Prevalence of Trichomonas, Salmonella, and Listeria ...

AVIAN DISEASES 61:000–000, 2017

Prevalence of Trichomonas, Salmonella, and Listeria in Wild Birds from Southeast Texas Britni Brobey,A Ashwini Kucknoor,AB and Jim ArmacostABC A

Department of Biology, Lamar University, Beaumont, TX 77710

Received 22 February 2017; Accepted 18 May 2017; Published ahead of print 20 June 2017 ?1

SUMMARY. Infectious diseases can be a major threat to wildlife populations, especially in human-modified habitats, but infection rates in populations of wild animals are often poorly studied. Trichomonas, Salmonella, and Listeria are all pathogens known to infect birds, but their infection rates in wild bird populations are not well documented. This study documents infection rates of the three pathogens in wild bird populations inhabiting a suburban to rural gradient in Southeast Texas. Various species of wild birds were sampled at five sites in Southeastern Texas representing rural (,1 house per ha), exurban (approximately 1 house per ha), and suburban (approximately 10 houses per ha) habitat types. Birds were captured in mist nets and samples were taken from the oral cavity, crop, and vent to detect the presence of pathogens. Samples were screened for Trichomonas by examining wet mounts under a light microscope, whereas samples were screened for Salmonella and Listeria by examining colonies grown on agar plates. Pathogens detected during the initial screening were further confirmed by PCR and DNA sequencing. Infection rates for Trichomonas, Salmonella, and Listeria were 9%, 17%, and 5%, respectively. The distributions of infection rates across habitats (i.e., rural, exurban, rural) did not differ significantly from the expected null distributions for any of the three pathogens; however, the data suggested some interesting patterns that should be confirmed with a larger dataset. Infection rates for Trichomonas and Salmonella were highest at the suburban sites, whereas the infection rate for Listeria was highest at the rural site. Feeder birds were more likely to be infected by all three pathogens than non-feeder birds. Small sample sizes prevent definitive conclusions regarding variation in infection rates along the suburban to rural gradient, but the results suggest that pathogens followed the predicted patterns. For many of the bird species sampled, this study presents the first report of infection rates by these three pathogens in wild populations. RESUMEN. Prevalencia de Trichomonas, Salmonella y Listeria en aves silvestres del sureste de Texas. Las enfermedades infecciosas pueden ser una gran amenaza para las poblaciones de vida silvestre, especialmente en los ha´bitats modificados por humanos, pero las tasas de infección en las poblaciones de animales salvajes a menudo son poco estudiadas. Trichomonas, Salmonella y Listeria son patógenos que se sabe que infectan a las aves, pero sus tasas de infección en las poblaciones de aves silvestres no estań bien documentadas. Este estudio documenta las tasas de infección de los tres patógenos en las poblaciones de aves silvestres que habitan gradientes de suburbano a rural en el sudeste de Texas. Varias especies de aves silvestres fueron muestreadas en cinco sitios en el Sureste de Texas que representan los tipos de ha´bitat rural (menos de una casa por hectarea), exurbano (aproximadamente 1 casa por ha) y suburbano (aproximadamente 10 casas por ha). Las aves fueron capturadas en redes de niebla y se tomaron muestras de la cavidad oral, del buche y de la cloaca para detectar la presencia de patógenos. Las muestras se examinaron para Trichomonas examinando montajes hu´ medos bajo un microscopio de luz, mientras que las muestras se examinaron para Salmonella y Listeria examinando las colonias crecidas en placas de agar. Los patógenos detectados durante la prueba de escrutinio inicial fueron confirmados por PCR y secuenciación del ADN. Las tasas de infección por Trichomonas, Salmonella y Listeria fueron 9%, 17% y 5%, respectivamente. Las distribuciones de las tasas de infeccio´ n entre los ha´bitats (es decir, rurales, exurbanos y rurales) no difirieron significativamente de las distribuciones nulas previstas para ninguno de los tres patógenos; Sin embargo, los datos sugieren algunos patrones interesantes que deben ser confirmados con un conjunto de datos ma´s grande. Las tasas de infección por Trichomonas y Salmonella fueron ma´s altas en los sitios suburbanos, mientras que la tasa de infección por Listeria fue ma´s alta en el sitio rural. Las aves alimentadoras ten´ıan ma´s probabilidades de ser infectadas por los tres pato´ genos que las aves no alimentadoras. El tamaño pequeño de las muestras no proporciona conclusiones definitivas sobre la variación de las tasas de infección a lo largo del gradiente suburbano a rural, pero los resultados sugieren que los patógenos siguieron los patrones predichos. Para muchas de las especies de aves muestreadas, este estudio presenta el primer informe de tasas de infección por estos tres pato´ genos en poblaciones silvestres. Key words: Infectious diseases, Trichomonas, Salmonella, Listeria, Wild birds, Southeast Texas, epidemiology, PCR Abbreviations: TE ¼ Tris–Ethylenediaminetetraacetic acid

