in absorbance at 340 nm. The rate of decrease in the A340 is directly proportional to the GPx activity in the sample.
4.4.8 Pierce BCA protein assay
Protein analysis in vitreous homogenates was performed using a kit and reagents manufactured by Thermo-Scientific (Thermo-Scientific, Pierce BCA Protein Assay Kit
Manual No. 23225, 2015). This method combines the reduction of Cu+2 to Cu+1 by protein in an alkaline medium with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu+1). The purple colored reaction product of this assay is formed by the chelation of two molecules of BCA with one cuprous ion which was measured colorimetrically at 550 nm.
4.4.9 Experimental design and post-laboratory analysis criteria
Studies have suggested that the proportion of pheomelanin plumage in different bird species may be correlated to their susceptibility to oxidative stress (Galvan et al., 2012),
106 Texas Tech University, Jordan Hunter, December 2016
(Galvan & Alonso-Alvarez, 2009). It was reported by (Galvan et al., 2012) that avian species with a larger proportion of plumage containing pheomelanin had higher prevalence of cataracts likely due to their reduced capability to protect the crystalline lens of the eye from ROS attack and oxidative stress leading to oxidative stress and damage. This may be due to the processes of pheomelanogenesis requiring the same cysteine amino acids for which glutathione (GSH) is the main physiological reservoir
(Meyskens et al., 1999). Along with ascorbic acid, GSH is one of the primary antioxidants found in the eye, and is important in maintaining redox balance in the intraocular tissues and crystalline lens, by keeping protein thiols in their reduced state, preventing harmful protein aggregation which causes lens opafication associated with cataractous eyes (Reddy, 1990). A physiological competition between the processes of pheomelanogenesis and endogenous antioxidant capability in the eye may exist (Galvan et al., 2012).
Based on studies by (Galvan et al., 2012), oxidative stress induced by the parasitic eye worm O. petrowi could also play a factor in depleting glutathione levels in birds while this antioxidant concentration is simultaneously influenced by the pheomelanin content of their plumages, with birds displaying brown to red phenotypes also having lower physiological levels of GSH due to the sequestering of cysteine in feathers. Northern bobwhite quail have a sexual dichromism in their plumage phenotype, as females exhibit more brown and auburn coloration in the plumages around their body and eyes, while males have more prominent white feathers around their eyes and throat areas which may grant them more resistance to oxidative stress than females.
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Eye disorders and cataractous changes in the eye lens are also known to come with age
(Taylor et al., 1995). We have attempted to account for both sex and age factors that could influence oxidative stress and eye disease progression by limiting the subjects of our study to adult males and females, which were sexed per coloration on the head and neck and aged based on molting patterns at the primary wing coverts (Koerth et al.,
2016).
4.5 Results
4.5.1 GSH analysis of ocular tissue
Globe A total of (n=25) pairs of eye globes from northern bobwhite quail comprised of 9 O.
petrowi infected (5 males, and 4 females), and 16 uninfected quail (7 males and 9
females) were used for comparative analysis of glutathione (GSH) in globe tissue. The
average concentration of GSH in infected male and female infected globe based on wet
weight was 1.45E-05 µmol/mg and 1.13E-05 µmol/mg respectively. The average
concentration of GSH in uninfected male and female globe based on wet weight was
2.26E-05 µmol/mg, and 2.96E-05 µmol/mg respectively.
A series of two-tailed t-tests using unequal variances was performed comparing average
eye globe GSH (µmol/mg) (1) infected samples from both sexes against uninfected
sample from both sexes (p=0.010), 2) infected males against uninfected males
(p=0.143), 3) infected females against uninfected females (p=0.022), 4) infected males
against infected females (p=0.271), and 5) uninfected males against uninfected females
(p=0.239).
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Lens A total of (n=23) pairs of eye lenses from northern bobwhite quail comprised of 8 O. petrowi infected (4 males, and 4 females), and 15 uninfected quail (6 males and 9 females) were used for comparative analysis of glutathione (GSH) in lens tissue. The average concentration of GSH in infected male and female infected lens based on wet weight was 1.58E-05 µmol/mg and 4.04E-05 µmol/mg respectively. The average concentration of GSH in uninfected male and female lens based on wet weight was
2.71E-05 µmol/mg, and 3.02E-05 µmol/mg respectively.
A series of two-tailed t-tests using unequal variances was performed comparing average eye lens GSH (µmol/mg) for 1) infected samples from both sexes against uninfected sample from both sexes (p=0.087), 2) infected males against uninfected males
(p=0.0022), 3) infected females against uninfected females (p=0.433), 4) infected males against infected females (p=0.270), and 5) uninfected males against uninfected females
(p=0.089).
4.5.2 TBARS analysis of ocular tissue and vitreous
Globe A total of (n=29) pairs of eye globes from northern bobwhite quail comprised of 14 O. petrowi infected (7 males, and 7 females), and 15 uninfected quail (6 males and 9 females) were used for comparative analysis of thiobarbituric acid reactive substances
(TBARS) in globe tissue. The average concentration of TBARS in infected male and female infected globe based on wet weight was 3.36E-02 µmol/mg and 6.63E-02
µmol/mg respectively. The average concentration of TBARS in uninfected male and
109 Texas Tech University, Jordan Hunter, December 2016 female globe based on wet weight was 1.10E-02 µmol/mg, and 1.03E-02 µmol/mg respectively.
A series of two-tailed t-tests using unequal variances was performed comparing average eye globe TBARS (µmol/mg) for 1) infected samples from both sexes against uninfected sample from both sexes (p=0.034), 2) infected males against uninfected males
(p=0.179), 3) infected females against uninfected females (p=0.016), 4) infected males against infected females (p=0.057), and 5) uninfected males against uninfected females
(p=0.241).
Lens A total of (n=33) pairs of eye lenses from northern bobwhite quail comprised of 16 O. petrowi infected (8 males, and 8 females), and 17 uninfected quail (7 males and 10 females) were used for comparative analysis of thiobarbituric acid reactive substances
(TBARS) in lens tissue. The average concentration of TBARS in infected male and female infected lens based on wet weight was 1.27E-02 µmol/mg and 1.33E-02
µmol/mg respectively. The average concentration of TBARS in uninfected male and female lens based on wet weight was 6.79E-03 µmol/mg, and 6.86E-03 µmol/mg respectively. A series of two-tailed t-tests using unequal variances was performed comparing average eye lens TBARS (µmol/mg) for 1) infected samples from both sexes against uninfected sample from both sexes (p=0.114), 2) infected males against uninfected males (p=0.199), 3) infected females against uninfected females (p=0.075),
4) infected males against infected females (p=0.126), and 5) uninfected males against uninfected females (p=0.081).
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Vitreous A total of (n=12) aliquots of eye vitreous from northern bobwhite quail comprised of 6
O. petrowi infected (3 males, and 3 females), and 6 uninfected quail (3 males and 3 females) were used for comparative analysis of thiobarbituric acid reactive substances
(TBARS) in eye vitreous. The average concentration of TBARS in infected male and female infected eye vitreous based on wet weight was 1.35E-06 µmol/mg and 1.28E-07
µmol/mg respectively. The average concentration of TBARS in uninfected male and female globe based on wet weight was 1.00E-08 µmol/mg, and 3.70E-07 µmol/mg respectively. A series of two-tailed t-tests using unequal variances was performed comparing average eye vitreous TBARS (µmol/mg) for 1) infected samples from both sexes against uninfected sample from both sexes (p=0.117), 2) infected males against uninfected males (p=0.078), 3) infected females against uninfected females (p=0.276),
4) infected males against infected females (p=0.090), and 5) uninfected males against uninfected females (p=0.201).
4.5.3 CAT analysis of ocular tissue and vitreous
Globe
A total of (n=20) pairs of eye globes from northern bobwhite quail comprised of 10 O. petrowi infected (5 males, and 5 females), and 10 uninfected quail (5 males and 5 females) were used for comparative analysis of catalase (CAT) activity in globe tissue.
The average activity per milligram of CAT in infected male and female infected globe based on wet weight was 2.52E-05 µmol/min/mg and 1.73E-05 µmol/min/mg respectively. The average activity per milligram of CAT in uninfected male and female
111 Texas Tech University, Jordan Hunter, December 2016 globe based on wet weight was 1.13E-05 µmol/min/mg, and 1.05E-05 µmol/min/mg respectively.
A series of two-tailed t-tests using unequal variances was performed comparing average eye globe CAT (µmol/min/mg) for 1) infected samples from both sexes against uninfected sample from both sexes (p=0.027), 2) infected males against uninfected males
(p=0.082), 3) infected females against uninfected females (p=0.128), 4) infected males against infected females (p=0.259), and 5) uninfected males against uninfected females
(p=0.429).
Vitreous A total of (n=20) aliquots of eye vitreous from northern bobwhite quail comprised of 10
O. petrowi infected (5 males, and 5 females), and 10 uninfected quail (5 males and 5 females) were used for comparative analysis of catalase (CAT) activity in eye vitreous.
