DOI: 10.7589/2014-05-128
Journal of Wildlife Diseases, 51(2), 2015, pp. 341–347 # Wildlife Disease Association 2015
SURVEILLANCE AND CHARACTERIZATION OF RIEMERELLA ANATIPESTIFER FROM WILD BIRDS IN SOUTH KOREA Se-Yeoun Cha,1,3 Hye-Suk Seo,1,3 Bai Wei,1 Min Kang,1 Jae-Hee Roh,1 Ran-Hee Yoon,1 Ji-Hyuk Kim,2 and Hyung-Kwan Jang1,4 1 Department of Infectious Diseases and Avian Diseases, College of Veterinary Medicine and Korea Zoonosis Research Institute, Chonbuk National University, 79 Gobongro, Iksan, 570-752, South Korea 2 Poultry Science Division, National Institute of Animal Science, Rural Development Administration, 114 Shinbang 1-gil, Cheonan, 331-808, South Korea 3 These authors contributed equally to this study 4 Corresponding author (email: [email protected])
We conducted surveillance for Riemerella anatipestifer (RA) in wild birds along the East Asian-Australasian flyway in South Korea. Detected RA were characterized by serotype, antibiotic susceptibility, and sequence analysis of the 16S rRNA gene. We collected 944 wild birds of 34 species from 19 of South Korea’s major migratory wild bird habitats between 2011 and 2012. We identified RA by PCR and rRNA gene sequence in 71/102 (69.6%) pharyngeal swabs and 19/ 944 (2.0%) cloacal swabs of wild birds. Most RA positives (71/75 [95%] pharyngeal and 19/704 [(2.6%] cloacal) were from three duck species (family Anatidae): Mallard Duck (Anas platyrhynchos), Northern Pintail (Anas acuta), and Spot-billed Duck (Anas poecilorhyncha). Thirty-three RA isolates obtained and examined were highly resistant to aminoglycosides: kanamycin (100%), gentamicin (94%), amikacin (91%), neomycin (88%), and streptomycin (82%). Six isolates were identified as serotype 4 by agar gel precipitation. Serotypes 1 and 7, which are known virulent serotypes, were also identified in three isolates from wild duck species. Key words: Prevalence, Riemerella anatipestifer, South Korea, wild bird. ABSTRACT:
isolated from five of seven juvenile Whistling Swans (Olor columbianus) found sick or dead on the lakes during the autumn migration in Saskatchewan, Canada (Wobeser and Ward 1974). Moreover, RA and RA-like field isolates have been described from wild bird species in Germany, (e.g., Carolina Wood Ducks (Aix sponsa), Mallard Ducks (Anas platyrhynchos), and Greater Eider Ducks (Somateria mollissima) with signs and lesions typical of exudative septicemia or neurologic disorders (Hinz et al. 1998). These observations indicate that RA could cause large losses of wild migrating birds or resident waterfowl. The East Asian-Australasian flyway is one of the eight migratory bird flyways in the world where about 250 bird species pass through 22 nations from Russia, Mongolia, and China to Korea, Japan, and South Asia. South Korea is located in this flyway and is geographically important as a stopover site for Charadriidae and wintering habitats of Anatidae. According to a study by the Ministry of Environment of South Korea, 1,259,717 birds of 204
INTRODUCTION
Riemerella anatipestifer (RA) is a gramnegative, nonmotile, nonspore-forming, rod-shaped bacterium. Infected birds are characterized by respiratory symptoms, septicemia, pericarditis, perihepatitis, meningitis, and salpingitis (Sandhu 2008). The bacterium is distributed worldwide and is an important cause of infectious disease among waterfowl, turkeys, and chickens (Sandhu 2008; Rubbenstroth et al. 2009; Li et al. 2011). It has also been isolated from wild birds, including wild ducks (Anatidae), geese (Anatidae), black swans (Cygnus atratus), pheasants (Phasianidae), guinea fowl (Numididae), quail (Galliformes), guillemot (Uria aalge), and gulls (Laridae) (Bruner et al. 1970; Eleazer et al. 1973; Pierce and Vorhies 1973; Hinz et al. 1998; Sandhu 2008). In Canada, an outbreak of RA disease in young, wild waterfowl at Niska Waterfowl Research Center, Guelph, Canada resulted in losses of approximately 100 wild ducks and geese (Karstad et al. 1970). Riemerella anatipestifer was also 341
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species were observed during the winter season of 2011 in migratory bird habitats of South Korea. Among them, Baikal Teal (Anas formosa) held the largest wintering population (34.6%), followed by the Mallard Duck (10.0%), Rook (Corvus frugilegus) (7.1%), White-fronted Goose (Anser albifrons) (6.2%), Spot-billed Duck (Anas poecilorhyncha) (4.8%), and Bean Goose (Anser sp.) (4.6%). The top five species, except Rook, belong to the order Anseriformes and account for 60.2% of the total winter migratory birds (Ministry of Agriculture, Food and Rural Affairs [MAFRA] 2012). Because birds of the order Anseriformes are highly susceptible to RA, these observations indicate that $60% of migratory birds may be infected with RA. According to the Food and Agricultural Organization of the United Nations (2012), China and Vietnam were home to 75% of the world’s duck populations. Outbreaks of RA have caused economic losses on duck farms in many Asian countries (Loh et al. 1992; Pathanasophon et al. 2002; Wang et al. 2012). In addition, there are seasonal migrations of wild birds that overwinter in these duck production areas, often mingling with farmed wild birds and ducks. Thus, wild birds in this area may be risk for transmission of RA to domestic poultry. Although there are wildlife health and economic implications for RA in wild birds, the prevalence of RA in Asia has not been reported. We investigated the prevalence, host-species diversity, antibiotic susceptibility, and serotypes of RA isolates in wild birds that migrate across South Korea, China, and Southeast Asia. MATERIALS AND METHODS Sample sources and collection
