Microbial Infections Are Associated with Embryo Mortality in ArcticNesting Geese Cristina M. Hansen,a Brandt W. Meixell,b Caroline Van Hemert,b Rebekah F. Hare,a Karsten Huefferc Department of Biology and Wildlife, University of Alaska Fairbanks, Fairbanks, Alaska, USAa; U.S. Geological Survey, Alaska Science Center, Anchorage, Alaska, USAb; Department of Veterinary Medicine, University of Alaska Fairbanks, Fairbanks, Alaska, USAc

D

espite the importance of egg hatchability on fecundity in birds, the mechanisms of embryo mortality remain poorly understood. Viability of avian eggs declines as the time to incubation onset increases (1–3), and microbial infection likely represents an important mechanism of embryo mortality (4–6). Investigations of microbial processes acting on wild bird eggs first focused on cavity-nesting species in tropical climates (4, 5, 7) but have been expanded to microbes found in temperate climates and open-cup nests (6, 8, 9). Potentially pathogenic bacteria may be transferred to the surface of the eggshell from nesting females, or may originate in the environment, and they are able to enter the egg through pores in the shell and membrane (10). A variety of bacteria are commonly present on eggshells shortly after laying and increase in abundance thereafter (6). The presence of water on eggshells increases the abundance and diversity of bacteria and appears to play an important role in sustaining bacterial growth (11). Godard et al. (6) reported considerably higher bacterial loads on eggs in open-cup nests than on eggs in cavity nests, likely a result of increased exposure to water in the former. In tropical environments, there is a positive relationship between trans-shell infection and humidity and temperature, and incubation inhibits bacterial growth and trans-shell penetration by reducing moisture on shells (5, 12). In contrast, incubation may not hinder bacterial growth or penetration of eggshells in temperate climates with cooler ambient temperatures (9, 13). Less commonly, vertical transmission from the nesting female may result in infection or mortality of embryos. For example, in chickens, there is no direct relationship between the presence of Salmonella enterica serovar Enteritidis on the eggshell and infection of egg contents, indicating that transmission occurs in the reproductive tract; infection of the albumen occurs as the egg passes through the oviduct (14). Campylobacter spp. (reviewed in

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reference 15) and Mycoplasma spp. (16) are also vertically transmitted. Hatching failure resulting from nonviable eggs has been reported in temperate- and Arctic-breeding waterfowl (17); however, the effects of microbial processes on egg hatchability in northern regions are unknown. During the summers of 2011 and 2012, we identified at least one nonviable egg in approximately 10% of Greater white-fronted goose (Anser albifrons; here, whitefronted goose) nests monitored on the Arctic Coastal Plain of Alaska (B. W. Meixell, unpublished data). While no previous reports of egg viability exist for this nesting population, the proportion of nonviable eggs was several times higher than rates reported for white-fronted geese and other species of geese nesting in Alaska (18). Elevated rates of nonviable eggs combined with a high nest density in the area provided an opportunity to investigate the mechanisms responsible for hatching failure in Arctic-nesting geese. White-fronted geese breed in Alaska and northern Canada and winter in the southern and western United States and in Mexico (19). On the Arctic Coastal Plain of Alaska, at the northern extent of their breeding range, white-fronted geese historically nested in low densities, but the population has more than tripled in abun-

Received 2 March 2015 Accepted 4 June 2015 Accepted manuscript posted online 5 June 2015 Citation Hansen CM, Meixell BW, Van Hemert C, Hare RF, Hueffer K. 2015. Microbial infections are associated with embryo mortality in Arctic-nesting geese. Appl Environ Microbiol 81:5583–5592. doi:10.1128/AEM.00706-15. Editor: H. Goodrich-Blair Address correspondence to Karsten Hueffer, [email protected]. Copyright © 2015, American Society for Microbiology. All Rights Reserved. doi:10.1128/AEM.00706-15

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To address the role of bacterial infection in hatching failure of wild geese, we monitored embryo development in a breeding population of Greater white-fronted geese (Anser albifrons) on the Arctic Coastal Plain of Alaska. During 2013, we observed mortality of normally developing embryos and collected 36 addled eggs for analysis. We also collected 17 infertile eggs for comparison. Using standard culture methods and gene sequencing to identify bacteria within collected eggs, we identified a potentially novel species of Neisseria in 33 eggs, Macrococcus caseolyticus in 6 eggs, and Streptococcus uberis and Rothia nasimurium in 4 eggs each. We detected seven other bacterial species at lower frequencies. Sequences of the 16S rRNA genes from the Neisseria isolates most closely matched sequences from N. animaloris and N. canis (96 to 97% identity), but phylogenetic analysis suggested substantial genetic differentiation between egg isolates and known Neisseria species. Although definitive sources of the bacteria remain unknown, we detected Neisseria DNA from swabs of eggshells, nest contents, and cloacae of nesting females. To assess the pathogenicity of bacteria identified in contents of addled eggs, we inoculated isolates of Neisseria, Macrococcus, Streptococcus, and Rothia at various concentrations into developing chicken eggs. Seven-day mortality rates varied from 70 to 100%, depending on the bacterial species and inoculation dose. Our results suggest that bacterial infections are a source of embryo mortality in wild geese in the Arctic.