The average activity per milligram of CAT in infected male and female infected eye vitreous based on wet weight was 1.11E-07 µmol/min/mg and 5.43E-08 µmol/min/mg respectively. The average activity per milligram of CAT in uninfected male and female globe based on wet weight was 1.44E-08 µmol/min/mg, 5.43E-08 and µmol/min/mg respectively.
A series of two-tailed t-tests using unequal variances was performed comparing average eye vitreous CAT (µmol/min/mg) for 1) infected samples from both sexes against uninfected sample from both sexes (p=0.073), 2) infected males against uninfected males
(p=0.113), 3) infected females against uninfected females (p=0.279), 4) infected males against infected females (p=0.248), and 5) uninfected males against uninfected females
(p=0.156).
112 Texas Tech University, Jordan Hunter, December 2016
4.5.4 SOD analysis of ocular tissue
Globe A total of (n=20) pairs of eye globes from northern bobwhite quail comprised of 10 O. petrowi infected (5 males, and 5 females), and 10 uninfected quail (5 males and 5 females) were used for comparative analysis of superoxide dismutase (SOD) activity in globe tissue. The average activity per milligram of SOD in infected male and female infected globe based on wet weight was 2.24E-03 µmol/min/mg and 2.87E-03
µmol/min/mg respectively. The average activity per milligram of SOD in uninfected male and female globe based on wet weight was 1.86E-03 µmol/min/mg, and 2.04E-03
µmol/min/mg respectively.
A series of two-tailed t-tests using unequal variances was performed comparing average eye globe SOD (µmol/min/mg) for 1) infected samples from both sexes against uninfected sample from both sexes (p=0.008), 2) infected males against uninfected males
(p=0.071), 3) infected females against uninfected females (p=0.020), 4) infected males against infected females (p=0.041), and 5) uninfected males against uninfected females
(p=0.229).
Lens
A total of (n=20) pairs of eye lens’ from northern bobwhite quail comprised of 10 O. petrowi infected (5 males, and 5 females), and 10 uninfected quail (5 males and 5 females) were used for comparative analysis of superoxide dismutase (SOD) activity in eye lens. The average activity per milligram of SOD in infected male and female infected eye lens based on wet weight was 3.35E-03 µmol/min/mg and 5.98E-03 µmol/min/mg respectively. The average activity per milligram of SOD in uninfected male and female
113 Texas Tech University, Jordan Hunter, December 2016 globe based on wet weight was 2.61E-03 µmol/min/mg, and 1.65E-03 µmol/min/mg respectively.
A series of two-tailed t-tests using unequal variances was performed comparing average eye lens SOD (µmol/mg) for 1) infected samples from both sexes against uninfected sample from both sexes (p=0.009), 2) infected males against uninfected males
(p=0.177), 3) infected females against uninfected females (p=0.020), 4) infected males against infected females (p=0.074), and 5) uninfected males against uninfected females
(p=0.132).
4.5.5 GPx analysis of ocular tissue
Globe A total of (n=20) pairs of eye globes from northern bobwhite quail comprised of 10 O. petrowi infected (5 males, and 5 females), and 10 uninfected quail (5 males and 5 females) were used for comparative analysis of glutathione peroxidase (GPx) activity in globe tissue. The average activity per milligram of GPx in infected male and female infected globe based on wet weight was 2.35E-02 nmol/min/mg and 2.93E-02 nmol/min/mg respectively. The average activity per milligram of GPx in uninfected male and female globe based on wet weight was 4.66E-02 nmol/min/mg and 1.65E-01 nmol/min/mg respectively.
A series of two-tailed t-tests using unequal variances was performed comparing average eye globe GPx (nmol/min/mg) for 1) infected samples from both sexes against uninfected sample from both sexes (p=0.007), 2) infected males against uninfected males
(p=0.189), 3) infected females against uninfected females (p=0.004), 4) infected males against infected females (p=0.279), and 5) uninfected males against uninfected females
114 Texas Tech University, Jordan Hunter, December 2016
(p=0.006).
Lens A total of (n=24) pairs of eye lens’ from northern bobwhite quail comprised of 12 O. petrowi infected (6 males, and 6 females), and 12 uninfected quail (6 males and 6 females) were used for comparative analysis of glutathione peroxidase (GPx) activity in eye lens. The average activity per milligram of GPx in infected male and female infected eye lens based on wet weight was 3.16E-02 nmol/min/mg and 1.38E-01 nmol/min/mg respectively. The average activity per milligram of GPx in uninfected male and female globe based on wet weight was 9.33E-02 nmol/min/mg and 9.41E-02 nmol/min/mg respectively.
A series of two-tailed t-tests using unequal variances was performed comparing average eye lens GPx (nmol/min/mg) for 1) infected samples from both sexes against uninfected sample from both sexes (p=0.413), 2) infected males against uninfected males
(p=0.077), 3) infected females against uninfected females (p=0.262), 4) infected males against infected females (p=0.069), and 5) uninfected males against uninfected females
(p=0.493).
4.5.6 General assessment of antioxidant and lipid peroxidation status
Analysis of antioxidant and lipid peroxidation content in all ocular tissue exhibited statistically significant difference (p<0.05) across all assays performed. GSH concentration in globe tissues was significantly higher in uninfected versus infected samples that included all sexes (p=0.010), and GSH was higher in uninfected females
115 Texas Tech University, Jordan Hunter, December 2016 versus infected females exclusively (p=0.022). Lens GSH concentrations were significantly higher (p=0.0022) in uninfected males versus infected males as well (Figure
4.1).
TBARS concentrations in the eye globe were at significantly higher levels in infected and lower in uninfected quail samples amongst all sexes (p=0.034), and lipid peroxidation was also higher in infected females more so than uninfected females exclusively (p=0.016) (Figure 4.6).
Catalase in the globe tissue displayed a statistically significant higher level of activity in infected tissue and was lower on average in uninfected tissue of all sexes (p=0.027)
(Figure 4.3). Infected males displayed higher on average catalase activity than infected females, and all uninfected sexes, however these differences were not statistically significant (Figure 4.4).
SOD activity was significantly higher in infected quail globe tissue versus uninfected quail both within the globe (p=0.008), and lens (p=0.009) factoring in all sexes. There were also significant differences between females in this assay as both globe (p=0.020) and lens (p=0.020) SOD activity for this sex was higher in infected female birds (Figure
4.2).
GPx activity in the globe was significantly different between infection status of all sexes
(p=0.007) as uninfected quail had on average higher GPx activity in this tissue, which was also the case when observing uninfected female globe tissue exclusively (p=0.004)
(Figure 4.5).
116 Texas Tech University, Jordan Hunter, December 2016
4.5.7 Comparing oxidative status between sexes
Analysis of antioxidant and lipid peroxidation content between male and female bobwhite quail ocular tissue exhibited statistically significant difference (p<0.05) in only two of the assays performed.
Infected males had on average lower SOD activity than infected females in the globe tissue (p=0.041) (Figure 4.2). Within uninfected globe tissue, uninfected females have a statistically significant higher level of GPx activity when compared to uninfected males
(p=0.006) (Figure 4.5).
Infected males had much higher on average TBARS in the vitreous compared to females, however these differences were not determined to be statistically significant (p=0.090).
Uninfected females also had much higher vitreous TBARS than males, with no statistical significance (p=0.201) (Figure 4.7).
4.6 Discussion
Ocular helminthiasis in humans and animals is known to cause various types of injury to ophthalmic tissues depending on the type of parasite species, and location of infestation
(Otranto & Eberhard, 2011). In our analysis of antioxidant and lipid peroxidation status of ophthalmic tissue taken from O. petrowi infested northern bobwhite quail, eye worm infested quail displayed significantly less glutathione, glutathione peroxidase activity, but significantly more catalase and superoxide dismutase activity. The level of oxidative damage as measured by TBARS was significantly higher in infected birds as well. These trends were often seen in both the crystalline lens by itself and the globe which was comprised of the sclera, cornea, and uveal tract tissues.
117 Texas Tech University, Jordan Hunter, December 2016
The focus of this study is to examine physiological differences in antioxidants between eye worm infected quail and uninfected quail within the intraocular tissues, therefore any potential pathology occurring based upon antioxidant status from this study is focused in this environment of the ophthalmic space.
The lower average glutathione and glutathione peroxidase availability in infected tissue is a primary point of concern for O. petrowi infected bobwhite quail as GSH is an important molecule for lenticular protein maintenance (Zhang et al., 2008) and as a co- substrate of GPx. The GSH redox system in the uvea and particularly the crystalline lens is also one of the primary methods to protect these tissues from oxidative damage
(Cabrera & Chihuailaf, 2011; Taylor et al., 1995).
The higher on average concentrations of TBARS in both the globe and the lens is a direct indicator of increased oxidative damage through reactive species degradation on lipids, proteins, and nucleic acids within these tissues. The correlation between decreasing levels of glutathione and increasing levels of TBARS products in these tissues would suggest that an oxidative stress state is occurring within the intraocular environment of the eye in O. petrowi infected quail.
Oxidative stress can be an etiologic cause of inflammation in ocular tissues (Wakamatsu et al., 2010), and this condition can provide some insight into any potential dysfunction within the intraocular environment. The choroid within the uveal tissue is the vascular layer of the eye located between the retina and sclera, and supplies oxygen and nutrients to the components of the retina necessary to receive light and produce images.