Between November 2011 and December 2012, we studied wild birds from 19 rivers and marshes in South Korea, including Gyeonggi (36u539–38u179N, 126u229–127u519E), Jeonbuk (35u189–36u909N, 125u589–127u559E), Jeonnam (33u549–35u309N, 125u049–127u549E), Chung-
buk (36u109–37u209N, 127u289–128u289E), Chungnam (35u589–37u709N, 125u319-127u289E), and Gyeongnam (34u299–35u549N, 127u359– 129u139E) provinces. These locations are on the migration route of bird species to Southeast Asia. A total of 944 wild bird samples of 34 species were collected (Table 1). All live wild birds sampled in this study were trapped in collaboration with the Animal and Plant Quarantine Agency (QIA) of South Korea. Birds were caught by netting, ground traps, and wild waterfowl roundups. We swabbed the pharynx or cloaca so that we could return the birds into the wild. Of the 944 birds sampled, 102 were sampled from both the pharynx and cloaca and 842 from the cloaca alone. All swab samples were transported in bacterial transport medium and placed in a box at 4 C until delivered to the laboratory. Characterization of RA
Pharyngeal and cloacal swab samples were spread over blood agar plates (Hanil Komed, Seung-nam, South Korea) containing 5% sheep’s blood. After incubation at 37 C for 36 h with 5% CO2, the cultures that contained mixed bacteria were collected in 2-mL microcentrifuge tubes with phosphate-buffered saline (PBS). A 200-mL aliquot was used for DNA isolation. Cells were resuspended in 200 mL of double-distilled H2O, frozen at 280 C for 10 min, and boiled at 100 C for 5 min. The DNA concentration was adjusted to 200 ng/mL using ASP-2680 (ACTGene, Piscataway, New Jersey, USA). We detected RA by PCR, as described previously, and subsequent sequencing was performed to avoid false-positive results in related bacterial species (Qu et al. 2006; Christensen et al. 2010; Wei et al. 2013). The forward primer 190f (59GTATTGAAAGCTCTGGCGG-39) and reverse primer 843r (59-TCGCTTAGTCTCTGAACCC-39) were used to amplify a 654-base pair segment of the 6SrRna gene of RA within the region of 190-843. The amplified PCR product was purified using a Gel Extraction Kit (Genomed, Lo¨hne, Germany) and sequenced with an ABI Autosequencer (Perkin Elmer Applied Biosystems, Foster City, California, USA). The similarities and divergence of the sequences were analyzed using the Genetyx version 7 (Genetyx, Tokyo, Japan). The RA isolates were also biochemically characterized by API-20NE and API-ZYM test kits (BioMerieux, Marcy l’Etoile, France). Growth tests were performed on MacConkey’s agar.
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TABLE 1. Prevalence of Riemerella anatipestifer-detected pharyngeal or cloacal swabs of wild bird species captured in South Korea, 2011–12.a No. positive/total (%) Family
Anatidae
Scolopacidae
Charadriidae Laridae Turdidae
Corvidae Paridae Aegithalidae Pycnonotidae Sturnidae Timaliidae Picidae
Columbidae Alcedinidae 14 families a
Species
Mallard Duck Northern Pintail Duck Spot-billed Duck Green-winged Teal Northern Shoveller Eurasian Wigeon Baikal Teal Dunlin Broad-billed Sandpiper Bar-tailed Godwit Whimbrel Great Knot Red-necked Stint Mongolian Plover Grey Plover Black-tailed Gull Tree Sparrow Grey-backed Thrush Pale Thrush White’s Ground Thrush Azure-winged Magpie Eurasian Jay Marsh Tit Great Tit Long-tailed Tit Brown-eared Bulbul Grey Starling Parrotbill Grey-headed Woodpecker Great Spotted Woodpecker White-backed Woodpecker Japanese Pygmy Woodpecker Rufous Turtle Dove Common Kingfisher 34 species
Scientific name
Anas platyrhynchos Anas acuta Anas poecilorhyncha Anas carolinensis Anas clypeata Anas penelope Anas formosa Calidris alpina Limicola falcinellus Limosa lapponica Numenius phaeopus Calidris tenuirostris Calidris ruficollis Charadrius mongolus Pluvialis squatarola Larus crassirostris Passer montanus Turdus hortulorum Turdus pallidus Zoothera dauma Cyanopica cyanus Garrulus glandarius Poecile palustris Parus major Aegithalos caudatus Hypsipetes amaurotis Sturnus cineraceus Sinosuthora webbiana Picus canus Dendrocopos major Dendrocopos leucotos Dendrocopos kizuki Streptopelia orientalis Alcedo atthis
Pharynx
Cloaca
31/32 (96.8) 8/532 (1.5) 22/23 (95.6) 4/120 (3.3) 18/20 (90.0) 7/52 (13.4) — 0/20 — 0/1 — 0/35 — 0/17 0/6 0/6 0/6 0/6 0/3 0/3 0/1 0/1 0/1 0/1 0/4 0/4 0/4 0/4 0/2 0/2 — 0/52 — 0/2 — 0/2 — 0/6 — 0/4 — 0/11 — 0/2 — 0/1 — 0/3 — 0/1 — 0/8 — 0/7 — 0/24 — 0/1 — 0/8 — 0/1 — 0/1 — 0/5 — 0/1 71/102 (69.6) 19/944 (2.0)
Em dash indicates no sample available.
Serotyping
We tested RA isolates against the three known RA serotypes, 1, 4, and 7, to determine if there were virulent isolates in wild birds. These serotypes are the most prevalent and frequently related to outbreaks of RA disease on duck farms in South Korea (QIA 2008). We serotyped RA isolates using a modification of the agar gel precipitation (AGP) test described by Brogden et al. (1982). Briefly, 16-wk-old specific-pathogen-free chicken antisera against the reference serotypes 1, 4, and 7 were obtained from QIA, South Korea, and were prepared, as previously described (Pathanasophon et al. 2002; QIA 2008). Antigens for the
AGP test were grown for 18–24 h on blood agar. Growth from the blood agar plates was collected, washed twice with PBS, resuspended in PBS, and boiled at 100 C for 1 h for the AGP test. Antibiotic susceptibility test
The susceptibility test of RA isolates to 26 antibiotics was tested by agar disc diffusion using BD BBL Sensi-Discs (Becton Dickinson and Company, Sparks, Maryland, USA; Table 2). Bacteria, grown to an optical density of 1.0 at 600 nm in tryptic soy broth, were streaked onto Mueller-Hinton agar (Oxoid Ltd., Basingstoke, UK) with 5% sheep’s blood. The antibiotic
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TABLE 2. Prevalence of antibiotic resistance in 33 Riemerella anatipestifer isolates from wild birds in South Korea.
