Isolation of Arcobacter spp. and identification of isolates by multiplex PCR from various domestic poultry and wild avian species

The purpose of the present study was to determine the extent and seasonal prevalence of Arcobacter spp. in domestic poultry and wild birds in the Kars region of Turkey using multiplex polymerase chain reaction (m-PCR). In this study, 1570 samples were collected from domestic poultry and wild avian species. The numbers of collected samples were as follows: 182 fecal samples from chickens, geese, and turkeys from family farms in the Kars region in Turkey; 1089 cloacal swab samples from chickens, geese, ducks, turkeys, and quails from family farms in this region; and 299 fecal samples from wild pigeons, crows, and owls in the same region. Arcobacter spp. were isolated from 17.43%, 35.77%, 3.63%, 6.87%, and 3.33% of the cloacal swab samples obtained from geese, ducks, chickens, turkeys, and quails, respectively. In the stool samples, Arcobacter spp. were isolated from 9.62%, 13.33%, and 4% of chicken, goose, and turkey samples, respectively. In wild birds, the isolation rates of Arcobacter spp. were 6.6%, 12.15%, and 0% in pigeons, crows, and owls, respectively. Using m-PCR, among 171 Arcobacter spp. isolates obtained from poultry and wild birds, 67, 78, 24, and 2 were identified as Arcobacter cryaerophilus, Arcobacter butzleri, Arcobacter skirrowii, and Arcobacter cibarius, respectively. Both poultry and wild avian species exhibited variable rates of Arcobacter species positivity. The presence of Arcobacter spp. in the digestive tracts of healthy poultry and wild birds may serve as a potential reservoir for the dissemination of these microbes in the environment and their transmission to other animals and humans.


Introduction
Arcobacter spp. are small (0.2-0.9 × 0.5-3 μm), spiralshaped, and Gram-negative bacteria. They are non-spore forming and show corkscrew-like motility, aided by nonshielded polar flagella (Kayman 2012). In contrast to the genus Campylobacter, Arcobacter species are mostly aerotolerant and able to grow at temperatures below 30°C (Brückner et al. 2020), although Arcobacter anaerophilus is an obligate anaerobe in the genus (Sasi Jyothsna et al. 2013). Therefore, Arcobacter species are separated from campylobacters due to differences in their structural properties and fatty acid profiles (Gonulalan and Ertas Onmaz 2015), together with their ability to grow at temperatures of 15-30°C and under aerobic conditions (Kayman 2012). The Arcobacter genus is a member of the Campylobacteraceae family and rRNA superfamily VI in the Epsilon division of Proteobacteria De Ley 1991, On 2001).
To date, 28 Arcobacter species have been identified and characterized (Kim et al. 2019), which have been isolated from various hosts, foods, and environmental sources (Talay et al. 2016), such as sewage (Collado and Figueras 2011), streams and rivers (Laishram et al. 2016;Talay et al. 2016), drinking water, and municipal water (Ertas et al. 2010;Jalava et al. 2014). Arcobacter spp. have been isolated from various clinical samples from humans and animals (Petersen et al. 2007;Adesiji et al. 2011). They have been found in intestinal and fecal samples from a range of farm animals Piva et al. 2013;Shakira et al. 2012); cloacal swabs and fecal samples from domestic poultry such as chickens, ducks, geese, and turkeys (Collado and Figueras 2011;Goni et al. 2016); and intestinal and fecal samples from numerous wild avian species (Fernández et al. 2007;Wesley and Schroeder-Tucker 2011;Di Francesco et al. 2014).
Within the genus, Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii are the most common pathogenic species and are associated with various infections in humans and animals (Van den Abeele et al. 2014). These infections mostly appear clinically as abortion, enteritis, and mastitis in domestic animals (Patyal et al. 2011) and as gastroenteritis, bacteriemia, endocarditis, peritonitis, diarrhea, and septicemia in humans (Vandenberg et al. 2004;Samie et al. 2007). The primary potential transmission sources of these agents are various foods, especially poultry and products thereof, together with contaminated water (Shah et al. 2012).
Healthy domestic and wild avian species that harbor Arcobacter spp. in their digestive tracts serve as important hosts for these pathogens (Atabay et al. 1998;Van Driessche et al. 2003). Feces of avian Arcobacter reservoirs play a major role in the transmission of arcobacters to the environment, other animals, and humans. Therefore, both domestic poultry and wild birds are important in the carriage and spread of arcobacters (Di Francesco et al. 2014).
There have been no previous studies aimed at the isolation and identification of Arcobacter spp. in domestic poultry and wild birds in the Kars region in Turkey. The aim of the present study was to determine the extent and seasonal prevalence of Arcobacter spp. in domestic poultry (geese, chickens, ducks, turkeys, and quails) and wild birds (crows, pigeons, and owls) in the Kars region using the m-PCR protocol.