Infectious diseases, including emerging ones, are seen as a major threat to wildlife, livestock, and humans (13), but population declines due to infectious diseases are rarely well-documented in avian populations, because often little is known about emerging pathogens. Moreover, diagnosis and screening is challenging in wild bird populations (7,24,31,47). Furthermore, research about wildlife diseases, particularly in urban landscapes, is needed, because

understanding infectious diseases has become increasingly important to animal conservation (13,32,37). Trichomoniasis, salmonellosis, and listeriosis are infectious diseases that affect wild bird populations worldwide (1,20,23). Trichomonas gallinae is a flagellated protozoan that parasitizes a variety of birds around the world (1), including free-ranging columbiforms and raptors, as well as domestic poultry (8,11,14,29,30,34,36,43,49). It was first isolated in passerines only 15 yr ago (1) and is an emerging disease among passerines in the United Kingdom (33,41) and North America (18). The Trichomo-

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These two authors contributed equally Corresponding author. E-mail: [email protected]

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nas protozoan enters the digestive tract and depending upon the virulence of the organisms, can then migrate to the liver, lungs, heart, and pancreas via the circulatory system, leading to death (1). Trichomoniasis is known to cause population declines in wild birds, such as the band-tailed pigeon (Patagioenas fasciata) (19,45). Wild birds may carry various pathogenic bacteria within their intestines (10,38,48) including Salmonella spp. The prevalence of Salmonella in healthy wild birds is usually low (3,7,20,22,42), because the disease usually causes acute illness, killing the bird in a short period of time (17,27,46). Many studies have isolated Salmonella from live or dead wild birds, demonstrating the importance of wild birds to the epidemiology of salmonellosis in humans and livestock (26). Listeria monocytogenes is another pathogenic bacterium commonly carried in the intestines of wild birds (23). L. monocytogenes is contracted by the birds from their food (23), and the prevalence of the pathogen reflects the degree of contamination of their environment (12,16,35). In newly hatched chicks (Gallus gallus domesticus), Listeria can cause gross abnormalities and histological lesions in the liver, heart, spleen, and kidneys, as well as toe paralysis (2). This study will contribute to our knowledge of infectious diseases in wild birds by determining the prevalence of Trichomonas, Salmonella, and Listeria in wild bird populations inhabiting sites along a suburban to rural gradient in Southeastern Texas. MATERIALS AND METHODS Various species of wild birds were captured at five sites, representing a suburban to rural gradient in Southeast Texas. The Dujay Sanctuary is a rural site, with a housing density of , 1 house per ha in the surrounding area. It is an approximately 16.2 ha tract in Hardin County, Texas, which is privately owned by Lamar University. It is characterized by a mixed pine and hardwood forest, dominated by loblolly pine (Pinus taeda) and white oak (Quercus alba). The Armacost site is an exurban site, with a housing density of about 1 house per ha in the surrounding area. It is an approximately 2.0 ha tract in Hardin County, Texas. It includes a house, yard, and pastures with scattered trees and shrubs. Dominant trees include loblolly pine, water oak (Quercus nigra), sweet gum (Liquidambar styraciflua), and black tupelo (Nyssa sylvatica). The Whittle, Yoder, and Christensen residences are suburban sites in Jefferson County, Texas, with a housing density of about 10–12 houses per ha in the surrounding area. They are all approximately 0.1 ha residential lots that include a house and a yard landscaped with a mix of native and ornamental trees and other plants. Bird feeders were present at all sites. Fieldwork was conducted weekly between April 2012 and February 2013, rotating among the three habitat types (rural, exurban, or suburban). Mist nets and traps were used to capture the wild birds that were sampled for pathogens (15,39). No more than five nets were employed at a time, and they were left open from dawn until midday. The nets were checked every 30 min throughout the morning to prevent unnecessarily stressing the birds. The traps were constructed out of wire mesh and were baited with commercial bird seed. Two traps (30 3 30 3 30 cm and 30 3 30 3 20 cm) were typically employed at a time, with one hanging and the other placed on the ground. A kite string tied to the door of the cage allowed the bander to close the door behind any bird that entered the trap. Each bird was banded with a uniquely numbered aluminum band from the U.S. Fish and Wildlife Service, and standard banding data were collected (39). All birds were released at the site of capture. Three samples were collected from each bird. A sterile, micro-tipped cotton applicator was used to swab the oral cavity and crop of each bird