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dance since the 1990s, to become the most abundant nesting waterfowl species in the area (20). White-fronted goose nests usually contain 3 to 6 eggs, with the female initiating the 24-day incubation period upon laying the penultimate egg (19). The primary objective of this study was to investigate microbial infection as a source of embryo mortality in white-fronted geese on the Arctic Coastal Plain of Alaska. Specifically, we sought to identify bacteria in contents of nonviable eggs, compare bacteria present within the cloacae of nesting females, in nest materials, and on eggshells to those found in the contents of nonviable eggs to evaluate potential sources, and to assess the pathogenicity of bacteria isolated from nonviable eggs. MATERIALS AND METHODS Sample collection. We monitored white-fronted goose nests and collected samples of egg contents between 14 June and 14 July 2013 near Point Lonely, AK (70°54=45.49⬙N, 153°14=28.82⬙W). We located nests on foot beginning the second week of June, corresponding with the beginning of nest initiation, and recorded each location by using a handheld global positioning system unit. We candled each egg within a clutch to determine incubation stage (21) and individually labeled eggs on both ends with a permanent marker. We visited nests every 4 to 7 days, at which time we candled each egg to assess egg viability and estimate hatch date. Eggs that were noted to be addled (dead or not developing with a history of containing a viable embryo) were collected. Eggs suspected of being infertile (no indication of development after a known period of incubation) were collected for comparison. After disinfecting the surface of the eggshell with 70% isopropyl alcohol, we sampled egg contents by puncturing the air sac end with a syringe and 18-gauge needle and aspirating up to 2.0 ml. Whole eggs and aspirate samples were kept at ⫺20°C for up to 15 days until shipment to the University of Alaska Fairbanks (UAF), where they were stored at ⫺50°C until analysis. A subset of seven eggs collected in the 48 h prior to leaving the field site were transported chilled to UAF, where they were aspirated and cultured immediately upon arrival. Previously aspirated whole eggs were thawed, opened, and examined visually; each egg was classified as addled or infertile (Fig. 1). Eggs were classified as addled if they had lost the distinction between albumen and yolk and whether the albumen was opaque. Infertile eggs had transparent albumen and a distinct yellow to orange yolk. To evaluate potential bacterial sources, we collected swab samples from eggshells, nest contents, and cloacae of nesting females. During egg laying, prior to incubation onset (i.e., preincubation), we selected a random sample of nests from which we swabbed both eggshells and nest contents separately. During incubation, we used a single swab to sample nest contents and eggshells from nests identified as containing an addled or infertile egg; for comparison, we also selected and swabbed eggshells and nest contents from nearby nests that contained all viable eggs. We

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FIG 1 Appearance of the contents of an addled egg (a) and those of an infertile egg (b).

