Amplicon pyrosequencing of wild duck eubacterial microbiome from a fecal sample reveals numerous species linked to human and animal diseases

Our investigation into the composition of the wild duck, , Aythya americana eubacterial microbiome from a fecal sample using amplicon pyrosequencing revealed that the representative bacterial species were quite distinct from a pond water sample, and we were able to classify the major operational taxonomic units with , , Fusobacterium mortiferum Streptobacillus moniliformis , , Lactobacillus intermedius Actinomyces suimastitidis Campylobacter , , , ., Canadensis Enterococcus cecorum Lactobacillus aviarus Actimomyces spp . representing the majority of Pseudobutyrivibrio spp and Helicobacter brantae the eubacterial fecal microbiome. Bacterial species present in the analysis revealed numerous organisms linked to human and animal diseases including septicemia, rat bite fever, pig mastitis, endocarditis, malar masses, genital infections, skin lesions, peritonitis, wound infections, septic arthritis, urocystitis, gastroenteritis and drinking water diseases. In addition, to being known carriers of viral pathogens wild ducks should also be recognized as a potential source of a range of bacterial diseases. 1 2 1

Throughout the history of medicine there has been an awareness of animal to human transmission of disease, and the etiological pathogens have been collectively described as zoonoses 1 .Water fowl and wild birds have been identified as reservoirs for the virus Influenza A 2,3 ; a highly mutable and infectious pathogen that infects avian and mammalian species 4 .Ducks are observed in a multitude of fresh water sources including ponds, water fountains and pools where they can defecate; bacteria have been shown to be distributed through aerosols from ornamental fountains 5,6 and reclaimed water dispensed through an irrigation system 7 .Humans may also have direct contact with ducks and their excrement through the recreational sport of duck hunting 8 .Ducks can also shed pathogens near chicken farms or other animals-such as pigs-that have access to outside areas.An avian influenza A virus (H7N7) epidemic in the Netherlands in 2003 thought to be initiated from a migratory water fowl resulted in the culling of 30 million poultry in an area of the country where freerange poultry farming was common 9 .Due to the migratory nature and unrestrained behavior of the wild duck, Aythya Americana, our study set out to investigate the bacterial microbiome of a wild duck and to identify its bacterial flora relative to the same bacterial species that have been reported to cause disease in farm animals and humans.

Methods
Amplicon pyrosequencing (bTEFAP) was originally described by Dowd et al. 10 and has been used in describing a wide range of environmental and health related microbiomes including the intestinal populations of a variety of animals and their environments including cattle [11][12][13][14][15] .A fecal sample obtained from a wild duck, Aythya americana, that was killed during duck hunting season (December 2012) by a licensed hunter, was aseptically swabbed onto a Whatman FTA card (GE Healthcare Life Sciences) using a sterile swab and gloves being careful to avoid environmental contamination.The flap of the FTA card was placed over the FTA paper and placed into a sterile pouch, and the FTA card was stored at room temperature prior to DNA amplification.2 mm punches were washed with FTA reagent and TE (10 mM Tris-HCL, 1 mM EDTA, pH 8.0) according to the manufacturer's protocol, and the dried punches were used as template DNA for thermal cycling.DNA was also isolated from pond water as a negative comparison and sampled from a source of water visited by numerous avian species but not at the source of the fecal sampling but within the migratory range of Aythya americana.The pond water DNA was isolated using water RNA/DNA purification kit (0.45 µm) [Norgen Biotek Corp, Thorold, ON, Canada].For thermal cycling and DNA amplification we used the 16S universal Eubacterial primers 27f 5´-AGAGTTT-GATCCTGGCTCAG-3´ and 1492r primer 5´-ACGGCTACCTT-GTTACGACTT-3´ (Integrated DNA Technologies).A single-step 30 cycle PCR using EconoTaq PLUS 2X Master Mix (Lucigen, Middleton, WI) were used under the following conditions: 94°C for 2 minutes, followed by 30 cycles of 95°C for 120 seconds; 42°C for 30 seconds and 72°C for 4 minutes; after which a final elongation step at 72°C for 20 minutes was performed.Following PCR, DNA products were resolved in a 1% agarose, 1X TAE gel stained with ethidium bromide and 1.5 Kb products were excised from the gel purified using a cyclo-prep spin column (Amresco, Solon, OH).All the DNA products were purified using Agencourt Ampure beads (Agencourt Bioscience Corporation, MA, USA).Samples were sequenced using Roche 454 FLX titanium instruments and reagents following manufacturer's guidelines.The Q25 sequence data derived from the sequencing process was processed using a proprietary analysis pipeline (www.mrdnalab.com,MR DNA, Shallowater, TX).Sequences were depleted of barcodes and primers.Next, short sequences < 200bp, sequences with ambiguous base calls, and sequences with homopolymer runs exceeding 6bp were removed.Sequences were then denoised and chimeras removed.Operational taxonomic units (OTUs) were defined after removal of singleton sequences, clustering at 3% divergence--97% similarity 10,15 .OTUs were then taxonomically classified using BLASTn against a curated GreenGenes database 16,17 and compiled into each taxonomic level into both "counts" and "percentage" files.