Inflammation of this tissue can manifest and choroiditis and or choroiretinis, and has symptoms that include, blurred vision, floating black spots, pain and redness in the eye, sensitivity to light and excessive tearing (Berman, 2016).
118 Texas Tech University, Jordan Hunter, December 2016
The eye lens functions by focusing light onto the retina making it a critical component of healthy vision. The depletion of GSH and GPx seen in our analysis of infected quail could be an indicator of cataractogenesis, facilitating a condition resulting from the opafication the crystalline lens which obscures the passage of light within the eye. The pathogenesis of cataract is believed to be the result of the oxidation of lens proteins that make-up lens fibers. The degradation of these proteins forms high molecular weight adducts that aggregate and precipitate within areas of the lens creating opacity in the tissue (Taylor et al., 1995).
In contrast to GSH and GPx, both catalase and superoxide dismutase concentrations were on average higher in infected quail globe, lens and vitreous among both males and females. In a similar study of intestinal helminthiasis by (Siwela et al., 2013), the authors observed significant decreases in all antioxidants studied, including SOD and CAT. The differences seen in this study could be attributed to a few factors however, including host and parasite species, and differences in the organ types sampled in proximity to the parasite’s habitat in the host’s body—as the gastrointestinal tract is known to express more diversity in enzymatic antioxidant isozymes (Brigelius-Fiohe, 1999).
In research by Bhuyan & Bhuyan (1977) the authors observed a depletion in catalase activity in rabbit ocular tissue treated with 3-amino-1H-1,2,4-triazole. The authors found that this did not alter the activity of GPx, and treated subjects had increased incidence of cataract because of reduced catalase capability in the lens. In a study of rat liver in response to the organophosphate insecticide chlorfenvinphos exposure, Lukaszewicz
Hussain & Moniuszko-Jakoniuk (2004) observed significant increases in both CAT and
GPx activity compared to control groups. Ferreira et al., (2004) observed that total reactive antioxidant potential was decreased in human cataract patients, however SOD
119 Texas Tech University, Jordan Hunter, December 2016 and GSH activities were three-fold higher among glaucoma patients while observing no changes in catalase levels among these groups.
Another potential explanation is that the depletion and/or inhibition of associated antioxidant processes could cause the host’s body to compensate for the reduced capability of GSH redox ocular protection by upregulating other mechanisms of free radical scavenging including SOD and CAT. Tissue-specific biases in expression of immune response and antioxidant mediation are not uncommon in helminth infected animals (Menzies et al., 2010), and this could be the case among northern bobwhite quail ophthalmic tissues.
There were not many statistically significant differences in antioxidant status between male and females, however, female bobwhites generally had higher levels of enzymatic antioxidants and GSH than males with some exceptions. This could be due to physiological differences in sex and reproduction, but also in factors associated with pheomelanin production for plumage coloration, and GSH synthesis for ocular oxidant protection competing for the same cysteine residues (Galvan et al., 2012). Female bobwhites characteristically have more brown plumage around their bodies, particularly near the throat and eyes, than adult males do. It is possible more cysteine is sequestered in the feathers of females than males, however we found no statistically significant differences between male and female GSH in the globe (p=0.239) or lens (p=0.089) of uninfected birds. Although, males appeared to have higher GSH concentrations in the globe, and females had higher concentrations in the lens (Figure. 4.1).
In summary, analysis of one non-enzymatic, and three enzymatic antioxidants, as well as lipid peroxidation by-products (TBARS) in ophthalmic tissue exhibited statistically
120 Texas Tech University, Jordan Hunter, December 2016 significant differences between O. petrowi uninfected and infected northern bobwhite quail. Infected quail had less glutathione and glutathione peroxidase in their intraocular tissues, but more superoxide dismutase and catalase than uninfected quail. On average, all quail with known eye worm infestation had higher concentrations of TBARS than uninfected quail within their intraocular tissues indicating the presence of increased oxidative damage. The depletion of antioxidants specific to the GSH redox system and larger concentrations of other free radical scavenging enzymes could be a result of tissue specific biases in enzymatic antioxidant expression in ophthalmic tissue that are inherent to northern bobwhite infested by O. petrowi. The correlation between intraocular eye disease and the depletion of antioxidant capability with increased markers of oxidative damage is known through the work of previous researchers. The results of this study are not explicit indicators of eye disorders in eye worm infected quail, however the levels of antioxidants and TBARS observed in these tissues can provide suitable surrogate biomarkers that correspond to the etiology of many ophthalmic associated disorders which could prove detrimental to the visual acuity and overall fitness of wild quail with eye helminthiasis.
121 Texas Tech University, Jordan Hunter, December 2016
Mean GSH umol/mg in ocular tissue across sex and infection status 4.50E-05
4.00E-05
3.50E-05
3.00E-05
2.50E-05
2.00E-05
Mean GSH umol/mg Mean 1.50E-05
1.00E-05
5.00E-06
0.00E+00 Female Male Female Male Female Male Female Male Infected Uninfected Infected Uninfected GLOBE LENS
Figure 4.1 GSH concentration in globe tissues was significantly higher in uninfected versus infected samples that included all sexes (p=0.010), and GSH was higher in uninfected females versus infected females exclusively (p=0.022). Lens GSH concentrations were significantly higher (p=0.0022) in uninfected males versus infected males.
122 Texas Tech University, Jordan Hunter, December 2016
Mean SOD activity (U/mg) in ocular tissue across sex and infection status
0.007
0.006
0.005
0.004
0.003
0.002 Mean SOD activity U/mg activity SOD Mean 0.001
0 Female Male Female Male Female Male Female Male Infected Uninfected Infected Uninfected GLOBE LENS
Figure 4.2 SOD activity was significantly higher in infected quail globe tissue versus uninfected quail both within the globe (p=0.008), and lens (p=0.009) factoring in all sexes. There were also significant differences between females in this assay as both globe (p=0.020) and lens (p=0.020), SOD activity for this sex was higher in infected female birds.
Infected males had on average lower SOD activity than infected females in the globe tissue (p=0.041).
123 Texas Tech University, Jordan Hunter, December 2016
Mean CAT activity (umol/min/mg) in globe across sex and infection status 3.00E-05
2.50E-05
2.00E-05
1.50E-05
1.00E-05
5.00E-06 Mean CAT Activity (umol/min/mg) Activity CAT Mean 0.00E+00 Female Male Female Male Infected Uninfected Globe
Figure 4.3 Catalase in the globe tissue displayed a statistically significant higher level of activity in infected tissue and was lower on average in uninfected tissue of all sexes (p=0.027).
124 Texas Tech University, Jordan Hunter, December 2016
Mean CAT activity (umol/min/mg) in vitreous across sex and infection status
1.40E-07
1.20E-07
1.00E-07
8.00E-08
6.00E-08
4.00E-08
2.00E-08
Mean CAT Activity (umol/min/mg) Activity CAT Mean 0.00E+00 Female Male Female Male Infected Uninfected Vitreous
Figure 4.4 Infected males displayed higher on average catalase activity than infected females, and all uninfected sexes, however these differences were not statistically significant.
125 Texas Tech University, Jordan Hunter, December 2016
Mean GPx activity (nmol/min/mg) in ocular tissue across sex and infection status 0.2
0.18
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02 Mean (nmol/min/mg) GPx activity Mean
0 Female Male Female Male Female Male Female Male Infected Uninfected Infected Uninfected Globe Lens
Figure 4.5 GPx activity in the globe was significantly different between infection status of all sexes
(p=0.007) as uninfected quail had on average higher GPx activity in this tissue, which was also the case when observing uninfected female globe tissue exclusively (p=0.004).
Within uninfected globe tissue, uninfected females have a statistically significant higher level of GPx activity when compared to uninfected males (p=006).
126 Texas Tech University, Jordan Hunter, December 2016
Mean TBARS (umol/mg) in ocular tissue across
sex and infection status 0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0 Female Male Female Male Female Male Female Male Infected Uninfected Infected Uninfected Globe Lens
Figure 4.6 TBARS concentrations in the eye globe were at significantly higher levels in infected and lower in uninfected quail samples amongst all sexes (p=0.034), and lipid peroxidation was also higher in infected females more so than uninfected females exclusively
(p=0.016).
127 Texas Tech University, Jordan Hunter, December 2016
Mean TBARS (umol/mg) in vitreous across sex and infection status 1.60E-06
1.40E-06
1.20E-06
1.00E-06
8.00E-07
6.00E-07
4.00E-07
2.00E-07
Mean TBARS (umol/mg) TBARS Mean 0.00E+00 Female Male Female Male Infected Uninfected Vitreous
Figure 4.7 Infected males had much higher on average TBARS in the vitreous compared to females, however these differences were not determined to be statistically significant
(p=0.090). Uninfected females also had much higher vitreous TBARS than males, with no statistical significance.