used BD liquid Amies elution swabs (Eswab; BD, Franklin Lakes, NJ, USA), following the manufacturer’s instructions, and swabbed approximately 1/3 of the surface of each eggshell and multiple locations within the nest. We obtained cloacal swab samples from a subset of nesting females during late incubation. Birds were captured on nests by using bow-net traps (22), and cloacal swabs were inserted approximately 10 mm into the cloaca. Swabs were frozen at ⫺20°C within a few hours of collection and stored for up to 22 days, then shipped to UAF, where they were stored at ⫺50°C until thawed for analysis. All procedures were approved by the U.S. Geological Survey Alaska Science Center Animal Care and Use Committee (2013-09) and were authorized by the U.S. Fish and Wildlife Service and Bureau of Land Management (BLM) under permit numbers MB789758 and BLM AK FF09571, respectively. Microbiology. Sequencing and culture results from 3 eggs collected during a pilot study in 2011 demonstrated that an approximately 350-bp region of the16S rRNA gene closely matched 3 bacterial genera (Neisseria, Staphylococcus, and Macrococcus), two of which were grown in culture (Staphylococcus and Macrococcus). Because Neisseria species are fastidious and require specialized growth media (23), we tailored our culture protocols to target the putative Neisseria genus of bacteria for the current study. Egg aspirates were plated on chocolate agar and blood agar (Remel, Lenexa, KS, USA) and incubated at 37°C for 24 to 72 h. Single colonies were picked and replated to obtain pure cultures for samples in which mixed cultures were observed. Once pure cultures were grown on agar, a single colony was inoculated into tryptic soy broth and grown overnight for DNA extraction and archiving. Microscopy. We Gram stained each colony type and photographed colonies under a light microscope. For transmission electron microscopy (TEM), we grew bacterial cultures in tryptic soy broth. The culture was transferred to a Formvar-coated copper 200-mesh grid (SPI Supplies, West Chester, PA) for 1 min to allow bacteria to settle and then the culture was blotted with filter paper. The bacteria that adhered to grids were stained with 1% phosphotungstic acid for 1 min, which was removed by blotting with filter paper. Grids were washed with water and then air dried. Images were taken on a JEOL JEM-1200EX TEM operated at 85 kV and with AMT image capture software (version 5.4.2.247) and an Orca (Hamamatsu) 12-bit 1,024- by 1,024-bit charge-coupled-device camera. DNA extraction. We extracted DNA from 25 ␮l of egg aspirate samples by using a Qiagen DNeasy blood and tissue kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions for animal tissue samples. Following culture of isolates, we extracted DNA from 1 ml of overnight culture using the same kits but following the manufacturer’s instructions for Gram-negative bacteria. For swab samples, we placed the liquid medium in which the swabs had been collected in a microcentrifuge tube and centrifuged the contents at 5,000 ⫻ g for 10 min. The supernatant was discarded, and the remaining pellet was resuspended in 180 ␮l buffer ATL (tissue lysis buffer); DNA was extracted using the Qiagen kits, following the manufacturer’s instructions for tissue extraction, and stored at ⫺50°C until analysis. PCR and sequencing. We amplified the bacterial 16S rRNA gene by using universal primers (24, 25) to detect and identify bacteria present in egg aspirate and pure culture samples. These primers amplify the bacterial rRNA gene from a wide variety of bacterial species (8,786 of 15,681 fulllength bacterial 16S rRNA sequences as of 2/1/2015, using the Ribosomal Database Project’s Probematch [http://rdp.cme.msu.edu/probematch]). Thermal cycling parameters were as follows: 94°C for 180 s followed by 40 cycles of 94°C for 30 s, 65°C for 60 s, and 72°C for 100 s. A final 10-min, 72°C extension phase was followed by a 4°C indefinite hold. Bacteria were identified via sequencing of the PCR product and aligning using the National Center for Biotechnology Information’s basic local alignment search tool (BLAST). We performed additional PCR and sequencing of the chaperonin 60 (cpn-60) gene by using the primers H529 and H530 (26) to further genetically characterize the Neisseria isolates. This gene has a finer phylogenetic

Microbial Infection of Goose Eggs

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We determined the viability of the embryos via candling, and eggs were checked daily starting 1 day after infection for 7 days (i.e., until embryos were 14 days old). We scored embryos that lost the integrity of their capillary networks as dead (32). A sample of egg contents from each egg was plated on tryptic soy agar on the day of death, or on day 7 postinfection for survivors and controls. All plates were checked daily for bacterial growth for up to 72 h. Bacterial organisms recovered were Gram stained, and DNA was sequenced as described above. We calculated dosespecific mean survival times of embryos inoculated with Neisseria by using the Kaplan-Meier procedure in XLSTAT. Embryos that died within 24 h of inoculation were assumed to have suffered lethal trauma during the inoculation and were removed from the experiment (32). Histopathology. One embryo each from eggs that died at 3, 4, and 5 days postinfection with the novel Neisseria isolates and one viable control embryo were sent to the University of Minnesota Veterinary Diagnostic Laboratory for histopathologic examination. Nucleotide sequence accession numbers. The sequence data obtained in this study have been deposited in GenBank. Putative Neisseria isolate 16S rRNA sequences were assigned accession numbers KF995745 to KF995749, KF999688 to KF999690, KJ596479 to KJ596481, KF999694 to KF999695, KJ596482, and KF999697 to KF999705. Putative Neisseria isolate cpn-60 sequences were assigned accession numbers KJ508837 to KJ508856. Sequences obtained using apparent Neisseria-specific PCR (cpn-60) were assigned accession numbers KM233718 to KM233764. Other isolate sequences were assigned accession numbers KJ652676 to KJ652699. The Neisseria canis cpn-60 sequence can be found under GenBank accession number KJ872773.