Results
Due to the aquatic nature of the animal, we initially expected that the biodiversity of bacterial species in the duck feces would reflect numerous bacterial species present in the pond water, and since we observed multiple species of aquatic birds in the pond we expected to find eubacteria in common.Figure 1 is a modified heat map showing differences and similarities among the classes of eubacteria sequenced and identified.The figure demonstrates clear differences at the taxonomical level of Class with few common classes of bacteria namely Actinobacteria, Clostridia and Gammaproteobactera.
However, similarities at the level of Genus and species included only Agrobacterium tumefaciencs and a species of Porphyromonas and a species of Ruminococcaceae (Figure 2).This analysis indicated distinct differences between the eubacteria present in the duck fecal sample and the pond water sample, and it also indicated that our sampling of the duck feces was devoid of any obvious pond water eubacterial constituents.
The taxonomical classification of OTU at the level of genus and species was compiled in relation to percentages of the Eubacterial microbiome (Table 1).In Table 2, we referenced reported cases of diseases related to the bacteria sequenced from the duck's fecal sample reflecting the eubacterial microbiome's potential to cause disease in humans and other mammals.The largest representative bacterial species-relative to percentage-was Fusobacterium mortiferum at 31.6%.Fusobacterium mortiferum reports related to human disease are sparse, but Fusobacterium have been associated with rare but serious cases of bacteremia 18,19 , and a 6 year study of "other gram-negative anaerobic bacilli" (OGNAB) isolated from anaerobic infections at the Wadsworth Clinical Anaerobic Clinical Anaerobic Bacteriology Research Laboratory in Los Angeles, CA reported that most strains of Fusobacteria-outside of Fusobacterium nucleatum-were resistant to erythromycin 20 .The pathogen, Fusobacterium nucleatum, on the other hand, is well-known for its association with disease and its ability to adhere to Gram-positive and Gram-negative bacteria in dental biofilms such as plaque 21 .
Streptobacillus moniliformis was also identified as a major constituent of the duck fecal eubacterial microbiome at 30.1%.Several well-studied and documented cases of disease are attributed to S. moniliformis including rat bite fever or Haverhill disease 22 , osteomyelitis 23 , epidural abscesses 24 , fever and polyarthralgia 25 , bacteremia 26 and contaminated drinking water related disease 27 .Other organisms and their respective illnesses included Lactobacillus intermedius (11.02%) in a renal transplant infection 28 , Actinomyces suimastitidis (4.47%) in pig mastitis 29 and Campylobacter canadensis (3.69%) in drinking water related disease 27 .Enterococcus cecorum was another identified pathogen at 3.59% of the sequenced Eubacterial microbiome, and E. cecorum has been reported to cause disease in chicks 30,31 and humans including aortic valve endocardititis 32 , empyema thoracis 33 , septicemia in a malnourished adult 34 and recurrent bacteremic peritonitis in a patient with liver cirrhosis 35 .Actinomyces odontolyticus (0.70%) has recently been reported to cause bacteremia in immunosuppressed patients 36 , and members of the genus Actinomyces have been known to cause actinomycosis for some time.A. odonolyticus was reported by Michell, Hintz and Haselby in 1997 to be the cause of a malar mass in soft tissue in a human 37 .A species of the genus Leptotrichia (0.36%) was also identified, a genus that has been associated with bacteremia in multiple myeloma