Antibiotics
Resistant Prevalence isolates (n) (%)
Aminoglycosides Amikacin Gentamicin Kanamycin Neomycin Streptomycin
30 31 33 29 27
91 94 100 88 82
b-lactam/b-lactamase inhibitor combinations Amoxicillin/clavulanic acid Oxacillin Penicillin Piperacillin
0 33 0 0
0 100 0 0
0
0
0 0 0 0 0
0 0 0 0 0
0 7 0
0 21 0
0 3
0 9
2
6
5
15
1 0 0
3 0 0
0
0
Carbapenems Imipenem Cephems Cephalothin Cefepime Cefoperazone Ceftazidime Ceftiofur Fluoroquinolones Ciprofloxacin Norfloxacin Ofloxacin Folate pathway inhibitors Sulfisoxazole Trimethoprim/ sulfamethoxazole Phenicols Chloramphenicol Quinolones Nalidixic acid Tetracyclines Minocycline Tetracycline Doxycycline Macrolides Erythromycin
discs were placed on the inoculated plates and incubated for 24 h at 37 C with 5% CO2. Quality control isolates included Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923. Antibiotic resistance in RA and quality control strains was determined according to the Clinical and Laboratory Standards Institute (2012).
RESULTS Detection and isolation of RA
We detected RA in nearly 70% of pharyngeal swabs and 2% cloacal swabs of wild birds (Table 1). Thirty-three RA isolates were obtained from 90 PCR- and sequence-positive samples. Thus, 90 positive samples were analyzed for 16S rRNA sequence similarity, and 33 isolates were used for biochemical characterization, serotyping, and antibiotic susceptibility testing. Detection of RA among bird species and sampling sites
We analyzed RA prevalence from pharyngeal samples by species and location. All of the RA-positive samples were from duck species (Anseriformes: Anatidae; Table 1). Among 75 wild ducks examined, 71 (95%) had RA in the pharynx. The predominant species of wild birds infected were Mallard Ducks, Northern Pintails, and Spot-billed Ducks (Table 1). No evidence of infection was observed in other orders, such as Charadriiformes. We detected RA in two of the 19 areas studied. The prevalence was 96% (53/55) at Man-kyung River (35u899–35u919N, 126u949–126u969E) in Jeonbuk, and 90% (18/20) at Mu-sim River (36u589N, 127u519E) in Chungbuk. 16S rRNA sequence similarity
The 16S rRNA gene sequences of the wild birds and reference strains belonged to the family Flavobacteriaceae. The divergence of the nucleotide sequences of 90 PCR- and sequence-positive samples in wild birds was 0–2%. There was a 98– 100% similarity of RA from wild birds with three available RA sequences (AF118416.1, AF118417.1, and AF118418.1) of isolates from the pharynx of healthy domestic ducks obtained from GenBank. The similarity of the nucleotide sequences of reference Riemerella columbipharyngis (HQ286278, HQ286279, HQ286280, and HQ286282) isolates from the pharynx of pigeons
CHA ET AL.—RIEMERELLA ANATIPESTIFER IN WILD BIRDS IN SOUTH KOREA
(Columba livia domestica) was 95–98% and 93–96% with Riemerella columbina (AF181448.1, GU903272.1, GU903273.1, and JF797623.1). No association was observed between the 16S rRNA groups and serotype or location. Biochemical and physiologic characterization
The phenotypic characteristics of the 33 isolates were confirmed by API galleries. All isolates were confirmed by positive oxidase and catalase. Seventeen of 33 (52%) isolates were positive on gelatin liquefaction. Indole and nitrates were not produced, and only 3/33 (9%) isolates of the cultures were positive for urease. None of the 12 carbohydrates were fermented, including D-glucose, D-mannose, and Dmaltose. None of the isolates grew on MacConkey’s agar. The biochemical and physiologic properties were similar to those of type strain ATCC 11845 (Ryll et al. 2001; Rubbenstroth et al. 2011). Serotyping
The 33 RA isolates were tested to three serotypes: 1, 4, and 7. Six (18%) isolates were serotype 4 from Mallard or Northern Pintail Ducks, two (6%) were serotype 1 from the Spot-billed Duck, and one (3%) isolate belonged to serotype 7 from the Spot-billed Duck. Five serotype 4 isolates were from the pharynx, and one was isolated from the cloaca of a wild duck. Three isolates of serotypes 1 and 7 were isolated from the pharynx. Antibiotic susceptibility
We examined 26 antibiotics to confirm the distribution of RA antibiotic resistance (Table 2). All 33 isolates were resistant to at least one antimicrobial agent tested. Resistance was most prevalent to kanamycin and oxacillin, followed by gentamicin, amikacin, neomycin, and streptomycin. No resistance was observed to cefepime, cefoperazone, or imipenem. The GenBank (http://www.ncbi.nlm.nih. gov/nuccore) accession numbers for the
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16S rRNA sequences from 14 representative isolates are the following: No. KC878309 (A11-WB-221); KC878310 (A11-WB-223); KC878311 (A11-WB-225); KC878312 (A11-WB-227); KC878313 (A11-WB-229); KC878314 (A11-WB-230); KC878315 (A11-WB-231); KC878316 (A11-WB-239); KC878317 (A11-WB-254); KC878318 (A11-WB-257); KC878319 (A11-WB-235-cl); KC878320 (A11-WB236-cl); KC878321 (A11-WB-237-cl); and KC878322 (A11-WB-253-cl). DISCUSSION
Considering the large proportion of order Anseriformes among winter migratory birds and the prevalence of RA in the Anseriformes, many migratory birds might be susceptible to RA. Hubalek (2004) suggested that RA is one of the 28 most important pathogens associated with migratory birds. We hope our investigation will serve as a foundation for future studies that will increase our understanding of the role of RA in the health of wild birds. We studied the distribution of RA in 14 wild bird families and 34 species. Three duck species (Anatidae) had a 95% (71/75) prevalence in pharyngeal swabs. A similar result was reported in Pekin Duck (Anas platyrhynchos forma domestica) by Ryll et al. (2001), who identified 39 RA from the pharynx of 49 Pekin Ducks and suggested that RA colonizes and persists on the pharyngeal mucous membranes of healthy domestic Pekin Ducks. Our findings indicate that RA might be common in the pharynx of wild ducks, as well as domestic ducks. We also detected RA in the cloacae of 2% of wild ducks. Cloacal isolates of RA have also been reported (Seo et al. 2013), and Xie et al. (2010) detected RA in cloacae by real-time PCR 3 h to 5 days after subcutaneous inoculation. This suggests that RA could be shed in feces of wild birds. Additional studies, including cloacal sampling, are needed. Northern Pintail and Spot-billed Ducks are commonly seen in migratory bird