Reference strains
The Arcobacter butzleri (CIP 103493), A. cryaerophilus (CIP 104014), and A. skirrowii (CIP 103588) reference strains used during the stages of isolation and molecular identification were kindly provided by Prof. Francis Megraud (Bacteriology Laboratory of Victor Sagalen Bordeaux Hospital, France).

Isolation and identification of arcobacters
Cloacal swab and fresh fecal samples from domestic poultry and wild birds were placed in 5 ml of arcobacter selective broth (Fluka, 59848, India) containing cefoperazone, amphotericin B, and teicoplanin (CAT selective supplement) (Oxoid, SR174E, UK) and transferred to the microbiology laboratory within 3 to 4 h under cold chain conditions. Swabs were incubated at 30°C for 48 h under microaerobic conditions using an Anaerocult C kit (Merck, 1.16275, Germany) for pre-enrichment. After incubation, the membrane filtration method recommended by Atabay and Corry (1997) was performed for enriched samples. All plates were incubated at 30°C for 2 to 7 days in a microaerobic atmosphere.
Phenotypic tests such as indoxyl acetate hydrolyzation, nitrate reduction, and H 2 S reduction tests together with Gram staining, motility examination, and catalase, oxidase and urease tests were carried out on colonies grown on blood agar plates. Aerotolerance (25°C and O 2 , 30°C and O 2 -CO 2 , 37°C and O 2 -CO 2 , 42°C and CO 2 ), growth in MacConkey agar (Oxoid, CM1169, England), and growth in the presence of 2-3.5% sodium chloride were evaluated. Arcobacter spp. isolates were kept at − 20°C to be used for molecular identification.

DNA extraction
A modified version of the boiling method of Dashti et al. (2009) was used for DNA extraction from suspected Arcobacter isolates. For this purpose, a few colonies belonging to the isolates incubated at 30°C under microaerobic conditions in blood agar were maintained at 99.9°C for 10 min in Tris-EDTA buffer (Sigma, 93283, Germany), after which the tubes were placed at +4°C for 10 min and then centrifuged for 10 min at 10,000 rpm. The obtained supernatants were used as template DNA suspensions.
The m-PCR method defined by Houf et al. (2000) was used to identify the isolates obtained in the present study at the species level. As a result of this method, 84 A. cryaerophilus, 78 A. butzleri, and 7 A. skirrowii isolates were identified. Then, the m-PCR method proposed by Douidah et al. (2010) was applied to identify A. cryaerophilus isolates. As a result of this m-PCR method, 65 of the 84 A. cryaerophilus isolates were identified as A. cryaerophilus again, but 17 isolates were identified as A. skirrowii and 2 as A. cibarius.