RESULTS

Samples collected. We monitored a total of 237 white-fronted goose nests during the 2013 field season and identified at least one nonviable egg (addled or infertile) in 41 nests (17%); this equated to a total of 8.6% of all eggs. Among nests identified with nonviable eggs, the majority contained a single addled egg (58.5%) and the remainder contained either a single infertile egg (9.8%), multiple addled eggs (9.8%), multiple infertile eggs (12.2%), or a combination of addled and infertile eggs (9.8%). In all, 36 addled eggs were collected from 28 nests and 17 infertile eggs were collected from 13 nests. The contents of addled eggs differed markedly from infertile eggs in visual appearance (Fig. 1). Addled eggs showed failure of the perivitelline membrane resulting in loss of the distinction between yolk and albumen, and their texture varied from thin and serous to thick and caseous. The color of addled egg contents varied from yellow to green to gray in color, whereas infertile eggs were characterized by a distinct yellow to orange yolk and transparent albumen. Microbiology, PCR, and sequencing. In total, 29 of 36 addled eggs and 4 of 17 infertile eggs were positive by PCR and/or culture for one or more species of identifiable bacteria. Thirty of 36 aspirate samples from addled eggs were PCR positive for the bacterial 16S rRNA gene. Of those positives, 12 yielded sequence information and the remainder contained double peaks at most base pairs and were considered uninterpretable; in most of these, we later detected multiple bacterial sequences after culture (Table 1). Six of 17 aspirate samples from infertile eggs were PCR positive for 16S rRNA, but none yielded sequence information or grew in culture. Twenty-six addled eggs had at least one type of colony growth on blood and/or chocolate agar, and all 16S rRNA sequences generated from pure cultures were interpretable (Table 2). From these combined egg aspirate and culture PCR results, 23 Neisseria sequences were identified; 21 were detected from pure cultures and 2 were isolated directly from egg aspirates (eggs that

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resolution than the 16S rRNA gene (reviewed in reference 26). Thermal cycling parameters were as follows: 94°C for 180 s, followed by 40 cycles of 94°C for 30 s, 50°C for 60 s, and 72°C for 60 s. A final 10-min 72°C extension phase was followed by an indefinite 4°C hold. Additionally, we designed primers cpn2F (5=-AGCCGGTACCTGAA AAGTCA-3=) and cpn2R (5=-ACAGGCAGCAAATCACGGATA-3=) to amplify a 304-bp portion of the cpn-60 gene in the Neisseria isolates detected in this study. This Neisseria-specific PCR was used on the swab samples in an attempt to identify the source of the bacteria. All egg aspirate samples were also tested using this assay in order to provide greater detection sensitivity for Neisseria in cases in which other bacterial species may have dominated cultures or Neisseria was present at very low levels. Thermal cycling protocols were as follows: 94°C for 3 min, followed by 40 cycles of 94°C for 30 s, 68°C for 60 s, and 72°C for 30 s. A final 10-min 72°C extension step was followed by an indefinite 4°C hold. For all PCRs we used Illustra PuRe Taq ready-to-go PCR beads (GE Healthcare, Pittsburgh, PA, USA), and reactions were performed in an MJ Mini personal thermal cycler (Bio-Rad, Hercules, CA, USA). Escherichia coli DNA was used for the positive controls for 16S rRNA and cpn-60 gene PCRs. Putative Neisseria DNA (isolated from one of the sample eggs) was used as a positive control in the Neisseria detection PCRs. Nuclease-free water was used as a negative control for all reactions. The 16S rRNA gene was not amplified in any run of the Neisseria detection PCR. Sanger sequencing of PCR products was performed by Elim Biopharmaceuticals (Hayward, CA). The 16S rRNA gene PCR products were initially sequenced using primer R1 (5=-GWATTACCGCGGCKGCTG-3=), which amplifies approximately the first 500 bp of the 16S rRNA gene (27). We used additional primers to obtain a full-length 16S rRNA gene sequence for some of the Neisseria isolates: primers F1 (5=-GAGTTTGATC CTGGCTCAG-3=) (28), F2D (5=-GATTAGATACCCTGGTAG-3=) (29), and R2B (5=-CTTGTGCGGGCCCCCGTCAATTC-3=) (30). cpn-60 sequences were obtained using primers M13F and M14B (31). Sequences were manually inspected using Ridom Trace Edit (http://www.ridom.de /traceedit) and trimmed to remove poor-quality base scores at the ends of each sequence. Sequences were considered uninterpretable if more than one peak was present at each nucleotide position on the chromatogram. To improve coverage and generate consensus sequences for samples sequenced with multiple primers, sequences were aligned using Clustal omega (http://www.ebi.ac.uk/Tools/msa/clustalo/). Chaperonin 60 and 16S rRNA gene sequences from other species in the order Neisseriales were obtained from GenBank. Phylogenetic trees were generated using 2,000 bootstrap iterations after aligning and trimming sequences using SeaView (http://www.molecularevolution.org/software/alignment/seaview). Because cpn-60 sequence data are not available publicly for Neisseria canis (a close relative of the Neisseria isolate), we obtained and sequenced N. canis type strain H6 (ATCC 14687) from the American Type Culture Collection (Manassas, VA, USA) as described for all other isolates. Embryonated egg infections. We inoculated chicken embryos with the bacteria isolated most commonly from eggs by using methods described by Nix et al. (32). Two of the Neisseria isolates and one isolate each of Macrococcus caseolyticus, Streptococcus uberis, and Rothia nasimurium were grown to the late log phase (optical density at 600 nm, 0.95 to 1.05) and diluted in phosphate-buffered saline (PBS) for injection. Inoculating doses were determined through serial dilution and CFU counts; five 10fold dilutions, beginning with 100 CFU, were prepared from each isolate. Inoculating doses were within ranges reported to have been used by others (32, 33, 34). One-day-old fertilized White Leghorn chicken eggs obtained from Charles River Laboratories (Wilmington, MA) were incubated at 37°C with high humidity and mechanically tilted to a 45° angle every hour for 7 days prior to infection and throughout the experiment. Five eggs were inoculated with each dilution of each strain (125 total eggs), and 9 control eggs were inoculated with PBS. Eggshells were punctured at the air sac end, and 100 ␮l of inoculum was injected under the chorioallantoic membrane with a tuberculin syringe. After injection, the shells were sealed with a drop of Elmer’s School glue (Elmer’s Products Inc., Columbus, OH).