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habitats. The Northern Pintail is a widely occurring duck that breeds in northern areas of Europe, Asia, and North America. It winters mainly south of its breeding range, reaching almost to the equator in Panama, northern sub-Saharan Africa, and southern Asia, including China, Taiwan, and Korea (Madge and Burn 1988; Robinson 2002). The Spot-billed Duck is resident in the southern part of its range from Pakistan and India to Korea and southern Japan but is also migratory, wintering in Southeast Asia. The Northern Pintail, Spotbilled Duck, and Mallard are dabbling ducks feeding primarily along the surface of the water or foraging on farmland for seeds and insects (BirdLife International 2004; MAFRA 2012). Thus, there may be increasing opportunities for contact with and transmission to domestic poultry. Twenty-one serotypes of RA have so far been identified with no cross reactivity (Pathanasophon et al. 2002). We analyzed the occurrence of three serotypes in a wild bird community. Information concerning RA serotypes of wild birds is useful to evaluate potential risks for propagating virulent serotypes. Serotypes 1 and 7, which we identified from the pharynx in the Spot-billed Duck, are highly virulent and frequently related to outbreaks of RA disease in duck farms in China, Thailand, Singapore, Taiwan, and South Korea (Loh et al. 1992; Pathanasophon et al. 2002; QIA 2008). It is possible that these serotypes are shared among countries located on the same flyway. However, further studies are necessary to clarify the relationship between various isolates. Additionally, we isolated two serotypes from one Mallard Duck: serotype 4 was isolated from the pharynx, and another unidentified serotype was isolated from the cloaca. Pathanasophon et al. (2002) documented infection with multiple serotypes in one bird on a farm in Thailand. This observation suggests that several infections with multiple serotypes may be associated with a given outbreak. We expect that other
serotypes besides 1, 4, and 7 also exist in wild duck species. Our finding of high RA resistance to aminoglycosides is similar to results of Sun et al. (2012) and Rubbenstroth et al. (2013) for R. columbina and R. columbipharyngis. Sun et al. (2012) found that the MIC50 and MIC90 values of aminoglycosides such as streptomycin, kanamycin, gentamicin, amikacin, and neomycin were high (32 to $128 mg/mL) among 103 RA isolates. The birds in this study were apparently healthy; hence, they are free-flying, subclinical carriers across national boundaries. Additionally, high accessibility to domestic farms suggests the potential of exchanging RA between wild birds and domestic ducks. Surveillance to evaluate RA transmission from wild birds to domestic ducks is needed. ACKNOWLEDGMENTS
This research was supported by Agricultural Biotechnology Development Program (3140233), Ministry of Agriculture, Food and Rural Affairs, and Cooperative Research Program for Agriculture Science and Technology Development (PJ01011403 and PJ009314) Rural Development Administration, Republic of Korea. LITERATURE CITED Animal and Plant Quarantine Agency. 2008. Studies of pathogenesis against riemerellosis and development of Riemerella killed vaccine for ducks, www.qia.go.kr. Accessed July 2014. BirdLife International. 2014. IUCN Red List for birds, http://www.iucnredlist.org. Accessed May 2014. Brogden KA, Rhoades KR, Rimler RB. 1982. Serologic types and physiologic characteristics of 46 avian Pasteurella anatipestifer cultures. Avian Dis 26:891–896. Bruner DW, Angstrom CI, Price JI. 1970. Pasteurella anatipestifer infection in pheasants. A case report. Cornell Vet 50:491–494. Christensen H, Bisgaard M. 2010. Phylogenetic relationships of Riemerella anatipestifer serovars and related taxa and an evaluation of specific PCR tests reported for R. anatipestifer. J Appl Microbiol 108:1612–1619. Clinical and Laboratory Standards Institute (CLSI). 2012. Performance standards for antimicrobial
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susceptibility testing. Twenty-second informational supplement. Document M100-S22. CLSI, Wayne, Pennsylvania, 44 pp. Eleazer TH, Blalock HG, Harrell JS, Derieux WT. 1973. Case report: Pasteurella anatipestifer as a cause of mortality in semiwild pen-raised Mallard ducks in South Carolina. Avian Dis 17:855–857. Food and Agriculture Organization of the United Nations. 2012. FAO monitors pigs, poultry and waterfowl in hot spots for influenza, www.fao. org/avianflu/en/news/ept_plus.html. Accessed May 2014. Hinz KH, Ryll M, Kohler B, Glunder G. 1998. Phenotypic characteristics of Riemerella anatipestifer and similar micro-organisms from various hosts. Avian Pathol 27:33–42. Hubalek Z. 2004. An annotated checklist of pathogenic microorganisms associated with migratory birds. J Wildl Dis 40:639–659. Karstad L, Lusis P, Long JR. 1970. Pasteurella anatipestifer as a cause of mortality in captive wild waterfowl. J Wildl Dis 6:408–413. Li JX, Tang Y, Gao JY, Huang CH, Ding MJ. 2011. Riemerella anatipestifer infection in chickens. Pak Vet J 31:65–69. Loh H, Teo TP, Tan HC. 1992. Serotypes of ‘‘Pasteurella’’ anatipestifer isolates from ducks in Singapore: A proposal of new serotypes. Avian Pathol 21:453–459. Madge S, Burn H. 1988. Waterfowl: An identification guide to the ducks, geese, and swans of the world. Houghton Mifflin, New York, New York, 298 pp. Ministry of Agriculture, Food and Rural Affairs. 2012. The white book of highly pathogenic avian influenza in 2010–2011, www.mafra.go.kr. Accessed December 2013. Pathanasophon P, Phuektes P, Tanticharoenyos T, Narongsak W, Sawada T. 2002. A potential new serotype of Riemerella anatipestifer isolated from ducks in Thailand. Avian Pathol 31:267– 270. Pierce RL, Vorhies MW. 1973. Pasteurella anatipestifer infection in geese. Avian Dis 17:868–870. Qu FF, Cai C, Zheng XJ, Zhang DB. 2006. Rapid identification of Riemerella anatipestifer on the basis of specific PCR amplifying 16S rDNA. Wei Sheng Wu Xue Bao 46:13–17. Robinson J. 2002. Anas acuta. Animal Diversity Web. University of Michigan Museum of Zoology, www.animaldiversity.ummz.umich.edu. Accessed May 2014. Rubbenstroth D, Hotzel H, Knobloch J, Teske L, Rautenschlein S, Ryll M. 2011. Isolation and characterization of atypical Riemerella columbina strains from pigeons and their differentiation from Riemerella anatipestifer. Vet Microbiol 147:103–112.