Seasonal evaluation results
The isolation rates of Arcobacter spp. varied according to season and poultry type. In the cloacal swab samples from geese, the rate was highest in winter (23.53%), followed by summer (21.17%), autumn (18.52%), and spring (9.80%). In fecal samples from geese, the highest rate was observed in summer (20.75%), followed by spring (9.1%) and autumn (5%). No isolates were detected in winter. For ducks, sampling was performed only in spring and summer, and the isolation rate was higher in summer than in spring (37.74% and 33.33%, respectively). In the cloacal swab samples from chickens, the highest isolation percentage was detected in summer (6.12%), followed by winter (2.8%), and no isolation was carried out in autumn and spring. For turkeys, the bacterial isolation rate was highest in winter (17.39%), followed by summer (7.41%), and no Arcobacter isolates were detected in autumn and spring. In the fecal samples, isolation (10%) was carried out only in spring. For quails, sampling was performed only in autumn and winter periods. The isolation rate was 7.14% in autumn. Among wild birds, fecal samples were obtained from crows and pigeons only in summer and spring periods. The highest isolation rate was observed in summer (13.97% for crows and 7.4% for pigeons). The variation in the isolation rates of Arcobacter spp. according to the season and avian species are shown in Table 2.
The isolation rate observed in cloacal swab samples from geese was similar to that reported by Atabay et al. (2008) (18%) and higher than that reported by Bogantes et al. (2015) (0%). The Arcobacter isolation rate detected in duck cloacal swab samples in the present study (35.77%) was very close to that reported by Fernández et al. (2007) (40%), whereas it was much higher than that found by Bogantes et al. (2015) (5%) and Silha et al. (2015) (4.2%). In contrast, A. cibarius was identified in two goose cloacal swab samples by m-PCR. The differences in the isolation rates in geese and ducks may be attributed to the sampling times, feeding conditions, contact with other animals, and access to contaminated water. Nevertheless, compared with those in other domestic poultry species, the Arcobacter isolation rate was highest in samples from geese and ducks. This finding is in accordance with those of other studies (Atabay et al. 2006;Gonzalez et al. 2007) that have concluded that geese and ducks are potential reservoirs of arcobacters. However, these discrepancies may reflect different methodological aspects, such as the amount of sample analyzed, type of culture medium, incubation atmosphere, and even the geographic area where sampling was performed.
In the present study, the Arcobacter isolation rate obtained from cloacal swab samples from chickens (3.63%) was lower than the rate found by Van Driessche and Houf (2007) (10%). Ho et al. (2008) detected no Arcobacter isolates in chicken cloacal swab samples. The findings of the present study on the Arcobacter isolation rate from chicken fecal samples (9.62%) were compatible with those found by Atabay et al. (2006)  . Previous studies that have examined samples of chicken intestinal contents have reported differences in isolation rates ranging between 0% and 100% (Van Driessche and Houf 2007;Ho et al. 2008).
In this study, the Arcobacter isolation rates in turkey cloacal swabs and fecal samples were 6.87% and 4%,  respectively. These rates were higher than those found by Andersen et al. (2007) (2%), and the isolation rate in fecal samples was lower than that reported by Fernández et al. (2007) (28.6%).
Only a few studies have examined the presence of Arcobacter spp. in quails (Ok Anadut and Gumussoy 2005;Rahimi 2014). In the present study, the Arcobacter isolation rate in quails was 3.33%. The different isolation rates in samples obtained from various kinds of poultry may depend on geographic-and region-specific characteristics, farming styles, the isolation and identification methods used, sample collection, and transportation conditions and proximity to other animals. In the present study, Arcobacter isolation rates were considerably lower in quails than in ducks and geese (i.e., aquatic poultry). As noted in previous studies (Wesley and Miller 2010;Fernandez et al. 2015), this finding supports the hypothesis that arcobacters are not permanent commensals of the intestinal flora of poultry because of the high body temperatures of these animals (40.5-42°C).
In this study, the identification of Arcobacter isolates using two m-PCR assays in domestic poultry was performed, and the isolates were initially identified as A. cryaerophilus, followed by A. butzleri and A. skirrowii. Similar to the findings of other studies (Rahimi et al. 2012), in this study, the most commonly isolated species in all the samples was initially A. cryaerophilus and were subsequently A. butzleri and A. skirrowii. In accordance with the findings of previous studies (Rahimi et al. 2012), the results of species-specific m-PCR revealed that the most common species in the samples from wild birds was A. butzleri, followed by A. cryaerophilus and A. skirrowii. Only a few studies have detected the presence of A. skirrowii in wild birds (Van Driessche and Houf 2007;Adesiji et al. 2011). In the present study, A. skirrowii was isolated and identified, albeit at a low rate, in ducks and chickens compared to geese. The distribution of Arcobacter spp. in different organisms may change depending upon the relationships of the hosts with other organisms and their environment as well as the ecological features and environmental conditions to which they are adapted.