to obtain a sample to be screened for Trichomonas. The swab was then placed in a test tube containing tryptone yeast extract maltose (TYM) medium. A saline solution was injected into the oral cavity of each bird to obtain a second sample to be screened for Trichomonas. That sample was then placed in a sterile test tube. A sterile, micro-tipped cotton applicator was used to swab the cloaca to obtain a sample to be screened for Salmonella and Listeria. Any fresh droppings were also collected with the cotton applicator and added to the sample. That swab was then placed in a test tube containing nutrient medium. After being collected in the field, samples were transported to the laboratory and immediately screened for pathogens. The oral cavity and crop samples were screened for the presence of Trichomonas by preparing wet mounts, which were then viewed under a light microscope (1). This initial screening may fail to detect Trichomonas if the protozoan is present in low numbers. Therefore, a subsample was incubated in a Trichomonas growth medium at 37 C for four days, after which new wet mounts were prepared and viewed again under a light microscope. The cloacal and droppings samples were incubated at 37 C for 24–48 hr in nutrient broth, after which they were streaked on nutrient agar plates and incubated again at 37 C for 24 hr. Any bacterial colonies present were then stained with Gram stain. To further confirm the presence of specific pathogens, DNA isolation and PCR amplification were conducted, following the methods of Robinson et al. (41). A boiling lysis method was used for the isolation of DNA from the Trichomonas samples. A 1-ml sample was placed in a sterile 1.5-ml microcentrifuge tube and centrifuged for 10 min at 12,000 rpm. The pellet was resuspended in 100 ll of 13 Tris– Ethylenediaminetetraacetic acid (TE). The sample was then boiled for 10 min, placed on ice, and centrifuged for 5 min at 13,000 rpm. The supernatant was then separated from the pellet, and the supernatant was used as the PCR template. A boiling lysis method was also used for the isolation of DNA from the Salmonella and Listeria samples. A sterile pipette tip was used to transfer a single colony from each nutrient agar plate to a sterile 1.5-ml microcentrifuge tube containing 20 ll of 13 TE. The sample was then boiled for 10 min, placed on ice, and centrifuged for 10 min. The supernatant was used as the template for PCR. PCR was used to amplify a 290 bp region of the 16S rRNA gene from Trichomonas by using the published primers TFR1 and TFR2 (41), forward (Trich 1F: 5 0 -ATGAGTCAACACACGCCATCAG-3 0 ) and reverse (Trich 1R: 5 0 -CACCTGGACGTCTGTGACCTTC-3 0 ). Trichomonas PCR reactions were run with 10 ll of 23 RedTaq mix (Bioline), 2 ll of DNA template, 6 ll of sterile H2O, 1 ll forward primer, and 1 ll reverse primer, to make up 20 ll per reaction. After an initial 5 min denaturation at 95 C, 34 cycles of 95 C for 1 min, 50 C for 1 min, and 72 C for 1 min were carried out followed by extension at 72 C for 1 min by using a Biorad thermocycler. The annealing temperature was 50 C. For Salmonella and Listeria, PCR was carried out by using primers to amplify regions of the 16S rRNA genes. The primers Sal1 (5 0 GTGAAATTATCGCCACGTTCGGGCAA-3 0 ) and Sal2 (5 0 TCATCGCACCGTCAAAGGAACC-3 0 ) were used to amplify a 284 bp fragment from Salmonella (28). MonoA (5 0 -CAAACTGCTAACACAGCTACT-3 0 ) and LIS1B (5 0 -TTATACGCGACCGAAGCCAAC3 0 ) were used to amplify a 660 bp fragment from Listeria (5). The primer annealing temperatures for Salmonella and Listeria were 55 C and 58 C, respectively. The PCR products were separated on 1% agarose gel stained with ethidium bromide. The expected product sizes (290 bp for Trichomonas, 660 bp for Listeria, and 284 bp for Salmonella) were verified using a 1 kb ladder (Bioline). Because the PCR reactions produced multiple nonspecific bands in addition to the expected bands in many samples, the expected sized bands were excised from the gel, and DNA from the excised pieces was purified using a Qiagen Gel purification kit following the manufacturer’s protocol. The purified DNA extracted from gel pieces were sequenced (Functional Biosciences), and the sequences were used in a Basic Local Alignment of Sequence Tool (BLAST) search