128 Texas Tech University, Jordan Hunter, December 2016
5 CHAPTER V
IMMUNE RESPONSE IN QUAIL OCULAR TISSUE
5.1 Introduction
The decline of northern bobwhite quail (Colinus virginianus) populations across Texas
over the last few decades is a phenomenon of considerable ecological importance
(Brennan, 1999; Williams et al., 2004). Old world (Phasiandiae) and new world
(Odontophoridae) quail and related galliformes have been historically important birds as livestock, game, agriculture, and scientific study (Gust et al., 2015; Williams et al., 2000;
Stoddard, 1931). Understanding both the environmental and biological factors that may have contributed to the decline of C. virginianus is important to assist the conservation efforts for this species and other wildlife sharing the same or similar habitats (Brausch et al., 2010; Gust et al., 2015; Kitulagodage et al., 2011; Romijn et al., 1995; Ottinger et al.,
2005; Quinn et al., 2012). Identifying the significant environmental factors or pathogenic agents that influence this avian population may also benefit our broader understanding of how these factors influence wildlife and human populations on all trophic levels within ecoregions (Angelo et al., 2012).
Species such as the northern bobwhite and Japanese quail (Corturnix japonica) have been used as effective animal models in the study of toxicology and the physiological responses of vertebrates to pharmaceuticals, pesticides and pathogens (Romijn et al.;
1995, Newsted et al., 2007; Lusk et al., 2002; Quinn et al., 2012; Quinn et al., 2009).
The occurrence of parasitic and infectious diseases affecting northern bobwhite quail has involved many studies (Bruno et al., 2015; Dunham et al., 2014; Kellogg & Calpin, 1971;
129 Texas Tech University, Jordan Hunter, December 2016
Kocan et al., 1979). The parasitic eye worm Oxyspirura petrowi is a helminth frequently found in the eye orbit, and around the ocular surface, lacrimal tissues and nictitating membrane of ground dwelling birds including the northern bobwhite quail (Skrjabin,
1929). Existing knowledge of O. petrowi induced pathology on host quail and the impact of this parasite on overall visual acuity and fitness in the host is limited (Dunham et al.,
2014). Some infected quail have been reported to exhibit signs of inflammation around sites of worm infestation including the conjunctiva, and intraorbital lacrimal and harderian gland tissues (Bruno et al., 2015; Saunders, 1935). Studies have shown that pathogens and parasitic helminths can also induce harmful oxidative stress in animal and bird host’s tissues which could be linked to these signs of inflammation caused by increased immune response (Siwela et al., 2013; Mougeot et al., 2010; Sorci & Faivre,
2009).
The correlation between O. petrowi infections and their potential to induce
immunopathology and diseases of the eye through oxidative stress mechanisms in
northern bobwhite quail has not been completely investigated. The purpose of this study
is to identify through relative gene expression of cytokines and transcription factors
associated with Th2 pathway humoral immune response and proliferation of reactive
oxygen species for parasite elimination in O. petrowi infected northern bobwhite quail. In
addition, the expression of cytokines, proteins and antioxidants associated with the
mediation of reactive species affecting oxidative status have examined to determine the
presence of oxidative stress in ophthalmic tissues.
A comparative assessment of genes expression related to immune and oxidative stress
response between eye worm infected and uninfected wild northern bobwhite quail was
130 Texas Tech University, Jordan Hunter, December 2016
performed using nucleic acid sequences specific to avian cytokines, transcription factors,
enzymatic antioxidants and proteins.
O. petrowi infected northern bobwhite quail exhibited a higher expression of the target
genes IL-8, IL-10, IL-13, GATA-3 and avUCP as compared to quail with no noticeable
O. petrowi infection in our relative analysis of gene expression utilizing qRT-PCR. A
noticeable increase of IL-10 and avUCP expression among infected subjects compared to
uninfected quail suggests an alteration in oxidative status within the ophthalmic tissues of
infected hosts in response to O. petrowi infestation.
The use of these markers correlate to the general immune response and oxidative status
of ocular tissue and provide valuable insight into the host’s physiological response to eye
worm helminthiasis. The link between oxidative stress and disease progression in many
organs including the eye (Babizhayev & Costa, 1994) has been explored by many
researchers (Rahal et al., 2014; Rahma et al., 2012), and ocular oxidative stress induced
by immunopathology in infected birds has the potential create more severe visual acuity
and fitness related problems in these hosts (Galvan et al., 2012).
5.2 Eye Immunology
Intraorbital glands comprised of the lacrimal and harderian glands are responsible for maintaining normal hydration, nutrition and defense of the ocular surface tissues (Knop
& Knop, 2005). Aqueous secretions are provided by the lacrimal gland while the harderian gland plays a vital role in ocular immunity (Dimitrov & Genchev, 2011),
(Kozlu & Altunay, 2011) through secretion of immunoglobulins IgA, IgG, and IgM.
131 Texas Tech University, Jordan Hunter, December 2016
This gland also provides vascular distribution of immune cells including macrophages, lymphocytes and granulocytes for the eye orbit (Baba et al., 1990; Boydak & Mehmet,
2009; Bruno et al., 2015).
Ophthalmic tissues and the globe are capable of inducing innate and acquired immune responses to mitigate the effects of microorganisms (Akpek & Gottsch, 2003), however, the eye is an organ with immune privilege, and does not elicit the same systemic responses to foreign bodies or pathogen challenge as other organs in the body (Taylor,
2009). The exact reason for this organ’s immunological responses have not been completely determined, however research suggests that the difficulty required for immune-mediating cells to pass the blood ocular barrier tissues and the absence of direct lymphatic drainage required for antigens to pass onto lymphatic nodes and induce immune response play important parts in immune cell regulation out of the eye (Barker
& Billingham, 1968; Medawar, 1948; Taylor, 2009).
The intraocular microenvironment also contains large amounts of immunosuppressive molecules and processes which can mediate immune cell activity. Macrophages in the aqueous humor can present antigens to promote immune suppression (Wilbanks &
Streilein, 1992). Aqueous humor can suppress IFN-γ generation and promote TGF-β production by CD4 T cells (Taylor et al., 1992) and contains many neuropeptides including; α-MSH, CGRP, VIP, and SOM to suppress activation of inflammatory activity of macrophages (Taylor, 2005), cytokine TGF-β2 and the molecules, IDO and
FasL to suppress the activation of Th1 cells (Griffith et al., 1995; Ryu & Kim, 2007;
Taylor et al., 1994; Taylor et al., 1998). These features among others indicate that there is a high threshold and many conditions that must be met before the start of
132 Texas Tech University, Jordan Hunter, December 2016 inflammation and immunity within the normal ocular tissue microenvironment (Taylor,
2009).
Experimental studies have demonstrated that ocular immunosuppressive capability can be altered when antigens such lipopolysaccharide (LPS) are introduced into the ocular microenvironment as in the cases of endotoxin-induced uveitis (EIU) and endotoxin autoimmune uveitis (EAU). These studies have shown that before detection of retinal infiltration, the immunosuppressive capabilities of the aqueous humor are inactive and large amounts of pro-inflammatory cytokines in and T-cell subsets could be detected
(Kitaichi et al., 2005; Luger et al., 2008; Ohta et al., 2000). During these conditions the anterior chamber-associated immune deviation (ACAID) that induces immunosuppression when foreign antigens are present in the ocular microenvironment was inhibited at the onset of retinal inflammation—the microenvironment did not resume its ability to induce ACAID until after uveitis was resolved. These experiments indicate that a direct attack on ocular tissue causes an accelerated inflammatory response, and diminished immunosuppression for some time, and the resulting uveitis could be a way in which the ocular microenvironment attempts to recover its immune privilege (Taylor, 2009). This creates a potential pathway for intraocular pathogenesis through increased inflammatory response exacerbated under chronic antigen exposure from pathogens or parasites.
For regular maintenance (moistening, nutrition, and defense), the eye surface depends primarily on the conjunctiva and secretions from the lacrimal and Harderian glands, collectively called intraorbital glands (Knop and Knop, 2005). While the lacrimal gland is responsible for more aqueous secretions, the avian Harderian gland plays a vital role
133 Texas Tech University, Jordan Hunter, December 2016 in controlling local orbital immunity (Dimitrov & Genchev, 2011; Kozlu & Altunay,
2011) via immunoglobulins IgA, IgG, and IgM, as well as vascular released immune cells (macrophages, lymphocytes, granulocytes) onto the eye orbit (Baba et al., 1990;
Boydak and Mehmet, 2009; Bruno et al., 2015).
5.3 Th2 host immune response to helminths—interleukins, transcription factors
and avian uncoupling protein
Leukocytes consist of many subsets including; granulocytes, monocytes, and
lymphocytes, that are known to travel to the areas in proximity of infected tissue and
secrete metabolites. Injury to these tissues may occur as the immune response is either
misdirected or overexpressed, which can then cause immunopathology in the host.
Organisms have mechanisms to mediate this process and prevent inflammation and tissue
damage using certain cytokines like interleukin (IL-10), and transforming growth factor
beta (TGF-β) to mitigate and resolve this inflammation as the pathogen is eliminated
(Belkaid, 2007). Research has suggested that cases of chronic infection by pathogen or
parasite and an over-stimulated inflammatory response may cause more harm than the
direct effects of the pathogen (Graham et al., 2005; Mackintosh et al., 2004; Raberg et
al., 1998).