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Rubbenstroth D, Ryll M, Behr KP, Rautenschlein S. 2009. Pathogenesis of Riemerella anatipestifer in turkeys after experimental mono-infection via respiratory routes or dual infection together with the avian metapneumovirus. Avian Pathol 38: 497–507. Rubbenstroth D, Ryll M, Hotzel H, Christensen H, Knobloch JK, Rautenschlein S, Bisgaard M. 2013. Description of Riemerella columbipharyngis sp. nov., isolated from the pharynx of healthy domestic pigeons (Columba livia f. domestica), and emended description of the genus Riemerella, Riemerella anatipestifer and Riemerella columbina. Int J Syst Evol Microbiol 63:280–287. Ryll M, Christensen H, Bisgaard M, Christensen JP, Hinz KH, Kohler B. 2001. Studies on the prevalence of Riemerella anatipestifer in the upper respiratory tract of clinically healthy ducklings and characterization of untypable strains. J Vet Med B Infect Dis Vet Public Health 48:537–546. Sandhu TS. 2008. Riemerella anatipestifer infection. In: Diseases of poultry, 12th Ed., Saif YM, Barnes HJ, Fadly AM, Glisson JR, McDougald LR, Swayne DE, editors. Blackwell Publishing Ltd., Oxford, UK, pp. 758–764. Seo HS, Cha SY, Kang M, Jang HK. 2013. Chicken embryo lethality assay for determining the virulence of Riemerella anatipestifer isolates. Avian Pathol 42:387–392. Sun N, Liu JH, Yang F, Lin DC, Li GH, Chen ZL, Zeng ZL. 2012. Molecular characterization of the antimicrobial resistance of Riemerella anatipestifer isolated from ducks. Vet Microbiol 158:376–383. Wang XP, Zhu DK, Wang MS, Cheng AC, Jia RY, Chen S, Chen XY, Tang T. 2012. Development and application of specific polymerase chain reaction assay targeting the gyrB gene for rapid detection of Riemerella anatipestifer. Poult Sci 91:2450–2453. Wei B, Cha SY, Kang M, Park IJ, Moon OK, Park CK, Jang HK. 2013. Development and application of a multiplex PCR assay for rapid detection of 4 major bacterial pathogens in ducks. Poult Sci 92:1164–1170. Wobeser G, Ward GE. 1974. Pasteurella anatipestifer infection in migrating Whistling Swans. J Wildl Dis 10:466–470. Xie YP, Pan BJ, Chen ZX, Xu LG, Yang W, Wei ML. 2010. Establishment and application of real-time fluorescence qPCR assay for quick detection of Riemerella anatipestifer infection in ducks. China J Anim Husb Vet Med 37:87–92. Submitted for publication 19 May 2014. Accepted 8 August 2014.
Journal of Wildlife Diseases, 51(2), 2015, pp. 341–347 # Wildlife Disease Association 2015
SURVEILLANCE AND CHARACTERIZATION OF RIEMERELLA ANATIPESTIFER FROM WILD BIRDS IN SOUTH KOREA Se-Yeoun Cha,1,3 Hye-Suk Seo,1,3 Bai Wei,1 Min Kang,1 Jae-Hee Roh,1 Ran-Hee Yoon,1 Ji-Hyuk Kim,2 and Hyung-Kwan Jang1,4 1 Department of Infectious Diseases and Avian Diseases, College of Veterinary Medicine and Korea Zoonosis Research Institute, Chonbuk National University, 79 Gobongro, Iksan, 570-752, South Korea 2 Poultry Science Division, National Institute of Animal Science, Rural Development Administration, 114 Shinbang 1-gil, Cheonan, 331-808, South Korea 3 These authors contributed equally to this study 4 Corresponding author (email: [email protected])
We conducted surveillance for Riemerella anatipestifer (RA) in wild birds along the East Asian-Australasian flyway in South Korea. Detected RA were characterized by serotype, antibiotic susceptibility, and sequence analysis of the 16S rRNA gene. We collected 944 wild birds of 34 species from 19 of South Korea’s major migratory wild bird habitats between 2011 and 2012. We identified RA by PCR and rRNA gene sequence in 71/102 (69.6%) pharyngeal swabs and 19/ 944 (2.0%) cloacal swabs of wild birds. Most RA positives (71/75 [95%] pharyngeal and 19/704 [(2.6%] cloacal) were from three duck species (family Anatidae): Mallard Duck (Anas platyrhynchos), Northern Pintail (Anas acuta), and Spot-billed Duck (Anas poecilorhyncha). Thirty-three RA isolates obtained and examined were highly resistant to aminoglycosides: kanamycin (100%), gentamicin (94%), amikacin (91%), neomycin (88%), and streptomycin (82%). Six isolates were identified as serotype 4 by agar gel precipitation. Serotypes 1 and 7, which are known virulent serotypes, were also identified in three isolates from wild duck species. Key words: Prevalence, Riemerella anatipestifer, South Korea, wild bird. ABSTRACT:
isolated from five of seven juvenile Whistling Swans (Olor columbianus) found sick or dead on the lakes during the autumn migration in Saskatchewan, Canada (Wobeser and Ward 1974). Moreover, RA and RA-like field isolates have been described from wild bird species in Germany, (e.g., Carolina Wood Ducks (Aix sponsa), Mallard Ducks (Anas platyrhynchos), and Greater Eider Ducks (Somateria mollissima) with signs and lesions typical of exudative septicemia or neurologic disorders (Hinz et al. 1998). These observations indicate that RA could cause large losses of wild migrating birds or resident waterfowl. The East Asian-Australasian flyway is one of the eight migratory bird flyways in the world where about 250 bird species pass through 22 nations from Russia, Mongolia, and China to Korea, Japan, and South Asia. South Korea is located in this flyway and is geographically important as a stopover site for Charadriidae and wintering habitats of Anatidae. According to a study by the Ministry of Environment of South Korea, 1,259,717 birds of 204