Discussion
Numerous pathogenic eubacterial species have been identified in the fecal sample obtained from the wild duck, Aythya Americana, using amplicon pyrosequencing, a widely accepted method for analyzing the bacterial composition of microbial ecosystems.We were surprised to find that most of the species of eubacteria sequenced the duck feces were not present in a pond water sample from a water source that was known to be visited by numerous water fowl.Perhaps, the analyses of small samples from a pond or lake are not adequate when investigating the presence of avian contamination.that are produced when the reclaimed water is used as a source of irrigation such as in golf courses and gardens, a common practice that might warrant further inquiry.

The summary in
When determining the cause of disease it is difficult-if not impossible-to identify the source of infection, and whether it has indeed originated from an animal that is migratory or aquatic in nature.Many of the bacterial species that were cited to cause infections among humans were also found in the excrement of a migratory and aquatic bird that defecates in water supplies and around other animals.However, since our analysis was limited to the careful analysis of a single, wild duck's eubacterial microbiome, the disease potential was relative to that animal only and cannot be extrapolated to all ducks of the same species.Thus, the disease possible that many of the bacterial entities when disseminated to humans and other animals could also cause subclinical respiratory illnesses that are not reported due to patient resolution.
It is only prudent to recommend that immunocompromised humans and animals should limit their exposure to environments where ducks may have polluted the water source-this includes outdoor pools and fountains.That realization also supports the practice of adequately chlorinating or sanitizing artificial pools and fountains to prevent opportunistic infections through aerosols or breaks in the skin.Duck hunters should also be aware of the risk of bacterial contamination in addition to the risk posed by the influenza virus.Additionally, reclaimed water poses a threat to the elderly and other immunocompromised humans who might be exposed to aerosols

F1000Research
to know why these bacteria are in the bird, and what they do there -are they commensals, or pathogens?What has been found in other bird species?This is not discussed in the present version.
Taken together, the data should be considered with some caution as it is based on such a small sample set and conclusions from comparisons should therefore be toned down.Preferentially, the study should be extended to include more avian samples.
Minor things: Title: Very little in this article actually refers to animal disease, which makes the title slightly misleading.
Introduction: It would be good if the host could be put in a larger perspective here, and that previous work on ducks as carriers of human-associated bacterial pathogens are cited to a larger extent.The ornamental/decorative fountains are unlikely significant duck habitats.  .focuses on the description of the bacterial community of a duck fecal sample.In et al the study the authors analyzed 16S rDNA pyrosequencing data to determine the identity and diversity of the bacteria present in the sample.They also collected water samples and performed similar studies in order to determine if the water was influenced by the duck staging over the area were the sample was collected.The presence of pathogenic bacteria in avian feces is well documented, although most of the work has been conducted with a relatively limited number of species.It is also known that birds can influence the microbial quality of surface waters.Thus any additional data on the occurrence and relative F1000Research influence the microbial quality of surface waters.Thus any additional data on the occurrence and relative abundance of potential pathogens from birds and their relationship to human health risks is welcomed.
The study, as presented, suffers from some relevant limitations and in my opinion the data should be considered preliminary in nature.Perhaps the most important one is the low number of samples analyzed (n=1) for the fecal and water samples.Thus in this regard it would be difficult to determine if this sample is representative of the duck fecal microbiota or not.It is also not clear how many sequences were analyzed in this study and which area of the 16S rRNA gene was used in the analysis.The authors mentioned that they amplified the entire 16S rRNA gene but normally such large amplicons are not used for next generation sequencing.To this reviewer it seems that there is some details missing on the method, e.g.perhaps the sequencing facility performed a second amplification with primers that generated a smaller PCR product which was compatible with the sequencing technology.There are many other details on the methods used that are not clear, such as how long was the fecal sample at room temperature before it was processed, holding time for water sample before processed, the total volume of water filtered and how it was filtered.They also collected and analyzed one water sample on one data, which again it would be difficult to be representative of the pond.Typically, more samples would be needed to arrive at any conclusions.
There is also the issue of using relatively short fragments of the 16S rRNA to accurately identify a bacterium at the species levels.What was the length of the sequences used in each of the cases and what was the sequence identity for each of the sequences that were identified as closely related to the identified pathogens?The authors would benefit from performing additional assays to further confirm the presence of potentially pathogenic populations.16S rDNA-based assays might be available for some of the "pathogens" identified but preferably functional genes should be used if considered for such an exercise.The authors should also considered to compare this dataset (albeit limited in scope) with other studies in which 16S rDNA sequencing information has been used to describe avian fecal microbiota.
As a minor comment, there are two data points in Figure 1 (which actually is a table) for Fusobacteria.
I have read this submission.I believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.
No competing interests were disclosed.