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TABLE 1 Partial 16S rRNA gene PCR results from egg aspirate samplesa Database match (% identity)

PCR result

n

BLASTn

RDP

Addled

⫹

6

Addled

⫹

5

Neisseria animaloris or N. canis (95–96) N. animaloris (96 –97)

Addled Addled Addled Infertile Infertile

⫹ ⫹ ⫺ ⫹ ⫺

1 18 6 6 11

Helcococcus ovis (91) Uninterpretable – Uninterpretable –

Bacterium “New Zealand A” (99–100) Bacterium “New Zealand A” (99–100) H. ovis (68) Uninterpretable – Uninterpretable –

a

Primers F2C and R2C were used for PCR; primer R1 was used for sequencing. Sequences with more than one peak at each base pair were considered uninterpretable and likely resulted from the presence of more than one species of bacteria. ⫺, sample was PCR negative.

had no growth on blood or chocolate agar). BLAST alignment results using partial 16S rRNA gene sequences identified N. canis and N. animaloris as the closest matches to the Neisseria isolates from this study. The highest identity score for any of these isolates was 97%. The Neisseria-specific PCR assay results were positive for all samples from which we cultured Neisseria and/or obtained a Neisseria sequence. Neisseria DNA was also detected in six additional addled eggs and four infertile eggs with this assay; however, these eggs did not yield culturable Neisseria. Macrococcus caseolyticus was identified in six addled eggs, and Streptococcus uberis and Rothia nasimurium were each identified in four addled eggs; Shigella flexneri and Staphylococcus sciuri were each identified in two addled eggs, and the remaining five bacterial species were each detected in a single addled egg (Table 2). Cloacal and nest swabs. We analyzed a total of 91 swab samples using the Neisseria-specific assay (Table 3). Most nest and eggshell swabs collected during incubation were positive for Neisseria DNA, and there was no difference in frequency of Neisseria DNA occurrence in nest contents between nests containing addled eggs (20 of 24) and control nests without addled eggs (15 of

No. of addled eggs with Neisseria

Timing, type of sample, and swab result

n

Preincubation samples (eggshell/nest combination) Eggshell positive/nest positive Eggshell negative/nest positive Eggshell negative/nest negative

12b 4 1 7

Incubation samples (egg and nest material) Swabbed with addled egg in nestc Positive Negative Swabbed as control (no addled egg) Positive Negative

24 20 4 15 15 0

Hatching samples Cloacal swabsd Positive Negative

28 4 24

0 1 0

15 2 0 0

0 2

a The PCR assay amplified a 304-bp segment of the chaperonin 60 gene (cpn-60) and was specific for Neisseria isolated from addled eggs. b The preincubation samples were from 12 nests, 12 clutches. c Two nests were also swabbed during preincubation, and one was positive for Neisseria DNA. d Two cloacae swab samples were from nests that were swabbed during preincubation.