INTRODUCTION
Riemerella anatipestifer (RA) is a gramnegative, nonmotile, nonspore-forming, rod-shaped bacterium. Infected birds are characterized by respiratory symptoms, septicemia, pericarditis, perihepatitis, meningitis, and salpingitis (Sandhu 2008). The bacterium is distributed worldwide and is an important cause of infectious disease among waterfowl, turkeys, and chickens (Sandhu 2008; Rubbenstroth et al. 2009; Li et al. 2011). It has also been isolated from wild birds, including wild ducks (Anatidae), geese (Anatidae), black swans (Cygnus atratus), pheasants (Phasianidae), guinea fowl (Numididae), quail (Galliformes), guillemot (Uria aalge), and gulls (Laridae) (Bruner et al. 1970; Eleazer et al. 1973; Pierce and Vorhies 1973; Hinz et al. 1998; Sandhu 2008). In Canada, an outbreak of RA disease in young, wild waterfowl at Niska Waterfowl Research Center, Guelph, Canada resulted in losses of approximately 100 wild ducks and geese (Karstad et al. 1970). Riemerella anatipestifer was also 341
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species were observed during the winter season of 2011 in migratory bird habitats of South Korea. Among them, Baikal Teal (Anas formosa) held the largest wintering population (34.6%), followed by the Mallard Duck (10.0%), Rook (Corvus frugilegus) (7.1%), White-fronted Goose (Anser albifrons) (6.2%), Spot-billed Duck (Anas poecilorhyncha) (4.8%), and Bean Goose (Anser sp.) (4.6%). The top five species, except Rook, belong to the order Anseriformes and account for 60.2% of the total winter migratory birds (Ministry of Agriculture, Food and Rural Affairs [MAFRA] 2012). Because birds of the order Anseriformes are highly susceptible to RA, these observations indicate that $60% of migratory birds may be infected with RA. According to the Food and Agricultural Organization of the United Nations (2012), China and Vietnam were home to 75% of the world’s duck populations. Outbreaks of RA have caused economic losses on duck farms in many Asian countries (Loh et al. 1992; Pathanasophon et al. 2002; Wang et al. 2012). In addition, there are seasonal migrations of wild birds that overwinter in these duck production areas, often mingling with farmed wild birds and ducks. Thus, wild birds in this area may be risk for transmission of RA to domestic poultry. Although there are wildlife health and economic implications for RA in wild birds, the prevalence of RA in Asia has not been reported. We investigated the prevalence, host-species diversity, antibiotic susceptibility, and serotypes of RA isolates in wild birds that migrate across South Korea, China, and Southeast Asia. MATERIALS AND METHODS Sample sources and collection
Between November 2011 and December 2012, we studied wild birds from 19 rivers and marshes in South Korea, including Gyeonggi (36u539–38u179N, 126u229–127u519E), Jeonbuk (35u189–36u909N, 125u589–127u559E), Jeonnam (33u549–35u309N, 125u049–127u549E), Chung-
buk (36u109–37u209N, 127u289–128u289E), Chungnam (35u589–37u709N, 125u319-127u289E), and Gyeongnam (34u299–35u549N, 127u359– 129u139E) provinces. These locations are on the migration route of bird species to Southeast Asia. A total of 944 wild bird samples of 34 species were collected (Table 1). All live wild birds sampled in this study were trapped in collaboration with the Animal and Plant Quarantine Agency (QIA) of South Korea. Birds were caught by netting, ground traps, and wild waterfowl roundups. We swabbed the pharynx or cloaca so that we could return the birds into the wild. Of the 944 birds sampled, 102 were sampled from both the pharynx and cloaca and 842 from the cloaca alone. All swab samples were transported in bacterial transport medium and placed in a box at 4 C until delivered to the laboratory. Characterization of RA
Pharyngeal and cloacal swab samples were spread over blood agar plates (Hanil Komed, Seung-nam, South Korea) containing 5% sheep’s blood. After incubation at 37 C for 36 h with 5% CO2, the cultures that contained mixed bacteria were collected in 2-mL microcentrifuge tubes with phosphate-buffered saline (PBS). A 200-mL aliquot was used for DNA isolation. Cells were resuspended in 200 mL of double-distilled H2O, frozen at 280 C for 10 min, and boiled at 100 C for 5 min. The DNA concentration was adjusted to 200 ng/mL using ASP-2680 (ACTGene, Piscataway, New Jersey, USA). We detected RA by PCR, as described previously, and subsequent sequencing was performed to avoid false-positive results in related bacterial species (Qu et al. 2006; Christensen et al. 2010; Wei et al. 2013). The forward primer 190f (59GTATTGAAAGCTCTGGCGG-39) and reverse primer 843r (59-TCGCTTAGTCTCTGAACCC-39) were used to amplify a 654-base pair segment of the 6SrRna gene of RA within the region of 190-843. The amplified PCR product was purified using a Gel Extraction Kit (Genomed, Lo¨hne, Germany) and sequenced with an ABI Autosequencer (Perkin Elmer Applied Biosystems, Foster City, California, USA). The similarities and divergence of the sequences were analyzed using the Genetyx version 7 (Genetyx, Tokyo, Japan). The RA isolates were also biochemically characterized by API-20NE and API-ZYM test kits (BioMerieux, Marcy l’Etoile, France). Growth tests were performed on MacConkey’s agar.