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Table 1. Infection rates of the 24 species of birds sampled during the study, summed across study sites. Species are listed in taxonomic order. Feeder birds are those granivores that are known to commonly visit feeders with seed (i.e., the ruby-throated hummingbird is excluded). Infection rates Common name

feeder species white-winged dove mourning dove Inca dove blue jay Carolina chickadee tufted titmouse red-breasted nuthatch chipping sparrow northern cardinal house finch pine siskin American goldfinch house sparrow non-feeder species chimney swift ruby-throated hummingbird red-bellied woodpecker yellow-bellied sapsucker eastern phoebe white-eyed vireo eastern bluebird hooded warbler pine warbler yellow-rumped warbler yellow-breasted chat totals

Scientific name

n

Trichomonas

Salmonella

Listeria

Zenaida asiatica Zenaida macroura Columbina inca Cyanocitta cristata Poecile carolinensis Baeolophus bicolor Sitta canadensis Spizella passerina Cardinalis cardinalis Haemorhous mexicanus Spinus pinus Spinus tristis Passer domesticus

2 5 3 1 2 1 2 11 26 3 1 7 29

— — 33% 100% — — — 9% 12% 33% — — 14%

— 20% 33% 100% — — — — 23% — — 29% 21%

— — — — — — — — 8% — — — 7%

Chaetura pelagica Archilochus colubris Melanerpes carolinus Sphyrapicus varius Sayornis phoebe Vireo griseus Sialia sialis Setophaga citrina Setophaga pinus Setophaga coronata Icteria virens

1 2 4 2 1 2 2 2 3 3 1 116

— — — — — — — — — — — 9%

— — 50% 50% — — — — — — — 17%

— — — — — 50% — 50% — — — 5%

against specific databases: http://trichdb.org/trichdb/ for Trichomonas, http://www.sanger.ac.uk/resources/downloads/bacteria/salmonella.html for Salmonella, and http://genome.tbdb.org/annotation/genome/ listeria_group/Blast.html for Listeria. Sequences that matched the respective databases were considered confirmation of the presence of a particular pathogen.

RESULTS

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Trichomonas. The overall infection rate for Trichomonas was 9% (11 of 116 birds were infected; Table 1). The blue jay (Cyanocitta cristata) (100%), Inca dove (Columbina inca) (33%), and house finch (Haemorhous mexicanus) (33%) were the most commonly infected species, although the infection rate for the blue jay was based on a single sample (see Table 1 for scientific names of all birds captured during the study). All eleven birds infected with Trichomonas belonged to species classified as feeder birds (Table 1). The rate of infection by Trichomonas increased from the rural site (0%) to the exurban site (7%) to the suburban sites (14%) (Fig. 1), although the distribution was not significantly different from the expected null (v2 ¼ 2, df ¼ 2, P ¼ 0.37). Salmonella. The overall infection rate for Salmonella was 17% (20 of 116 birds were infected; Table 1). The most commonly infected species included the blue jay (100%), yellow-bellied sapsucker (Sphyrapicus varius) (50%), red-bellied woodpecker (Melanerpes carolinus) (50%), Inca dove (33%), American goldfinch (Spinus tristis) (29%), northern cardinal (Cardinalis cardinalis) (23%), house sparrow (Passer domesticus) (21%), and mourning dove (Zenaida macroura) (20%), although the infection rate for the blue jay was based on a single sample (Table 1). Seventeen of the 20 birds (85%)