15) (Table 3). Of the 12 paired swabs collected prior to the onset of incubation, 4 sets of both eggshell and nest material tested positive for Neisseria DNA. However, none of those originated from a nest that later contained a Neisseria-infected addled egg. One preincubation nest swab was Neisseria positive, and that nest later contained an addled egg from which Neisseria was isolated; the eggshell swab from that sample was negative. Of 28 cloacal swabs collected at hatch from white-fronted goose females, 4 swabs were positive for Neisseria DNA, but none was from females known to

TABLE 2 Pure culture partial 16S rRNA gene BLASTn sequence matches for isolates obtained from addled eggsa No. of addled eggsb 5 3 3 2 2 1 1 1 1 1 1 1 1 1 1 11 a b

BLASTn result (% identity) Match 1

Match 2

N. animaloris or N. canis (95–96) N. animaloris (96 –97) N. animaloris (96 –97) N. animaloris (96 –97) N. animaloris (97) N. animaloris or N. canis (96) N. animaloris or N. canis (97) N. animaloris or N. canis (96) N. animaloris (96) N. animaloris (96) N. animaloris or N. canis (96) S. uberis (100) M. caseolyticus (98) S. uberis (100) S. flexneri (99) No growth

M. caseolyticus (98) S. uberis (99 –100) R. nasimurium (98 –99) R. nasimurium (97) R. nasimurium (97) M. caseolyticus (97) M. caseolyticus (98) Paracoccus yeei (98) Stenotrophomonas rhizophila (100) S. sciuri or Staphylococcus vitulinus (99) Serratia fonticola (99) Shigella flexneri (99)

Match 3

Ottowia thioxydans (96) Moraxella cuniculi (98) Staphylococcus sciuri (99)

Primers F2C and R2C were used for PCR; primer R1 was used for sequencing. No bacterial growth was detected in infertile eggs. Number of addled eggs that contained the indicated species.

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Status

TABLE 3 Bacteriologic swab results for samples collected during laying (preincubation), incubation, or hatching of Greater white-fronted geese (Anser albifrons) at Point Lonely, AK, in 2013a

Microbial Infection of Goose Eggs

have incubated a Neisseria-infected addled egg. In two cases, we detected Neisseria-infected addled eggs from the nests of females with negative cloacal swabs. Morphology. Gram staining of the Neisseria isolates derived from culture revealed Gram-negative cocci (Fig. 2a). Transmission electron microscopy of the Neisseria-like organism revealed the morphology of the culture to be diplococcic (Fig. 2b) with a diameter of approximately 500 nm. Neisseria phylogenetics. The neighbor-joining tree constructed from full-length (1,298 to 1,448 bp) 16S rRNA gene sequences clearly showed the Neisseria isolates obtained from whitefronted goose eggs located in a distinct cluster but near N. canis and others in the family Neisseriales (Fig. 3). Similarly, the neighbor-joining tree derived from 406-bp cpn-60 sequences demonstrated clustering of the Neisseria isolates from this study (Fig. 4), and both trees indicated some genetic variation among the Alaskan isolates. Embryonated egg infections. The majority of embryos from eggs infected with the Neisseria isolates died by day 7 postinfection, although survival varied by inoculation dose; all embryos inoculated with greater than 10,000 CFU died by day 5, while 10% of embryos inoculated with 1,000 CFU and 30% of eggs infected with 100 CFU survived the 7-day trial (Fig. 5). All eggs inoculated with S. uberis (100 to 104 CFU) died by day 4 postinfection. All eggs inoculated with 106 CFU of R. nasimurium died by day 4; those inoculated with 105 or 106 CFU died by day 5, those inoculated with 103 CFU died by day 4, and 1 egg inoculated with 102 CFU R. nasimurium survived until 7 days postinfection. All eggs inoculated with 106 CFU of M. caseolyticus died by day 5. Eggs inoculated with all other dilutions of M. caseolyticus (105 to 102) died by day 7 postinfection. All control eggs survived the 7-day trial following inoculation with PBS. Bacteria were not recovered from embryos that survived infection with any of these bacterial species. Histopathology of embryos inoculated with Neisseria showed marked underdevelopment, tissue degeneration, and necrosis. Organ, tissue, and cellular details were obscured by cellular infil-

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trates. Bacterial colonization with Gram-negative cocci was present in 2 of 3 embryos. DISCUSSION

