Survey of zoonotic parasites and bacteria in faeces of Canada geese (Branta canadensis) in North‐Central Oklahoma

Abstract Background As a population of non‐migratory Canada geese (Branta canadensis) has been growing in residential and recreational areas, public concerns on potential acquisition of zoonotic pathogens from Canada geese and their faecal deposits have been increasing. Objectives The main study objective was to evaluate the prevalence of zoonotic microorganisms, Campylobacter spp., Cryptosporidium spp., Giardia spp. and Salmonella spp. and antimicrobial resistant Escherichia coli in faeces of Canada geese residing in North‐Central Oklahoma, United States. Methods A total of 204 faecal samples were collected from 11 locations in North‐Central Oklahoma, where public recreational areas such as lakes and ponds were located, and Canada geese were commonly inhabited. Faecal samples were examined by a centrifugal flotation to evaluate the prevalence of Cryptosporidium spp. and Giardia spp. infections. A total of 180 faecal samples were grouped into 36 pooled samples and cultured using standard culture methods to detect the prevalence of Campylobacter spp. and Salmonella spp. infections. The antimicrobial resistance profile was determined on 32 E. coli isolates recovered from the 36 sample pools, using the Kirby Bauer Disk Diffusion method. Results The targeted zoonotic pathogens were not identified by the faecal examinations performed. Of the 32 E. coli isolates, 17 isolates (53.1%) demonstrated resistance to ≥1 antimicrobial agent. Conclusions Targeted zoonotic pathogens were not detected among the examined resident Canada geese in North‐Central Oklahoma. The findings of multiple‐antimicrobial resistant E. coli infections are potentially a public health concern although the prevalence was low in this study. Further, larger scale surveys are recommended.


INTRODUCTION
Populations of resident (non-migratory) Canada geese (Branta canadensis) have been growing significantly among urban sites throughout North-Central Oklahoma, as well as many other regions of North America, due to several factors including the lack of predators and the availability of suitable habitats for geese, such as artificial ponds and lakes surrounded by well-maintained turfgrasses (Ayers et al., 2014;Cole et al., 2005;Gorham & Lee, 2016;Leonard, 2013;Rutledge et al., 2013;Vogt et al., 2018;Zhou et al., 2004). Environment Canada (2011) estimated that there are at least seven million Canada geese present in North America, and its population is stable or increasing. The faecal droppings of the waterfowl are commonly found in close proximity with populated public recreational areas, causing a source of environmental contamination and an increase of potential public health concerns for humans. A large number of microorganisms have been recovered from the faeces of Canada geese, including Campylobacter spp., Cryptosporidium spp., Giardia spp., Salmonella spp. and antimicrobial resistant Escherichia coli (Ayers et al., 2014;Cole et al., 2005;Converse et al., 1999;Fallacara et al., 2001;Fallacara et al., 2004;Gorham & Lee, 2016;Graczyk et al., 1998;Kassa et al., 2001;Kassa et al., 2004;Moriarty et al., 2011;Rutledge et al., 2013;Vogt et al., 2018;Zhou et al., 2004). Humans can contract these microorganisms through accidental ingestion from contaminated environments during leisure activities such as swimming, playing in sediments, fishing, water skiing and picnicking near beaches where Canada geese are often observed (Acha & Szyfres, 2003;Ayers et al., 2014; Center for Disease Control and Prevention, 2019; Center for Disease Control and Prevention, 2020;Rutledge et al., 2013).
Our main study objectives were to evaluate the prevalence of zoonotic pathogens, specifically focused on Campylobacter spp., Cryptosporidium spp., Giardia spp. and Salmonella spp., and antimicrobial resistant E. coli in the faeces of Canada geese inhabiting North-Central Oklahoma and to assess the concerns for Canada geese as a potential public health threat. Additionally, the prevalence of other parasitic and bacterial infections in the resident Canada geese in the same area was determined.

Sample collection
Between May and July 2019, faecal droppings were collected from Canada geese observed in 11 recreational places located in three counties within North-Central Oklahoma (OK): five locations (L-1 through L-5) in Stillwater, Payne county (Table 1); four locations (L-6 through L-9) in Oklahoma City and Edmond, Oklahoma county (Table 2) and two locations (L-10 and L-11) in Broken Arrow, Tulsa county (  or genus level when possible and recorded for data analysis (Soulsby, 1982;Zajac & Conboy, 2012). In addition to a centrifugal flotation with Sheather's sugar solution on each sample, three to seven faecal samples collected from the same location at the same date were pooled and examined by an additional centrifugal flotation with a 33% zinc sulphate solution (specific gravity of 1.18) to identify Giardia spp. trophozoites/cysts. All faecal slides were read by a board-certified parasitologist and a veterinary student with assistance from the parasitologist.