There are two sub-groups of T helper (Th) cells that encompass different types of
cytokine production cascades with different purposes (Cherwinski et al., 1987; Mosman
et al., 1986). Th1 cell types are associated with cells infected by viruses and other
intracellular microbes and have features that include; cytolytic activity, synthesis of IL-2
and interferon gamma (IFN-γ), as well as help B cells with IgG, IgM, IgA synthesis
134 Texas Tech University, Jordan Hunter, December 2016
(Degen et al., 2005). Th2 cells are not cytolytic but are known to generate IL-4 and IL-5 cytokines, assist in IgE helminth specific antibody synthesis, as well as IgG, IgM and
IgA antibody production (Everts et al., 2010). The immune system chooses what CD4
Th1 or CD4 Th2 cells are induced based upon the type of infection or autoimmune disease (Mosman et al., 1986). Inflammatory states with delayed type hypersensitivity reactions and low antibody generation leads to Th1 cell selection. Inflammatory reactions associated with constant antibody production such as allergic reactions or larger pathogenic organisms initiating significant amounts of IgE generation are typically associated with Th2 cells (Janeway, 1992). The cytokine profiles associated with Th cells dictate the intensity, duration and type of immune or inflammatory response the host produces and are important in the resulting impact of infectious diseases (Mosman et al.,
1986). Studies have shown that the Th1 response of hosts with schistosomiasis is linked to purging this parasite, and that Th2 responses will generally result in granuloma formation and disease development (Infante-Durante & Kamradt, 1999).
In most cases helminth infection, will initiate a Th2 immune response in the host that results in eosinophilia, goblet and mucosal mast cell hyperplasia and synthesis of noncomplement fixing antibodies (Gause et al., 2003).
The human eye worm onchocerca volvulus for example has been reported to cause increased CD4 cells and IL-4 production eventually leading to inflammatory response in the ocular tissue, a primary characteristic Th2 response (Akpek & Gottsch, 2003; Chan et al., 1993; Chan et al., 1989). IL-5 is another cytokine associated with Th2 response and is known to induce phagocytic eosinophils to purge parasites (Spencer & Weller, 2010).
IL-13 is a cytokine secreted by CD4 Th2 cells that is known to activate IgE in human B cells, and initiate physiological changes in tissue that enable parasite clearance (Wynn,
135 Texas Tech University, Jordan Hunter, December 2016
2003). IL-8 is a chemokine referred to as a neutrophil chemotactic factor, it is a key mediator of inflammation and induces movement of phagocytes to areas of tissue in proximity to infection and signals phagocytosis around these areas (Modi et al., 1990).
During an oxidative stress state IL-8 secretion increases, leading to more recruitment of inflammatory cells and oxidant stress mediators (Vlahopoulos et al., 1999). Studies have shown that there is a close relationship between Th1 and Th2 immune responses in mammals and birds, with factors upregulating or downregulating the activity of these cells in inverse proportion (Degen et al., 2005). Depletion of glutathione, an outcome of oxidative stress, has been shown to downregulate Th1 immunity and affect the polarity of immune response as oxidative stress skews the immune response towards
Th2 cells and suppresses Th1 cell differentiation (Kim et al., 2007).
IL-10 is known to have many different effects on the hosts immunoregulation and inflammation including downregulation of; Th1 cytokine expression, MHC class II antigens, and co-stimulatory molecules on macrophages (de Waal Malefyt et al., 1991).
The other purposes of this cytokine are to inhibit bacterial antigen product initiation of pro-inflammatory cytokines, facilitate B cell survival, and antibody production (Varma et al., 2001)
Other regulators of immune response and oxidative stress include GATA-3 and avian uncoupling protein. GATA-3 is a transcription factor that is known to induce Il-4, IL-5, and IL-13 secretion in Th2 cells and suppress the differentiation of naïve T-cells towards the Th1 cell type while facilitating differentiation of Th0 cells to Th2 cell subtypes (Yagi et al., 2011). The capabilities of GATA-3 are thought to be related to tissue type
(Wilson, 2008), and its transcriptional level expression closely linked to IL-4 receptor cytokines (Cook & Miller, 2010).
136 Texas Tech University, Jordan Hunter, December 2016
Uncoupling proteins are a part of a group of mitochondrial proteins that are involved in
uncoupling the oxidation of reduced substrates from the phosphorylation of ADP to ATP
through a leak of protons within the cellular inner membrane (Ricquier & Bouillaud,
2000). The role of avian uncoupling proteins (avUCP) has not been completely
characterized, but it is believed to be associated with facultative thermogenesis, and lipid
metabolism (Colin et al., 2003).
Studies by (Criscuolo et al., 2005) have reported that avUCP overexpression in yeast
instigates mediation of ROS, and one of primary purposes of this protein in birds is to
stop mitochondrial ROS proliferation and oxidative stress during periods of high
metabolic activity (Rey et al., 2010).
5.4 Oxidative stress and eye disease
Oxidative stress conditions are imposed on the eye from exposure to exogenous and/or endogenous sources, subsequently altering the redox balance in these tissues and potentially causing a variety of ophthalmologic disorders such as; cataract of the lens
(Varna et al. 1984, Varna et al., 2011), diabetic retinopathy (Lopes de Faria et al., 2011;
Yang et al., 2010; Yamagishi & Matsui, 2011,), age related macular degeneration
(AMD) (Ding et al., 2009; Kaarniranta et al., 2011; Liutkeviciene et al. 2010), and experimental retinal detachment (Mantopoulos et al., 2011; Roh et al., 2011).
The intraocular anterior and posterior portions of the eye and associated tissues are also
particularly susceptible to ROS attack and degradation. Oxidative stress is believed to be
a primary factor in the formation of cataractous changes in the crystalline lens of the eye
(Babizhayev et al., 2004; Babizhayev & Costa, 1994; Babizhayev, 2012; Babizhayev et
al., 1988; Spector, 1995). The structure and integrity of the lens epithelial cells can be
137 Texas Tech University, Jordan Hunter, December 2016 damaged by hydrogen peroxide, dehydrostatic acid, and lipid peroxides. This damage relates to the induction of protein disulfide crosslinks, reduced protein solubility and lower cellular adhesions (Babizhayev & Yegorov, 2014; Colitz et al. 2004, Spector,
2000).
Research has shown that oxidants and ROS can induce premature senescence in human lens epithelial cells, accelerating telomere shortening and reducing telomerase activity
(Babizhayev et al., 2011; Babizhavey & Yegorov, 2010; Huang et al., 2005). Data from studies by (Babizhayev & Yegorov, 2014) have demonstrated that human cataractogenesis involves senescence of lens cells, induced by oxidative stress linked to
DNA damage, inhibition of telomerase and marked telomere shortening. The ribonucleic protein telomerase in mammal lens epithelial cells has been suggested to assist damage telomeres during oxidative stress and preventing acceleration of cell senescence. The epithelial cell layer is the initial location of oxidative stress incidence, followed by attack on the lens fibers causing cortical cataract (Spector et al. 1995).
Glutathione (GSH) is important for normal maintenance of lens transparency and is the predominant reducing antioxidant molecule found in large concentration of the lens.
GSH in collaboration with the glutathione redox cycle in the lens epithelium and superficial cortex detoxifies many harmful reactive oxidants including H2O2 and dehydroascorbic acid (Babizhayev & Yegorov, 2014).
The mitochondrial respiratory chain of cellular mitochondria is a potent generator of reactive species including superoxide and hydrogen peroxide, and it contributes to a large degree of oxidative damage in vital cells. Oxidative damage from mitochondria is of large clinical importance, however direct treatment of mitochondria with antioxidants has
138 Texas Tech University, Jordan Hunter, December 2016 not been entirely successful likely due to their wide range of dispersal throughout the body, ophthalmic tissues and fluids (Babizhayev, 2016).
Cataracts are one of the most significant ophthalmologic diseases in veterinary medicine and ophthalmology (Babizhayev & Yegorov, 2014). The correlation between age related cataract and oxidative damage of lens epithelial cells and the internal lens in suggests that this could also potentially be used as general biomarker for assessing systemic damage done by reactive oxygen species and life expectancy in animals (Urfer et al., 2011),
(Babizhayev & Yegorov, 2014). Antioxidant and lipid peroxidation markers in the blood and tissue are generally viewed as the factors most indicative of redox state in an organism, however, there is currently no universally acknowledged method among researchers regarding the most consistent and accurate measure of oxidative stress
(Kadiiska et al., 2015).
A large amount of work has been done with different harmful microorganisms and pathology of the eye as well (Klotz et al., 2000; Mircheff, 2011). However, there is limited research in ocular helminthiasis of the type O. petrowi is known to impose on its host, and the subsequent immune response and/or oxidative stress induced by this infestation.
5.5 Materials and methods
5.5.1 Subject capture and sample harvesting
Northern bobwhite quail were captured in grain sorghum baited wire mesh traps placed around locations within west Texas and Oklahoma between 2011 and 2013. Several captured quail were randomly selected for euthanization via cervical dislocation and
139 Texas Tech University, Jordan Hunter, December 2016 immediately stored under freezing conditions. Birds were transported from each field location to our hub laboratory where necropsy was performed and study relevant organs were harvested. The remaining carcass and ocular tissue were stored at -80 °C until use.