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TABLE 1. Prevalence of Riemerella anatipestifer-detected pharyngeal or cloacal swabs of wild bird species captured in South Korea, 2011–12.a No. positive/total (%) Family
Anatidae
Scolopacidae
Charadriidae Laridae Turdidae
Corvidae Paridae Aegithalidae Pycnonotidae Sturnidae Timaliidae Picidae
Columbidae Alcedinidae 14 families a
Species
Mallard Duck Northern Pintail Duck Spot-billed Duck Green-winged Teal Northern Shoveller Eurasian Wigeon Baikal Teal Dunlin Broad-billed Sandpiper Bar-tailed Godwit Whimbrel Great Knot Red-necked Stint Mongolian Plover Grey Plover Black-tailed Gull Tree Sparrow Grey-backed Thrush Pale Thrush White’s Ground Thrush Azure-winged Magpie Eurasian Jay Marsh Tit Great Tit Long-tailed Tit Brown-eared Bulbul Grey Starling Parrotbill Grey-headed Woodpecker Great Spotted Woodpecker White-backed Woodpecker Japanese Pygmy Woodpecker Rufous Turtle Dove Common Kingfisher 34 species
Scientific name
Anas platyrhynchos Anas acuta Anas poecilorhyncha Anas carolinensis Anas clypeata Anas penelope Anas formosa Calidris alpina Limicola falcinellus Limosa lapponica Numenius phaeopus Calidris tenuirostris Calidris ruficollis Charadrius mongolus Pluvialis squatarola Larus crassirostris Passer montanus Turdus hortulorum Turdus pallidus Zoothera dauma Cyanopica cyanus Garrulus glandarius Poecile palustris Parus major Aegithalos caudatus Hypsipetes amaurotis Sturnus cineraceus Sinosuthora webbiana Picus canus Dendrocopos major Dendrocopos leucotos Dendrocopos kizuki Streptopelia orientalis Alcedo atthis
Pharynx
Cloaca
31/32 (96.8) 8/532 (1.5) 22/23 (95.6) 4/120 (3.3) 18/20 (90.0) 7/52 (13.4) — 0/20 — 0/1 — 0/35 — 0/17 0/6 0/6 0/6 0/6 0/3 0/3 0/1 0/1 0/1 0/1 0/4 0/4 0/4 0/4 0/2 0/2 — 0/52 — 0/2 — 0/2 — 0/6 — 0/4 — 0/11 — 0/2 — 0/1 — 0/3 — 0/1 — 0/8 — 0/7 — 0/24 — 0/1 — 0/8 — 0/1 — 0/1 — 0/5 — 0/1 71/102 (69.6) 19/944 (2.0)
Em dash indicates no sample available.
Serotyping
We tested RA isolates against the three known RA serotypes, 1, 4, and 7, to determine if there were virulent isolates in wild birds. These serotypes are the most prevalent and frequently related to outbreaks of RA disease on duck farms in South Korea (QIA 2008). We serotyped RA isolates using a modification of the agar gel precipitation (AGP) test described by Brogden et al. (1982). Briefly, 16-wk-old specific-pathogen-free chicken antisera against the reference serotypes 1, 4, and 7 were obtained from QIA, South Korea, and were prepared, as previously described (Pathanasophon et al. 2002; QIA 2008). Antigens for the
AGP test were grown for 18–24 h on blood agar. Growth from the blood agar plates was collected, washed twice with PBS, resuspended in PBS, and boiled at 100 C for 1 h for the AGP test. Antibiotic susceptibility test
The susceptibility test of RA isolates to 26 antibiotics was tested by agar disc diffusion using BD BBL Sensi-Discs (Becton Dickinson and Company, Sparks, Maryland, USA; Table 2). Bacteria, grown to an optical density of 1.0 at 600 nm in tryptic soy broth, were streaked onto Mueller-Hinton agar (Oxoid Ltd., Basingstoke, UK) with 5% sheep’s blood. The antibiotic
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TABLE 2. Prevalence of antibiotic resistance in 33 Riemerella anatipestifer isolates from wild birds in South Korea.
Antibiotics
Resistant Prevalence isolates (n) (%)
Aminoglycosides Amikacin Gentamicin Kanamycin Neomycin Streptomycin
30 31 33 29 27
91 94 100 88 82
b-lactam/b-lactamase inhibitor combinations Amoxicillin/clavulanic acid Oxacillin Penicillin Piperacillin
0 33 0 0
0 100 0 0
0
0
0 0 0 0 0
0 0 0 0 0
0 7 0
0 21 0
0 3
0 9
2
6
5
15
1 0 0
3 0 0
0
0
Carbapenems Imipenem Cephems Cephalothin Cefepime Cefoperazone Ceftazidime Ceftiofur Fluoroquinolones Ciprofloxacin Norfloxacin Ofloxacin Folate pathway inhibitors Sulfisoxazole Trimethoprim/ sulfamethoxazole Phenicols Chloramphenicol Quinolones Nalidixic acid Tetracyclines Minocycline Tetracycline Doxycycline Macrolides Erythromycin
discs were placed on the inoculated plates and incubated for 24 h at 37 C with 5% CO2. Quality control isolates included Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923. Antibiotic resistance in RA and quality control strains was determined according to the Clinical and Laboratory Standards Institute (2012).