In the present study, the samples were first assessed according to the presence of A. cryaerophilus, A. butzleri, and A. skirrowii. For this purpose, the m-PCR method described by Houf et al. (2000) was used for identification of the obtained Arcobacter isolate sat the species level. Nevertheless, according to data obtained from other studies, while this m-PCR method presents the advantage of 100% reliable identification of A. butzleri, it produces false-positive identification results for A. cryaerophilus . Therefore, an m-PCR assay developed by Douidah et al. (2010) that detects 5 Arcobacter species (A. butzleri, A. cryaerophilus, A. skirrowii, and A. cibarius and A. thereius) was used. This m-PCR assay was preferred in the second step in the current study. The confirmation of the A. cryaerophilus isolates identified in the first m-PCR assay was thus performed, and other species that might be in the samples were then identified. Nevertheless, it is thought that different assays are necessary for the correct identification of these species and other potential pathogenic Arcobacter spp.
Although many studies have examined the presence of arcobacters in domestic poultry (Bogantes et al. 2015;Hassan 2017), only a few such studies have included wild birds (Di Francesco et al. 2014;Giacometti et al. 2015). In a study in Italy where cloacal swab samples were taken from 95 Eurasian collared doves (Streptopelia decaocto), Arcobacter spp. were detected in 18 (19%) samples by nested PCR amplification of the 16S rRNA gene (Di Francesco et al. 2014). However, Giacometti et al. (2015) detected no Arcobacter spp. in a study of fecal samples from 47 wild pigeons (Columbia livia). In a study in Chile of fecal samples from 60 pelicans and 60 sparrows, Arcobacter spp. were isolated at a rate of 13.3% in pelicans and 6.7% in sparrows, and the obtained species was identified as A. butzleri (Fernández et al. 2007). In the present study, the analysis of fecal samples from wild birds detected no Arcobacter spp. isolates in samples from owls (n = 25). The isolation rates in crows and pigeons were 12.15% (13/107) and 6.6% (11/167), respectively. The differences among isolation rates may be derived from different avian species or their association with water.
In the present study, according to the results of the m-PCR assay, 10 wild pigeon isolates were identified as A. butzleri, and one isolate was identified as A. cryaerophilus, while 12 crow isolates were identified as A. butzleri, and one was identified as A. cryaerophilus. The bacterial isolation rate in wild pigeons in the present study was lower than that reported by Di Francesco et al. (2014) (19%). However, Di Francesco et al. (2014) utilized the direct PCR technique to identify Arcobacter spp. in cloacal swab samples from Eurasian collared doves, whereas the present study employed culture methods. There are difficulties in detecting Arcobacter spp. via culture methods. It was not possible to compare the findings regarding Arcobacter isolation rates in wild birds obtained in the present study with those in the literature due to the limited number of studies involving Arcobacter isolation and identification in wild birds (Di Francesco et al. 2014;Giacometti et al. 2015). However, the discovery of Arcobacter isolates in wild avian species in the Kars region suggests that they may play an important role in transferring arcobacters to humans and other birds or animals via the contamination of the environment and water.
In this study, it was not possible to compare the seasonal distribution of Arcobacter isolation rates across all four seasons, as samples could not be obtained from the studied poultry in each season. However, where seasonal isolation rates in domestic poultry could be evaluated, the highest values were found in winter for geese, in summer for ducks, summer for chickens, in winter for turkeys, and in autumn for quails. Therefore, the seasonal distribution of arcobacters in domestic poultry seems to vary. Such variation may be associated with the bird's age, growth stage, and type of poultry farming. Therefore, more extensive studies are needed to shed light on the seasonal distribution of Arcobacter isolation rates.
In wild birds (wild pigeon and crow), the seasonal distribution of Arcobacter could be examined only in spring and summer. No Arcobacter spp. were isolated from fecal samples collected in spring months, while Arcobacter was isolated from samples collected in summer.

Conclusion
The present study revealed that most domestic poultry (goose, duck, chicken, turkey, and quail) and some wild birds (crow and pigeon) carried Arcobacter, with varying bacterial isolation rates. By harboring Arcobacter species in their digestive systems, these birds may serve as potential reservoirs for the dissemination of Arcobacter species in the environment and their transmission to other birds, animals, and humans. Duck, goose, and chicken farming in the Kars region are mainly based on traditional practices, in which birds are housed with other farm animals. This may give rise to a significant risk of Arcobacter infections in domestic animals and humans in the region.