Eight Oxyspirura petrowi infected quail and eight quail with no detectable O. petrowi infestation were selected for gene expression analysis. The selected samples consisted of eight female birds including four O. petrowi infected, and four uninfected as well as eight male birds that consisted of four O. petrowi infected, and four uninfected for a total of
(n=16) samples.
5.5.2 Tissue Homogenization
Whole pairs of eyes were enucleated after thawing and each pair of ocular tissues, was consolidated into individual working samples. Eye vitreous was aspirated using a small syringe and transferred to a clean micro centrifuge tube for storage at -80 °C. A small incision was made on the perimeter of the globe and forceps were used to remove the lens for separate storage and homogenization. The remaining globe tissues were homogenized separately via a glass dounce homogenizer in pH 7.0 50 mM potassium phosphate (Fisher Chemical P288-100), 1mM EDTA (Ambion AM92605) buffer prepared in nuclease free water (Fisher Scientific # W5-1) to obtain an approximately
10% weight by volume homogenate (Saxena et al., 1992). Homogenates were centrifuged at 28,000 x g for 30 minutes at 4 °C, and the supernatants were transferred to a clean micro centrifuge tube in preparation for RNA extraction.
Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) using tissue samples in duplicate were performed for all assays using a Rotor Gene Q thermal
140 Texas Tech University, Jordan Hunter, December 2016
cycler and corresponding software. Instrument procedures were performed per the
manufacturers specifications.
5.5.3 RNA extraction and analysis
RNA extraction from ocular tissue homogenates was performed with the use of a kit and
reagents provided by Qiagen (RNeasy Mini Kit, Qiagen, Cat. No. 74104). Extracted
RNA in RNase free water was stored at -80 °C (stable for one month) until use. Extracted
RNA samples were analyzed for concentration and contamination using a Nano-Drop
ND-1000 at A260, and taking the absorbance ratio at A260/A280.
5.5.4 Gene expression analysis
Analysis of relative gene expression was done with the assistance of a one-step qRT-
PCR kit provided by Promega (GoTaq® 1-Step RT-qPCR System, Promega 2015).
One-step RT-PCR amplifications were performed in a 72-well rotor using Qiagen rotor gene optical reaction tubes. The thermal-cycler was programmed for reverse transcription at 50 °C for 15 min., denaturation and RT enzyme inactivation at 95 °C for
5 min., followed by 40 cycles of 10 sec. denaturation at 95 °C and 30 sec. annealing and extension at 60 °C. This was followed by melt curve analysis between 55 °C and 95 °C with 0.5 °C of temperature increments.
Samples were analyzed via real-time PCR using the sample maximization strategy with inter-run calibrators (Dervaux et al., 2010). This method determines the changes in mRNA level and expression of northern bobwhite quail genes corresponding to the regulation of antioxidants, cytokines, and transcription factors across multiple samples
141 Texas Tech University, Jordan Hunter, December 2016 and expresses them relative to an internal control or “housekeeping gene” (B-actin).
This provides a relative quantification of expressed genes corresponding to the physiological and immune related responses of quail infected by O. petrowi in relation to uninfected quail.
Threshold cycle number (Ct) was analyzed using Rotor-gene Q software (Qiagen). The
Ct value of each target gene was normalized to the Ct value of the reference/housekeeping gene β-actin from the same sample and the fold change in the expression, or normalized gene expression (NRQ) was calculated using the 2-ΔΔCt method (Livak & Schmittgen, 2001). Reaction for each sample target and housekeeping genes were performed in duplicate and a control with no RNA template was included in every analysis.
The primers sequences, and annealing temperatures used for the housekeeping gene and target genes were provided by (Merchan et al. 2011; Ojano-Dirain et al., 2007; Uno et al. 2012; Vesco & Gasparino, 2013; Yarru et al., 2009) (Table 5.1). Primer sequences used in qRT-PCR were created by Integrated DNA technologies. Lyophilized primers from the manufacturer were reconstituted in tris-EDTA (TE) buffer (Promega, V6231) for 10 uM primer solutions stored at -20 C (one-year stability).
142 Texas Tech University, Jordan Hunter, December 2016
Table 5.1 Target gene primer sequences and annealing temperatures
Name Primer Sequences Annealing
Temperature (⁰C)
GATA-3 5′-TGGAACCTCAGCCCTTTTTCC-3′. Forward 57 GATA-3 5′-GTTAAAGGAGCTGCTCTTGG-3′ Reverse
GPX7 5'-TTGTAAACATCAGGGGCAAA-3'
Forward 60 GPX7 5'-TGGGGCCAAGATCTTTCTGTAA-3' Reverse
avUCP 5'-GCAGCGGCAGATGAGCTT-3' Forward 60 avUCP 5'-AGAGCTGCTTCACAGAGTCGTAGA-3' Reverse
B-actin 5'-CTGGCACCTAGCACAATGAA-3' Forward 55 B-actin 5'-CTGCTTGCTGATCCACATCT-3' Reverse
IL-4 5'-GAGAGCATCCGGATAGTGAAG-3'
Forward 60 IL-4 5'-TTCGCATAAGAGCTGGGTTC-3'
Reverse
IL-13 5'-CTGCAAGAAGGACTATGAGCCC-3'
Forward 55 IL-13 5'-CAGTGCCGGCAAGAAGTT-3'
Reverse
IL-10 5'-CACAACTTCTTCACCTGCGAG-3'
Forward 55 IL-10 5'-CATGGCTTTGTAGATCCCGTTC-3'
Reverse
IL-8 5'-CTGAGGTGCCAGTGCATTAG-3'
Forward 58 IL-8 5'-AGCACACCTCTCTTCCATCC-3'
Reverse
143 Texas Tech University, Jordan Hunter, December 2016
5.5.5 Experimental design and post-laboratory analysis criteria
Studies have suggested that the proportion of pheomelanin plumage in different bird species may be correlated to their susceptibility to oxidative stress (Galvan et al., 2012;
Galvan & Alonso-Alvarez, 2009). It was reported by (Galvan et al., 2012) that avian species with a larger proportion of plumage containing pheomelanin had higher prevalence of cataracts likely due to their reduced capability to protect the crystalline lens of the eye from ROS attack and oxidative stress leading to oxidative stress and damage.
This may be due to the processes of pheomelanogenesis requiring the same cysteine amino acids for which glutathione (GSH) is the main physiological reservoir (Meyskens et al., 1999). Along with ascorbic acid, GSH is one of the primary antioxidants found in the eye, and is important in maintaining redox balance in the intraocular tissues and crystalline lens, by keeping protein thiols in their reduced state, preventing harmful protein aggregation which causes lens opafication associated with cataractous eyes
(Reddy, 1990). A physiological competition between the processes of pheomelanogenesis and endogenous antioxidant capability in the eye may exist (Galvan et al., 2012).
Based on studies by (Galvan et al., 2012), oxidative stress induced by the parasitic eye worm O. petrowi could also play a factor in depleting glutathione levels in birds while this antioxidant concentration is simultaneously influenced by the pheomelanin content of their plumages, with birds displaying brown to red phenotypes also having lower physiological levels of GSH due to the sequestering of cysteine in feathers. Northern bobwhite quail have a sexual dichromism in their plumage phenotype, as females exhibit more brown and auburn coloration in the plumages around their body and eyes, while
144 Texas Tech University, Jordan Hunter, December 2016 males have more prominent white feathers around their eyes and throat areas which may grant them more resistance to oxidative stress than females.
Eye disorders and cataractous changes in the eye lens are also known to come with age
(Taylor et al., 1995). We have accounted for both sex and age factors that could influence oxidative stress and eye disease progression by limiting the subjects of our study to adult males and females, which were sexed per coloration on the head and neck and aged based on molting patterns at the primary wing coverts (Koerth et al., 2016).
5.6 Results
5.6.1 Expression of all infected relative to uninfected
A total of (n=16) pairs of eye globes from northern bobwhite quail comprised of 8 O.
petrowi infected (4 males, and 4 females), and 8 uninfected quail (4 males and 4 females)
were used for comparative expression analysis of the target genes; IL-4, IL-8, IL-10,
IL13, GATA-3, GPX-7, and avUCP. The gene expression as determined by the mean Ct
value of the samples to calculate ΔCt(mean) for both the “uninfected/calibrator” and
“infected/treated” samples. All uninfected or “calibrator” samples were used as the
experimental control from which the baseline axis of mean gene expression was
determined. The fold change for each target gene in all O. petrowi infected or “treated”
samples was determined using the formula (2-ΔΔCt=NRQ).
The average gene expression of IL-10 (2.08 fold), IL-13 (2.62 fold), and avUCP (2.75
fold) was increased amongst all infected quail sampled. Average expressions of IL-4,
145 Texas Tech University, Jordan Hunter, December 2016
IL8, GATA-3, and GPX-7 were decreased on average in all infected quail by comparison
(fold < 1) (Figure. 5.1).