RESULTS Detection and isolation of RA
We detected RA in nearly 70% of pharyngeal swabs and 2% cloacal swabs of wild birds (Table 1). Thirty-three RA isolates were obtained from 90 PCR- and sequence-positive samples. Thus, 90 positive samples were analyzed for 16S rRNA sequence similarity, and 33 isolates were used for biochemical characterization, serotyping, and antibiotic susceptibility testing. Detection of RA among bird species and sampling sites
We analyzed RA prevalence from pharyngeal samples by species and location. All of the RA-positive samples were from duck species (Anseriformes: Anatidae; Table 1). Among 75 wild ducks examined, 71 (95%) had RA in the pharynx. The predominant species of wild birds infected were Mallard Ducks, Northern Pintails, and Spot-billed Ducks (Table 1). No evidence of infection was observed in other orders, such as Charadriiformes. We detected RA in two of the 19 areas studied. The prevalence was 96% (53/55) at Man-kyung River (35u899–35u919N, 126u949–126u969E) in Jeonbuk, and 90% (18/20) at Mu-sim River (36u589N, 127u519E) in Chungbuk. 16S rRNA sequence similarity
The 16S rRNA gene sequences of the wild birds and reference strains belonged to the family Flavobacteriaceae. The divergence of the nucleotide sequences of 90 PCR- and sequence-positive samples in wild birds was 0–2%. There was a 98– 100% similarity of RA from wild birds with three available RA sequences (AF118416.1, AF118417.1, and AF118418.1) of isolates from the pharynx of healthy domestic ducks obtained from GenBank. The similarity of the nucleotide sequences of reference Riemerella columbipharyngis (HQ286278, HQ286279, HQ286280, and HQ286282) isolates from the pharynx of pigeons
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(Columba livia domestica) was 95–98% and 93–96% with Riemerella columbina (AF181448.1, GU903272.1, GU903273.1, and JF797623.1). No association was observed between the 16S rRNA groups and serotype or location. Biochemical and physiologic characterization
The phenotypic characteristics of the 33 isolates were confirmed by API galleries. All isolates were confirmed by positive oxidase and catalase. Seventeen of 33 (52%) isolates were positive on gelatin liquefaction. Indole and nitrates were not produced, and only 3/33 (9%) isolates of the cultures were positive for urease. None of the 12 carbohydrates were fermented, including D-glucose, D-mannose, and Dmaltose. None of the isolates grew on MacConkey’s agar. The biochemical and physiologic properties were similar to those of type strain ATCC 11845 (Ryll et al. 2001; Rubbenstroth et al. 2011). Serotyping
The 33 RA isolates were tested to three serotypes: 1, 4, and 7. Six (18%) isolates were serotype 4 from Mallard or Northern Pintail Ducks, two (6%) were serotype 1 from the Spot-billed Duck, and one (3%) isolate belonged to serotype 7 from the Spot-billed Duck. Five serotype 4 isolates were from the pharynx, and one was isolated from the cloaca of a wild duck. Three isolates of serotypes 1 and 7 were isolated from the pharynx. Antibiotic susceptibility
We examined 26 antibiotics to confirm the distribution of RA antibiotic resistance (Table 2). All 33 isolates were resistant to at least one antimicrobial agent tested. Resistance was most prevalent to kanamycin and oxacillin, followed by gentamicin, amikacin, neomycin, and streptomycin. No resistance was observed to cefepime, cefoperazone, or imipenem. The GenBank (http://www.ncbi.nlm.nih. gov/nuccore) accession numbers for the
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16S rRNA sequences from 14 representative isolates are the following: No. KC878309 (A11-WB-221); KC878310 (A11-WB-223); KC878311 (A11-WB-225); KC878312 (A11-WB-227); KC878313 (A11-WB-229); KC878314 (A11-WB-230); KC878315 (A11-WB-231); KC878316 (A11-WB-239); KC878317 (A11-WB-254); KC878318 (A11-WB-257); KC878319 (A11-WB-235-cl); KC878320 (A11-WB236-cl); KC878321 (A11-WB-237-cl); and KC878322 (A11-WB-253-cl). DISCUSSION
Considering the large proportion of order Anseriformes among winter migratory birds and the prevalence of RA in the Anseriformes, many migratory birds might be susceptible to RA. Hubalek (2004) suggested that RA is one of the 28 most important pathogens associated with migratory birds. We hope our investigation will serve as a foundation for future studies that will increase our understanding of the role of RA in the health of wild birds. We studied the distribution of RA in 14 wild bird families and 34 species. Three duck species (Anatidae) had a 95% (71/75) prevalence in pharyngeal swabs. A similar result was reported in Pekin Duck (Anas platyrhynchos forma domestica) by Ryll et al. (2001), who identified 39 RA from the pharynx of 49 Pekin Ducks and suggested that RA colonizes and persists on the pharyngeal mucous membranes of healthy domestic Pekin Ducks. Our findings indicate that RA might be common in the pharynx of wild ducks, as well as domestic ducks. We also detected RA in the cloacae of 2% of wild ducks. Cloacal isolates of RA have also been reported (Seo et al. 2013), and Xie et al. (2010) detected RA in cloacae by real-time PCR 3 h to 5 days after subcutaneous inoculation. This suggests that RA could be shed in feces of wild birds. Additional studies, including cloacal sampling, are needed. Northern Pintail and Spot-billed Ducks are commonly seen in migratory bird
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habitats. The Northern Pintail is a widely occurring duck that breeds in northern areas of Europe, Asia, and North America. It winters mainly south of its breeding range, reaching almost to the equator in Panama, northern sub-Saharan Africa, and southern Asia, including China, Taiwan, and Korea (Madge and Burn 1988; Robinson 2002). The Spot-billed Duck is resident in the southern part of its range from Pakistan and India to Korea and southern Japan but is also migratory, wintering in Southeast Asia. The Northern Pintail, Spotbilled Duck, and Mallard are dabbling ducks feeding primarily along the surface of the water or foraging on farmland for seeds and insects (BirdLife International 2004; MAFRA 2012). Thus, there may be increasing opportunities for contact with and transmission to domestic poultry. Twenty-one serotypes of RA have so far been identified with no cross reactivity (Pathanasophon et al. 2002). We analyzed the occurrence of three serotypes in a wild bird community. Information concerning RA serotypes of wild birds is useful to evaluate potential risks for propagating virulent serotypes. Serotypes 1 and 7, which we identified from the pharynx in the Spot-billed Duck, are highly virulent and frequently related to outbreaks of RA disease in duck farms in China, Thailand, Singapore, Taiwan, and South Korea (Loh et al. 1992; Pathanasophon et al. 2002; QIA 2008). It is possible that these serotypes are shared among countries located on the same flyway. However, further studies are necessary to clarify the relationship between various isolates. Additionally, we isolated two serotypes from one Mallard Duck: serotype 4 was isolated from the pharynx, and another unidentified serotype was isolated from the cloaca. Pathanasophon et al. (2002) documented infection with multiple serotypes in one bird on a farm in Thailand. This observation suggests that several infections with multiple serotypes may be associated with a given outbreak. We expect that other
serotypes besides 1, 4, and 7 also exist in wild duck species. Our finding of high RA resistance to aminoglycosides is similar to results of Sun et al. (2012) and Rubbenstroth et al. (2013) for R. columbina and R. columbipharyngis. Sun et al. (2012) found that the MIC50 and MIC90 values of aminoglycosides such as streptomycin, kanamycin, gentamicin, amikacin, and neomycin were high (32 to $128 mg/mL) among 103 RA isolates. The birds in this study were apparently healthy; hence, they are free-flying, subclinical carriers across national boundaries. Additionally, high accessibility to domestic farms suggests the potential of exchanging RA between wild birds and domestic ducks. Surveillance to evaluate RA transmission from wild birds to domestic ducks is needed. ACKNOWLEDGMENTS
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