infected with Salmonella belonged to species classified as feeder birds (Table 1). The rate of infection by Salmonella increased from the rural site (14%) to the exurban site (15%) to the suburban site (20%) (Fig. 1), but the distribution was not significantly different from the expected null distribution (v2 ¼ 0.21, df ¼ 2, P ¼ 0.90). Listeria. The overall infection rate for Listeria was 5% (6 of 116 birds were infected; Table 1). The white-eyed vireo (Vireo griseus) (50%) and hooded warbler (Setophaga citrina) (50%) were the most commonly infected species (Table 1). Four of the six birds (67%) infected with Listeria belonged to species classified as feeder birds (Table 1). The rate of infection by Listeria was highest at the rural site (29%), intermediate at the suburban site (4%), and lowest at the exurban site (3%) (Fig. 1), but the distribution was not significantly different from the expected null distribution (v2 ¼ 1.33, df ¼ 2, P ¼ 0.51). DISCUSSION

Infection rates for Trichomonas, Salmonella, and Listeria were 9%, 17%, and 5%, respectively, indicating that the populations of wild birds sampled during the study had a relatively low prevalence of these particular diseases at sites across the suburban to rural gradient. Schulz et al. (44) found that 6% of hunter-killed mourning doves sampled in Missouri tested positive for Trichomonas, whereas Girard et al. (19) found that 11% of hunter-killed and 4% of live-captured band-tailed pigeons from California were positive for Trichomonas. Krawiec et al. (29) found Salmonella in 15 of 40 species of wild birds sampled in Poland, with an average infection rate of individuals of 6%. Quessy & Messier (38) reported that 9% of trapped or shot


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Fig. 1. Infection rates by habitat type.

ring-billed gulls (Larus delawarensis) sampled near Montreal, Canada tested positive for Salmonella, whereas 10% tested positive for Listeria. Hellström et al. (23) found that 36% of avian fecal samples from in and around Helsinki, Finland, tested positive for Listeria. Trichomonas was most prevalent in Inca doves and house finches, which is in agreement with other studies that have found Trichomonas to be most common in wild doves and finches (1,29,36), and trichomonaisis has been implicated in the decline of the greenfinch (Carduelis chlorus) and chaffinch (Fringilla coelebs) in the United Kingdom (33). Trichomonas is likely spread to house finches through water and food sources shared with columbiforms (1). The infected birds captured during this study showed no visible gross lesions, suggesting that the birds were carrying an avirulent strain of the pathogen (50). Salmonella was most prevalent in Inca doves, mourning doves, red-bellied woodpeckers, yellow-bellied sapsuckers, American goldfinches, and house sparrows. American goldfinches and house sparrows were among commonly infected bird species in Ohio (9). Salmonella has been detected in a wide variety of wild birds, and has been reported to have caused epizootics in wild passerine species in Great Britain (40). Grigar et al. (21) found Salmonella in 3% of great-tailed grackles (Quiscalus mexicanus) sampled in East Texas. Infected birds often exhibit puffiness, anorexia, and greenish

diarrhea; house sparrows in particular exhibit swollen, matted eyelids (9). The infected birds captured during this study were not visibly sick and appeared to be asymptomatic. The prevalence of Salmonella in healthy wild birds is typically low (3,7,20,22,42), because the disease usually causes acute illness, killing the bird in a short period (17,27,46), but some birds can be asymptomatic carriers (9). Listeria was most prevalent in white-eyed vireos and hooded warblers, which were only captured at the rural site. Listeria is known to be present in a wide variety of wild birds, but it is most common in rural environments, presumably because Listeria is widespread in farm soil and vegetation (38). In birds, Trichomonas, Salmonella, and Listeria are spread via contaminated droppings, and transmission rates are increased when birds concentrate at feeders or other anthropogenic food sources (1,9,16,23,40,46), so it was expected that feeder birds would be more likely to be infected by all three pathogens than non-feeder birds. As predicted, the results of this study indicate that infection rates for all three pathogens were more prevalent among feeder birds than non-feeder birds. The data suggest some interesting patterns that should be confirmed with a larger data set. Host animals serve as the main reservoirs for Trichomonas (11,14) and Salmonella (25,26), so it was expected that infection rates for these pathogens would be higher at