5.6.2 Expression of male infected relative to male uninfected
A total of (n=8) pairs of eye globes from northern bobwhite quail comprised of 4 O. petrowi infected (4 males), and 4 uninfected quail (4 males) were used for comparative expression analysis of the target genes; IL-4, IL-8, IL-10, IL-13, GATA-3, GPX-7, and avUCP. The gene expression was determined by the mean Ct value of the samples to calculate ΔCt (mean) for both the “uninfected/calibrator” and “infected/treated” samples.
Male uninfected or “calibrator” samples were used as the experimental control in this analysis from which the baseline axis of mean gene expression was determined.
The fold change for each target gene in the O. petrowi infected males or “treated” samples was determined using the formula (2-ΔΔCt=NRQ).
The average gene expressions of IL-8 (2.33 fold), IL-10 (4.05 fold), IL-13 (4.86 fold),
GATA-3 (1.89 fold), and avUCP (7.80 fold) were increased amongst male infected quail sampled. Average expressions of IL-4, and GPX-7 were decreased on average in male infected quail by comparison (fold < 1) (Figure 5.2).
5.6.3 Expression of female infected relative to female uninfected
A total of (n=8) pairs of eye globes from northern bobwhite quail comprised of 4 O. petrowi infected (4 females), and 4 uninfected quail (4 females) were used for comparative expression analysis of the target genes; IL-4, IL-8, IL-10, IL-13, GATA-3,
GPX-7, and avUCP. The gene expression was determined by the mean Ct value of the
146 Texas Tech University, Jordan Hunter, December 2016 samples to calculate ΔCt (mean) for both the “uninfected/calibrator” and
“infected/treated” samples. Female uninfected or “calibrator” samples were used as the experimental control in this analysis from which the baseline axis of mean gene expression was determined. The fold change for each target gene in the O. petrowi infected females or “treated” samples was determined using the formula (2-ΔΔCt=NRQ).
The average gene expressions of IL-10 (1.06 fold) and IL-13 (1.41 fold), were increased amongst the female O. petrowi infected quail sampled. Average expressions of IL-4, IL8,
GATA-3, GPX-7, were decreased on average relative to control in female infected quail by comparison (fold < 1). The target gene avUCP was observed to have a fold change of
0.972, which is close to the base line expression for control (1.00) (Figure 5.3).
5.6.4 Expression of male infected relative to female infected
A total of (n=8) pairs of eye globes from northern bobwhite quail comprised of 4 O. petrowi infected male quail, and 4 O. petrowi infected female quail were used for comparative expression analysis of the target genes; IL-4, IL-8, IL-10, IL-13, GATA-3,
GPX-7, and avUCP. The gene expression was determined by the mean Ct value of the samples to calculate ΔCt(mean) for both the “female infected/calibrator” and
“male infected/treated” samples. Female infected or “calibrator” samples were used as the experimental control in this analysis, from which the baseline axis of mean gene expression was determined. The fold change for each target gene in the O. petrowi infected males or “treated” samples was determined using the formula (2-ΔΔCt=NRQ).
The average gene expressions of all target genes were increased amongst the male O. petrowi infected quail sampled. These included; IL-4 (2.42 fold), IL-8 (2.02 fold), IL-10
147 Texas Tech University, Jordan Hunter, December 2016
(3.34 fold), IL-13 (2.16 fold), GATA-3 (2.28 fold), GPX-7 (1.50 fold), and avUCP (3.44 fold) (Figure 5.4).
5.6.5 Expression of male uninfected relative to female uninfected
A total of (n=8) pairs of eye globes from northern bobwhite quail comprised of 4 uninfected male bobwhite quail, and 4 uninfected female quail were used for comparative expression analysis of the target genes; IL-4, IL-8, IL-10, IL-13, GATA-3,
GPX-7, and avUCP. The gene expression was determined by the mean Ct value of the samples to calculate ΔCt (mean) for both the “female uninfected/calibrator” and “male uninfected/treated” samples. Female uninfected or “calibrator” samples were used as the experimental control from which the baseline axis of mean gene expression was determined. The fold change for each target gene in the uninfected males or “treated” samples was determined using the formula (2-ΔΔCt=NRQ).
The average gene expressions of all target genes were decreased (fold change < 1) amongst the male uninfected quail sampled. These included; IL-4 (0.22 fold), IL-8 (0.16 fold), IL-10 (0.87 fold), IL-13 (0.63 fold), GATA-3 (0.12 fold), GPX-7 (0.25 fold), and avUCP (0.43 fold) (Figure 5.5).
5.6.6 General assessment of immune response
Three comparative analysis of relative gene expression were performed with no distinct comparison between sexes: 1) Target gene expression of all infected quail relative to a baseline y-axis of expression in all uninfected quail, 2) Target gene expression of infected male quail relative to a baseline y-axis of expression in uninfected male quail,
148 Texas Tech University, Jordan Hunter, December 2016 and 3). Target gene expression of infected female quail relative to a baseline y-axis of expression in uninfected female quail. From these analysis, the general trend of genes with higher expression in infected quail were IL-10, IL-13, and avUCP. Increased expression of IL-8 (2.33 fold), and GATA-3 (1.90 fold), was observed amongst infected male bobwhite quail when compared to uninfected male quail. Expression of avUCP was noticeably increased in analysis of all infected quail, and amongst male infected relative uninfected males, however its expression amongst females infected quail relative to uninfected females (0.972 fold) was very close to the baseline uninfected female control expression value of 1. Avian uncoupling protein sequences had on average higher expression in O. petrowi infected birds, as determined by fold change, than any of the genes surveyed in this study.
5.6.7 Comparing immune response between sexes
Two comparative analysis of relative gene expression were performed with comparison between sexes: 1) Target gene expression of infected male quail relative to a baseline y- axis of expression in infected female quail, and 2) Target gene expression of uninfected male quail relative to a baseline y-axis of expression in uninfected female quail. From these analysis, all target genes exhibited higher expression in infected male quail as compared to infected female quail, and lower expression in uninfected male quail relative to uninfected female quail. Avian uncoupling protein sequences were more expressed in
O. petrowi infected male birds (3.44 fold) than the other target genes surveyed, followed by IL-10, IL-4, GATA-3, IL-13, IL-8, and GPX-7 in decreasing order of fold change.
GATA-3 was the least expressed target gene (0.12 fold), and IL-10 (0.88 fold) was the most expressed target gene in uninfected male bobwhites relative to uninfected females.
149 Texas Tech University, Jordan Hunter, December 2016
5.7 Discussion
Ocular helminthiasis in humans and animals is known to cause various types of injury to ophthalmic tissues depending on the type of parasite species, and location of infestation
(Otranto & Eberhard, 2011). In our qRT-PCR gene expression analysis of immune response and oxidative status of ophthalmic tissue taken from O. petrowi infested northern bobwhite quail, eye worm infested quail generally displayed increased expression of the cytokines IL-10, IL-13, and the avian uncoupling protein, avUCP.
Increased expressions of the cytokine IL-8 and transcription factor GATA-3 were observed primarily in infected male quail relative to uninfected male quail. All target genes were higher expressed in infected male quail relative to infected female quail, and these same genes were expressed lower in uninfected male quail than uninfected female quail on average.
Interleukin-10 (IL-10) is an anti-inflammatory cytokine associated with immunoregulation and inflammation. It has the function to downregulate; Th1 cytokine expression, MHC class II antigens, and co-stimulatory molecules on macrophages (de
Waal Malefyt et al., 1991), as well as inhibit antigen initiation of pro-inflammatory cytokines from macrophages and Th1 T cells, facilitate B cell survival, and antibody production (Varma et al., 2001). Studies have shown that IL-10 can be produced by mast cells to counteract inflammation associated with these cells in proximity to the site of allergic reaction (Grimbadeston et al., 2007).
Interleukin-13 (IL-13) is considered a central mediator of the host’s physiological response to allergic inflammation. This cytokine is secreted by CD4+ Th2 cells in the processes of activating the antibody IgE in human B cells, and initiating physiological changes in tissue that enable organs in the host to expel parasites (Wynn, 2003). IL-4 is
150 Texas Tech University, Jordan Hunter, December 2016 often associated with IL-13 due to their similarity in function, however the former cytokine is considered to have less importance in these processes by comparison. It has been suggested that IL-13 may inhibit Th1 response pathways in favor of cell recruitment for mounting a Th2 response necessary to mitigate intracellular infections (Degen et al.,
2005). This would affect the ability of host immune cells to degrade intracellular pathogens.
Avian uncoupling protein (avUCP) has been associated with facultative thermogenesis, and lipid metabolism (Colin et al., 2003) and is believed to also function as a mediator of
ROS in birds to stop mitochondrial ROS proliferation and oxidative stress during periods of high metabolic activity (Rey et al., 2010).
The increased expression of IL-10, IL-13, and avUCP in O. petrowi infected quail is evidence that the ocular tissues of the avian host are responding to eye worm infiltration by upregulating physiological processes associated with parasite identification and clearance with IL-13 production. Infected quail could be attempting to resolve inflammation associated with ROS generation from the physical presence of parasitic infestation, or mast cell release of reactive oxygen/nitrogen species, by increasing production of anti-inflammatory cytokines (ex: IL-10). Oxidative stress in these tissues could be occurring, and either induced by inflammation potentially caused by phagocytic release of ROS, or aerobic mitochondrial ROS production due to heightened metabolic activity as indicate by elevated avUCP expression.