Competing Interests:
Author Response 06 Nov 2013 , Barry University, USA

Jonathan Coffman
The method for pyrosequencing including the variable regions of the 16S rRNA gene was referenced and previously described by: We have obtained additional samples from and sequenced the samples using Aythya americana ion torrent sequencing and made a rarefaction graph.
We understand we had a limited sample size and are trying to increase it, despite being hindered by rather limited resources and time constraints.

Figure 1 .
Figure 1.Comparison of Classes of Eubacteria present in the Duck to the Classes of Eubacteria present in pond water using a modified heat map.Darker colors represent a higher representation of the bacterial class.

Figure 2 .
Figure 2. Bacterial species present in both duck feces and pond water.

Figure
Figure 1: Are the numbers referring to percentages?And is it not more a table than a figure?

Table 1 . Taxonomical classification of operational taxonomic units into the Genus/species level with representative percentages of the Eubacterial Microbiome.
Another Actinomyces present in the wild duck eubacterial microbiome was Actinomyces turicensis at 0.3%, a bacterium associated with a spectrum of diseases including genital infections, urinary tract infections, skin infections, post-operative wound infection, abscesses, appendicitis, ear and nose and throat infection and bacteremia 39 .In addition, Actinomyces europaeus (0.14%) was reported in human abscesses40, Actinomyces neuii (0.03%) was reported to cause endophthalmitis 41 and periprosthetic infection 42 , Actinomyces vaccimaxillae (0.01%) was isolated from a cow jaw lesion43and Actinomyces hongkongensis (0.004%) was reported to cause high-mortality bacteremia in humans44.0.24% of the eubacterial population was composed of Plesiomonas shigelloides a well-documented pathogen associated with Travelers' diarrhea, dysentery and gastroenteritis[45][46][47][48].Arcanobacterium pyogenes was also present (0.18%), a pathogen reported to cause soft tissue infections in humans49.Atopobium vaginae (0.12%) was reported to cause bacterial vaginosis in a human50and Varibaculum cambriense (0.01%) was reported to cause complications with intrauterine devices and vaginal infections in Hong Kong 51 , Parvimonas micra (0.08%) was associated with odontogenic infection 52 and human bacteremia was reported with Atopobium rima 53 , Fusobacterium nucleatum 54 , Corynebacterium freneyi 55 and Streptococcus suis 56 .Finally, Veillonella dispar (0.02%) was reported in a case of septic arthritis 57 and Porphyromonas gingivalis (0.02%) is a well-studied pathogen reported decades earlier and associated with periodontitis 58 .

1 :
Are the numbers referring to percentages?And is it not more a table than a figure?

doi:10.5256/f1000research.2554.r2348 Jorge Santo Domingo Microbial
Contaminants Control Branch, US Environmental Protection Agency, Cincinnati, OH, USA The article of Strong