Antimicrobial susceptibility
The antimicrobial resistance profile was determined on 32 Escherichia coli isolates recovered from the 36 sample pools, using the Kirby Bauer Disk Diffusion method (Hudzicki, 2009

Antimicrobial susceptibility
Of the 32 E. coli isolates examined for antimicrobial resistance, 17 isolates (

DISCUSSION
To the best of our knowledge, this is the first study investigating the prevalence of zoonotic pathogens, Cryptosporidium spp., Campylobacter spp., Giardia spp. and Salmonella spp. and antimicrobial resistant  (Converse et al., 1999;Fallacara et al., 2004;Vogt et al., 2018). This could be due to a low interaction of bird populations with contaminated environments, such as infected livestock, and a generally higher sanitary condition of locations sampled in the current study (dos Santos et al., 2020;Hernandez et al., 2003). Similarly, the prevalence of Campylobacter spp. in wild birds has been highly variable depending on geographic regions (Kaakoush et al., 2015) and between indigenous birds compared to migratory birds (Kwon et al., 2017). It has also been reported that the prevalence of Campylobacter spp. can be influenced by the feeding characteristics of birds, with very low levels found in ground foraging granivores and arboreal/aerial insectivores (Waldenström et al., 2002). Additionally, in this study, for Campylobacter spp.  (Vogt et al., 2018). In addition to bacteria, yeast colonies (Candida spp.) were detected in one of the faecal sample pools from location L-11.
The detection of yeast in low frequency from faeces of Canada geese as well as other avian species has been reported (Buck, 1990;Glushakova et al., 2020). Yeasts, such as Candida spp., are commensal organisms but can cause opportunistic infections and pose potential risks to immunecompromised humans (Blinkhorn et al., 1989;Rahmati et al., 2020;Wawrysiuk et al., 2018).
One of the interesting findings in our study with potential zoonotic significance was the identification of schistosome eggs in a faecal sample collected in Stillwater, Payne county. Schistosomes are parasitic flatworms that can cause cercarial dermatitis, also commonly known as 'swimmer's itch' in humans (Horák et al., 2015). The most frequently identified schistosome in Canada geese is Anserobilharzia brantae, which was formerly classified as Trichobilharzia brantae (Brant et al., 2013;Horák et al., 2015). There is considerable diversity among schistosomes that may initiate cercarial dermatitis; four schistosome genera with about 30 described species from mammals and 10 genera with about 67 species from avian have been reported. Of the diversified family of Schistosomatidae, species of Trichobilharzia appear to be the primary etiological agents for dermatitis outbreaks globally (Horák et al., 2015). However, an outbreak of cercarial dermatitis caused by A. brantae (called as T. brantae at that time) transmitted by Canada geese and the freshwater snail, Gyraulus parvus, has been reported in Colorado Springs, Colorado (Brant & Loker, 2009 the infection has been reported and appears to be a common finding (Fallacara et al., 2001;Greiner et al., 1981;Skene et al., 1981;Soulsby, 1982;Tuggle & Crites, 1984). Three different genera of intestinal coc-cidia, Eimeria, Isospora and Tyzzeria, have been reported in Canada geese (Skene et al., 1981). In order to differentiate the three coccidian genera morphologically, oocysts need to be cultured with 2.0-2.5% potassium dichromate solution to facilitate sporulation (Berto et al., 2007;Greiner et al., 1981). Although one sample obtained in Oklahoma City, Oklahoma county contained coccidian oocysts with two sporocysts, a morphological characteristic of Isospora spp. (Table 2) (Skene et al., 1981;Zajac & Conboy, 2012), further identification of coccidia was not performed in this study. None of these coccidian parasites in Canada geese are considered as zoonotic agents.
Escherichia coli isolates were generally susceptible to most antimicrobials tested. No consistent multidrug resistance patterns were observed among isolates collected from the different counties. There was a single E. coli isolate from Stillwater, Payne County that showed resistance to multiple antimicrobials including potentiated beta-lactam, macrolide, phenicol and tetracycline classes, highlighting possible risks that these birds can pose as a source of potential harmful microbes. Most of the E. coli isolates showed resistance to azithromycin. Azithromycin is a macrolide antibiotic that has been suggested as a treatment for certain kinds of E. coli infections (Nitschke et al., 2012). In the current study, most of the resistant isolates (13/32) had a zone diameter of 12 mm which is the higher end of the range to be categorised as 'resistant' . Zone diameters above 12 mm are considered 'susceptible' . Further diagnostic work should be conducted to determine the genetic basis of the resistance to azithromycin.
There are some limitations in this study. Faecal samples were pooled for Giardia spp. and bacterial examinations due to financial and time constraints although it was not an ideal procedure to investigate the prevalence of microorganisms in geese as pool testing could provide only the prevalence of a flock or sampling location, instead of individual geese. In the current study, identification or tracking of individual Canada geese was not conducted. At every sampling visit, a single collection of faeces per goose was encouraged; however, multiple collections from the same goose could have occurred as Canada geese were capable of traveling from a location to another freely. Since faecal collection was conducted only during summer months (May through July) in 2019, our data in the current study did not consider seasonal or yearly differences. Additionally, one time faecal examination may not be sufficient to determine the prevalence of microorganisms in Canada geese due to intermittent shedding of Giardia spp. cysts (Zajac & Conboy, 2012), non-patent infections and size of the faecal sample. Although specialised growth enrichment media are preferred for detecting fragile bacterial species such as Campylobacter spp., a direct culture approach was used in this study, which could have resulted in the failure to isolate these potential pathogens. Lastly, since only aerobic and microaerophilic culture protocols were used, detection of obligate anaerobic bacteria was not achieved. Metagenome or targeted genome sequencing-based approaches will help in getting a more comprehensive understanding of the different pathogenic bacterial species.
In conclusion, the current study suggests that resident Canada geese observed in North-Central Oklahoma may not play a major role in the transmission of zoonotic pathogens, such as Campylobacter spp., Cryptosporidium spp., Giardia spp. and Salmonella spp. The findings of multiple-antimicrobial resistant E. coli and schistosome infections are potentially a public health concern although the prevalence was low in this study. As this study was relatively a small-scale and seasonal study, additional larger scale studies over a longer period of time are recommended in the interest of keeping the public residential and recreational areas safe and monitoring the prevalence of zoonotic and antimicrobial resistant agents.