Interleukin-8 (IL-8) is a chemokine generated by macrophages, epithelial and endothelial cells and induces chemotaxis in target cells, enabling granulocytes to transition to the proximity of infection and begin phagocytosis. Secretion of IL-8 is increased during
151 Texas Tech University, Jordan Hunter, December 2016 oxidative stress, recruiting inflammatory cells, and creating conditions that continue to increase localized inflammation (Vlahopoulos et al., 1999).
GATA-3 is a transcription factor capable of inducing IL-4, IL-5, and IL-13 secretion in
Th2 cells, as well as suppressing the differentiation of naïve T-cells towards the Th1 cell type while facilitating differentiation of Th0 cells to Th2 cell subtypes (Yagi et al.,
2011).
Both IL-8 and GATA-3 expression were both noticeably increased in O. petrowi infected male quail in our relative analysis and comparison to uninfected male quail. The level of expression of these target genes was not observed in our comparison of females or our general comparison excluding sex as a factor. Expression of these genes provides evidence that the eye worm infected hosts induce a shift of naïve T-cell production towards Th2 type immune cells and response. In addition, these hosts induce increased cellular signaling of granulocytes to migrate towards the locations of parasite infestation in ocular tissue and begin phagocytic processes of attacking pathogens via respiratory bursts of reactive oxygen species (ROS)— and that these mechanisms occur more among adult male bobwhites than females.
Our observations indicate there are distinct differences in immune response that are associated with bobwhite quail gender. Earlier studies of birds and other animals have reported that males and females often mount different levels of immune response
(Moreno et al., 2001), with males typically mounting less immune response than females due to higher levels of immune function suppressing testosterone (Folstad & Karter,
1994). However, in a study by (Cram et al., 2015) the authors reported that wild whitebrowed sparrow weavers (Plocepasser mahali) did not acquire systemic oxidative stress after immune challenge from phytohaemagglutinin (PHA), and that there was no
152 Texas Tech University, Jordan Hunter, December 2016 significant effect of sex on immune response as determined by wing-web swelling. In a study of immune challenge of Zebra finches (Taeniopygia guttata) using PHA, the authors reported that females mounted a significantly higher cell mediated response than males, however males mounted significantly higher humoral response than females
(McGraw & Ardia, 2005).
It has been suggested that activating immune responses in wild animals can potentially create consequences related to fecundity and survivability (Uller et al., 2006; Zuk &
Soehr, 2002). There are distinct environmental, physiological, and behavioral conditions among northern bobwhite quail populations that may require females to sacrifice mounting a high immune response in favor of providing the body more resources needed for breeding (Mueller & Dabbert, 2002). Alternately, there may exist conditions that increase immunity in male birds, including carotenoids in plumage (McGraw & Ardia,
2005).
Higher expression of immune related target genes surveyed in our study of infected male quail subjects could be indicative of increased humoral immunity in this gender, which is a response necessary to remove soluble antigens and degrade extracellular microorganisms like O. petrowi. There is the potential that female bobwhites could be expressing higher humoral immunity which is necessary for elimination of intracellular pathogens. This could occur from a polarized shift from a Th2 to Th1 immune pathway due to changes involving oxidative status and increased availability of glutathione in females relative to males (Kim et al., 2007) or a noticeably decreased expression of IL-4, and IL-13 (Degen et al., 2005), which are both conditions we have observed to some degree in our studies of oxidative status and immune response in bobwhite quail.
153 Texas Tech University, Jordan Hunter, December 2016
Interleukin-4 (IL-4) and GPX-7 (glutathione peroxidase) were the only genes that exhibited reduced expression in comparison to their controls in all our analysis, excluding the relative comparison between infected males and females. However, the analysis is only an indicator that this cytokine and antioxidant were expressed more in infected males relative to infected females and not the degree of actual expression in either sex.
The relationship between GPx and IL-4 has been examined in previous studies (Schnurr et al., 1999). Schnurr and colleagues (1999) reported the down-regulation of glutathione peroxidase in response to IL-4 and IL-13 cytokine presence in lung, liver and spleen of transgenic mice. A study of the effects of IL-4 on enzymatic antioxidants by
(Kalinichenko et al., 2013) provided evidence that intraperitoneal injection of IL-4 in rats induced an increase in glutathione peroxidase and superoxide dismutase activities in the hypothalamus and a decrease in these activities in the amygdala. Further studies have determined that IL-4 accelerates the decay of phospholipid hydroperoxide glutathione peroxidase (PHPGPx) mRNA, and that downregulation of GPx expression as mediated by IL-4 is a “post-transcriptional phenomenon” (Hattori et al., 2005; Kuhn & Borchert,
2002).
From these studies, it can be determined that a relationship between IL-4 and GPX exists-with IL-4 primarily influencing GPx activity or expression on some level.
However, none of the referenced studies have examined this relationship in ocular tissues, and there could exist some tissue specific biases in northern bobwhite quail that may cause expression of these genes to be correlated in circumstances of parasitic infection and/or oxidative stress.
154 Texas Tech University, Jordan Hunter, December 2016
In summary, O. petrowi infected northern bobwhite quail exhibited a higher expression of the target genes IL-8, IL-10, IL-13, GATA-3 and avUCP as compared to quail with no noticeable O. petrowi infection in our relative analysis of gene expression utilizing qRT-
PCR. Taken together, this provides evidence that eye worm infected quail are mounting a
Th2 pathway associated humoral immune response in ophthalmic tissue by inducing chemotaxis of polymorphonuclear leukocytes to the site of infection and signaling phagocytosis and respiratory burst of reactive oxygen species. The host could be attempting to mitigate potential oxidative stress in the ocular tissue through the expression of anti-inflammatory cytokines and high metabolic ROS mediators. The increased expression of IL-10 and avUCP is an indication that the oxidative status of the quail host has changed in response to O. petrowi infestation around the intraocular and extraocular globe tissue analyzed in this study. A decrease in the expressions of GPX-7, and IL-4 in the tissues of infected quail relative to uninfected subjects could be the result of a post-transcriptional relationship between the cytokine and enzymatic antioxidant.
Decreased expression of glutathione peroxidase among infected quail could be indicative of oxidative stress status in infected hosts, and it is possible that O. petrowi infected hosts could mediate their immune response per this alteration in oxidative status (Cram et al.,
2015).
155 Texas Tech University, Jordan Hunter, December 2016
Log(NRQ) fold change of all Infected relative
to Uninfected quail 10
1
0.1
0.01
0.001
IL-4 IL-13 IL-10 GATA-3 IL-8 GPX-7 avUCP
Figure 5.1 The average gene expression of IL-10 (2.08 fold), IL-13 (2.62 fold), and avUCP (2.75 fold) was increased amongst all infected quail sampled. Average expressions of IL-4, IL-
8, GATA-3, and GPX-7 were decreased on average in all infected quail by comparison
(fold < 1).
156 Texas Tech University, Jordan Hunter, December 2016
Log(NRQ) fold change of Infected male
relative to Uninfected male quail 10
1
0.1
0.01
IL-4 IL-13 IL-10 GATA-3 IL-8 GPX-7 avUCP
Figure 5.2 The average gene expressions of IL-8 (2.33 fold), IL-10 (4.05 fold), IL-13 (4.86 fold),
GATA-3 (1.89 fold), and avUCP (7.80 fold) were increased amongst male infected quail sampled. Average expressions of IL-4, and GPX-7 were decreased on average in male infected quail by comparison (fold < 1).
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Log(NRQ) fold change of Infected female
relative to Uninfected female quail 10
1
0.1
0.01
0.001
IL-4 IL-13 IL-10 GATA-3 IL-8 GPX-7 avUCP
Figure 5.3 The average gene expressions of IL-10 (1.06 fold) and IL-13 (1.41 fold), were increased amongst the female O. petrowi infected quail sampled. Average expressions of IL-4, IL8,
GATA-3, GPX-7, were decreased on average relative to control in female infected quail by comparison (fold < 1). The target gene avUCP was observed to have a fold change of
0.972, which is close to the base line expression for control (1.00).
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Log(NRQ) fold change of Infected male
relative to Infected female quail 10
1
IL-4 IL-13 IL-10 GATA-3 IL-8 GPX-7 avUCP
Figure 5.4 The average gene expressions of all target genes were increased amongst the male O. petrowi infected quail sampled. These included; IL-4 (2.42 fold), IL-8 (2.02 fold), IL-10
(3.34 fold), IL-13 (2.16 fold), GATA-3 (2.28 fold), GPX-7 (1.50 fold), and avUCP (3.44 fold).
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Log(NRQ) fold change of Uninfected male
relative to Uninfected female quail 1
0.1
IL-4 IL-13 IL-10 GATA-3 IL-8 GPX-7 avUCP
Figure 5.5 The average gene expressions of all target genes were decreased (fold change < 1) amongst the male uninfected quail sampled. These included; IL-4 (0.22 fold), IL-8 (0.16 fold), IL-10 (0.87 fold), IL-13 (0.63 fold), GATA-3 (0.12 fold), GPX-7 (0.25 fold), and avUCP (0.43 fold).
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