Our results suggest that embryo mortality in white-fronted goose eggs on the Arctic Coastal Plain of Alaska is caused by pathogenic bacteria. Of the 11 species of bacteria detected, the most prevalent was a single species of Gram-negative cocci in the Neisseria genus. Macrococcus, Streptococcus, and Rothia were also somewhat common and often occurred as coinfections with Neisseria. Most of the eggs from which we isolated bacteria were observed in normal development (1 to 18 days incubation) prior to becoming addled, and experimental egg inoculation using bacterial isolates obtained from our samples resulted in high rates of mortality in developing chicken embryos. Microbial infection can cause avian embryo mortality, and bacteria from eggshells and contents of nonviable eggs have been isolated from a range of avian species (10, 33–36). Our identification of a Neisseria species as the predominant bacterium present in addled eggs appears to be novel, although species in the Neisseria genus have been associated with waterfowl on several occasions. Neisseria animalis was isolated from mallard (Anas platyrhynchos) eggs in the Czech Republic (37). Three isolates that most closely matched N. mucosa, N. canis, and N. meningitidis were isolated from duck feces in New Zealand (38), and N. animaloris was detected in the liver of a shelduck (Tadorna sp.) in China (39). Furthermore, “goose gonorrhea” outbreaks in domestic geese in Hungary may be relevant (40, 41). The family Neisseriales occupies a wide range of habitats, including oral, gastrointestinal, and reproductive tracts of many species (43–47). BLAST results of Neisseria sequences from addled whitefronted goose eggs identified these isolates to be within the Neisseria genus, but species distinction was unclear (42). We also detected some variability between the egg isolates, implying that they did not recently expand from a single clone. This pattern, along with our alignment scores, suggests that the Neisseria we have

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FIG 2 (a) Gram stain of aspirate from an addled egg with small Gram-negative diplococci. (b) Transmission electron micrograph of a Neisseria isolate, showing approximately 500-nm diplococcic organisms with spherical to coffee bean shapes.

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isolated may be a previously undescribed species and deserves further investigation. In addition to the commonly identified Neisseria isolates, three other species of bacteria were isolated from ⬎3 addled eggs. The first, Macrococcus caseolyticus, is typically found in cow’s milk and is not considered a pathogen (48); Macrococcus species have been isolated from the surfaces of eggshells but have not been identified in the contents of nonviable eggs (49). The second, Streptococcus uberis, is a cause of mastitis in cattle (50) and has not previously been detected in bird eggs, although other streptococci are known to be associated with nonviable eggs (4, 35). Finally, Rothia

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nasimurium is most commonly isolated from the upper respiratory tract of pigs and mice (51), and other species in the Rothia genus occasionally cause disease in humans (52). We are unaware of any reports of Rothia species in bird eggs. All of these bacterial isolates are or are related to organisms that are most commonly commensals or opportunistic pathogens (except S. uberis). Given that white-fronted geese spend the nonbreeding season amid landscapes dominated by agriculture in Canada and the continental United States, it is plausible that these isolates originated in those locations, perhaps from association with domesticated animals. Most addled eggs were infected by one or more species of bac-

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FIG 3 Neighbor-joining tree based on full (1,243-bp) 16S rRNA gene sequences. Bootstrap values are shown at nodes and are based on 2,000 replicates. Neisseria sp. isolates from eggs collected at Point Lonely, AK (in red) are denoted by both the GenBank accession number (begins with KF) and our sample number (begins with KH). Additional sequences from the family Neisseriales were obtained from the ribosomal database project (http://rdp.cme.msu.edu).

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Downloaded from http://aem.asm.org/ on July 31, 2015 by UNIV OF TORONTO FIG 4 Neighbor-joining tree based on 406 bp of the cpn-60 gene. Bootstrap values are shown at nodes and are based on 2,000 replicates. Point Lonely, AK, Neisseria isolates (in red) are denoted by both GenBank accession number (begins with KJ) and our sample number (begins with KH). Additional sequences from the family Neisseriales were obtained from the Cpn database (http://www.cpndb.ca/cpnDB/home.php).

teria, and several (n ⫽ 4) did not yield bacterial DNA or bacterial growth upon culture. This implies that other causes may be responsible for some of our documented embryo mortality or that bacteria were present but occurred below levels of detection. It is also plausible that in some instances only one compartment of an egg was infected (yolk, albumen, or embryo) and, due to the lim-

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ited volume aspirated, bacteria may not have been sampled. For example, Cook et al. (4) isolated bacteria from different egg compartments, and not all species were present in the same compartments. Our inoculation experiment demonstrated the apparent pathogenicity of this Neisseria isolate and Macrococcus caseolyticus, Streptococcus uberis, and Rothia nasimurium to chicken em-

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bryos. Even at high doses, inoculation with nonpathogenic bacteria is not lethal to embryos (32, 33, 55, 56); in contrast, most embryos (70 to 100%, depending upon isolate and dose) in our experiment did not survive the 7-day experimental period. Although laboratory inoculations did not exactly match the conditions under which white-fronted goose embryos are exposed in the wild, our results demonstrate the ability of bacterial isolates from addled goose eggs to kill developing embryos. Most studies of avian embryo mortality from microbial pathogens have focused on trans-shell infection as the primary route of transmission (4–7). However, some species of bacteria (e.g., Salmonella, Campylobacter, and Mycoplasma species) are known to infect embryos prior to laying via direct contamination with infected reproductive organs (15, 16, 53). We attempted to identify the possible source and route of transmission of the commonly isolated Neisseria sp. by analyzing swab samples from eggshells, nest contents, and cloacae of nesting females. Nearly all eggshell and nest swab samples obtained during incubation tested positive for Neisseria, indicating that this bacterium is widespread in the nest environment. Based on this finding, we suspect trans-shell infection is a probable mechanism. However, we also detected Neisseria in some cloacal swab samples, meaning that female geese could infect eggs prior to laying, that they may transmit bacteria to the nest environment, or both. Thus, the route of infection with Neisseria is unclear, but our results, combined with the fact that other species in the same genus tend to be oral and gastrointestinal tract commensals, imply that nesting white-fronted geese are a likely source. However, we did not observe perfect concordance between the presence of Neisseria in nesting females and addled eggs in their nests. In two instances, cloacal swabs were negative for Neisseria DNA but Neisseria-infected eggs were identified in these nests