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suburban sites, where feeders attracts birds and increase transmission rates (4,6). In contrast, soil and vegetation are significant reservoirs for Listeria (16,38), so it was expected that the infection rate for this pathogen would be higher at the rural site, where agricultural practices on surrounding land may have contaminated the local soil. As predicted, the results of this study indicate that infection rates for Trichomonas and Salmonella were highest (though not significantly so) at the suburban sites, while the infection rate for Listeria was highest (though not significantly so) at the rural site. This study presents the first reports of rates of infection by Trichomonas, Salmonella, and Listeria in wild bird populations in Southeast Texas and the first reports anywhere of the presence of these diseases in some of the bird species sampled. The three pathogens occurred at relatively low prevalence rates in the populations of wild birds sampled during this study, indicating seemingly healthy populations, with regard to these particular pathogens. REFERENCES 1. Anderson, N., C. Johnson, S. Fender, S. Heckly, M. Metzler, P. Nave, and J. Yim. Clinical signs and histopathologic findings associated with a newly recognized protozoal disease (Trichomonas gallinae) in free-ranging house finches (Carpodacus mexicanus). J. Zoo Wildl. Med. 41:249–254. 2010. 2. Basher, H., D. Fowler, F. Rodgers, A. Seaman, and M. Woodbine, M. Role of haemolysin and temperature in the pathogenesis of Listeria monocytogenes in fertile hens’ eggs. Zbl. Bakt.-Int. J. Med. M. 258:223–231. 1984. 3. Bigland, C., G. Wilton, H. Vance, and H. Carlson. Salmonellosis of animals in Alberta, 1949 to 1960. J. Am. Vet. Med. Assoc. 140:251–253. 1962. 4. Boal, C., and R. Mannan. Comparative breeding ecology of Cooper’s hawks in urban and exurban areas of southeastern Arizona. J. Wildl. Man. 63:77–84. 1999. 5. Border, P.M., J. J. Howard, G. S. Plastow, and K. W. Siggens. Detection of Listeria species and Listeria monocytogenes using polymerase chain reaction. Lett. Appl. Microbiol. 11:158–162. 1990. 6. Bradley C., and S. Altizer. Urbanization and the ecology of wildlife diseases. Trends Ecol. Evol. 22:95–102. 2006. 7. Brittingham, M., S. Temple, and R. Duncan. A survey of the prevalence of selected bacteria in wild birds. J. Wildl. Dis. 24:299–307. 1988. 8. Bunbury, N., C. Jones, A. Greenwood, and D. Bell. Trichomonas gallinae in Mauritian colombids: implications for an endangered endemic. J. Wildl. Dis. 43:399–407. 2007. 9. Burton, D. L., and K. A. Doblar. Morbidity and mortality of urban wildlife in the midwestern United States. In: Proc. 4th International Symposium on Urban Wildlife Conservation, Tuscon, AZ. pp. 171–181. 2004. 10. Casanovas, L., M. de Simon, M. Ferrer, J. Arques, and G. Monzon. Intestinal carriage of campylobacters, salmonellas, yersinias and listerias in pigeons in the city of Barcelona. J. Appl. Bacteriol. 78:11–13. 1995. 11. Conti, J. A. Diseases, parasites and contaminants. In: Ecology and management of the mourning dove. T. S. Baskett, M. W. Sayre, R. E. Tomlinson, and R. E. Mirarchi, eds. Stackpole Books, Harrisburg, PA, pp. 225–230. 1993. 12. Coulson, J., J. Butterfield, and C. Thomas. The herring gull Larus argentatusas a likely transmitting agent of Salmonella montevideo to sheep and cattle. J. of Hyg. 91:437–443. 1983. 13. Daszak, P., A. Cunningham, and A. Hyatt. Emerging infectious diseases of wildlife-threats to biodiversity and human health. Science 287:443–449. 2000. 14. Dolan, K. Avian trichomoniasis. J. Wildl. Rehab. 28:30–32. 2006. 15. Fair, J., E. Paul, and J. Jones, eds. Guidelines to the use of wild birds in research. Ornithological Council, Washington, D.C. 2010. 16. Fenlon, D. Wild birds and silage as reservoir of Listeria in the agricultural environment. J. Appl. Bacteriol. 59:537–543. 1985.

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ACKNOWLEDGMENTS

We wish to thank the various landowners for access to their properties, including Lamar University and the Armacost, Christensen, Whittle, and Yoder families. We wish to thank the Biology Department of Lamar University for financial support for this research, primarily through a Dujay Research Assistantship. Finally, we wish to thank the graduate and undergraduate students who provided assistance and support. This research was conducted under the proper state and federal bird banding permits.


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