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earlier in the nesting season. This may mean that infected females pass bacteria to their eggs, either vertically or through contamination of the eggshell, but can later clear the infection (in this study, cloacal swabs were collected up to a month after egg laying). Or, in the case of possible vertical transmission, the bacteria may originate higher in the reproductive tract and thus not be available for detection via swabs of cloacae. Conversely, not all eggs associated with Neisseria-contaminated nests or females were addled, implying that transfer to eggs or through the eggshell is not ubiquitous or that, in some cases, white-fronted goose eggs may be resistant to low levels of infection. Based on our inoculation study, postinoculation survival time was negatively related to inoculation doses, and some eggs infected at low doses did survive. Survival to hatching of bacteriainfected embryos has been observed in both captive and wild waterfowl (14, 16, 37, 40), and surviving young can sometimes act as carriers of pathogenic bacteria (14, 40). It is also possible that white-fronted goose eggs are more resistant to bacterial infection than the chicken eggs used in our study, or that our inoculation doses were higher than is typical in the wild. Our observed rate of nonviable eggs (8.6% of all eggs monitored) in white-fronted geese on the Arctic coast of Alaska appears unusually high compared to rates for waterfowl at northern latitudes (18). Recent results from a study using experimental mallard nests suggested that bacterial infection of waterfowl eggs may have no effect on hatchability (37). In contrast, our results provided evidence that bacterial infection is a mechanism of embryo mortality and may have been responsible for the elevated rates of nonviable eggs reported here. However, it is unclear whether our findings are unique to the Arctic and to white-fronted geese, or if our results represent a more widespread phenomenon. It is plausible that the high rate of embryo mortality we observed is related

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FIG 5 Kaplan-Meier survival curves and mean survival data for embryonated chicken eggs infected with two different strains of Neisseria isolates from Point Lonely, AK. Percent survival is shown on the y axis, and days postinfection are shown on the x axis. N2 to N6 indicate the Neisseria inoculation dose (N2, 102 CFU; N3, 103 CFU; N4, 104 CFU; etc.). Five eggs were inoculated with each dilution of each strain of Neisseria.

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10.

11.

12.

13.

14.

ACKNOWLEDGMENTS This work is part of the U.S. Geological Survey (USGS) Wildlife Disease and Environmental Health program and the USGS Changing Arctic Ecosystem Initiative and is supported by funding from the USGS Ecosystem and Environmental Health mission areas. Any use of trade names is for descriptive purposes only and does not imply endorsement by the U.S. Government. We thank the field crew at Point Lonely (Mike Johnson, Tim Spivey, and Curtis Twellmann) for help collecting samples and Ryan Adam for technical support in the lab. Paul Flint provided valuable assistance with project design. John Pearce and Paul Flint provided helpful comments that improved the manuscript. Histopathology was conducted by Anibal Armién at the University of Minnesota Veterinary Diagnostic Laboratory, and transmission electron microscopy was performed at the Advanced Instrumentation Laboratory at the University of Alaska Fairbanks.

16.

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18. 19.

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to the recent 3-fold increase in abundance of white-fronted geese (and simultaneous increase in abundance of other goose species) on the Arctic Coastal Plain (20). This dramatic increase in density may have facilitated the maintenance and spread of novel pathogens to which preexisting immunity was lacking (54). Alternatively, potential bacterial egg pathogens may be normally present in white-fronted geese, but conditions unique to the Arctic promote increased egg mortality. Elucidation of these hypotheses requires further investigation into the source(s) of bacteria and identification of the route (i.e., trans-shell versus vertical) of embryo infection. Sampling for bacteria in addled eggs over a broader geographic area, and from additional avian taxa, would provide useful information for determining the current geographic and host distribution of Neisseria and other potentially pathogenic egg bacteria. Further, genetic characterization of additional Neisseria isolates may provide critical information for understanding the evolutionary history of this bacterium in the Arctic and among host species.

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