Nasal Bacterial Microbiome: Probing a Healthy Porcine Family

Upper respiratory tract (URT) infection caused the leading and devastating diseases in pigs. It was believed that the normal microbiome of URT plays a vital role in health and disease development. As the entry point of the URT, little knowledge of bacterial microbiome in porcine nasal was known. A cultivation-independent approach directly to 16s ribosomal RNA genes enabled us to reveal the nasal bacterial community, structure and diversity. Here, we found that an unprecedented 207 phylotypes were characterized from 933 qualified clones, indicating the variable, species richness but particularly dominant bacterial microbiome. The dominant species were from genus Comamonas and Acinetobacter, which constitute core normal bacterial microbiome in porcine nasal. Moreover, a set of swine specific pathogens and zoonotic agents were detected in the swine nasal microbiome. Collectively, we provided a snapshot of our current knowledge of the community structure of porcine nasal bacterial ecosystem in a healthy family that will further enhance our view to understand URT infection and public health.


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Respiratory disease has consistently been reported as one of the most 80 important causes of both morbidity and mortality in post-weaning pigs, as one 81 of the limiting factors of production, causing decreased weight gain, and 82 decreased feed efficiency, an overuse of antibiotics and ultimately respiratory 83 distress and death (Moorkamp et al., 2008;Palzer et al., 2008). There are 84 numerous organisms, both bacterial and viral, that can contribute to respiratory 85 distress in pigs, many can act as opportunistic agents on an already 86 compromised immune system, or can themselves become exacerbated by 87 infection with other opportunistic pathogens. This can result in a more 88 complicated aetiology, which may be associated with higher mortality. Hence, 89 knowledge of aetiology of respiratory disease, as well as host-pathogen 90 interaction, is crucial to understand Upper Respiratory Tract (URT) infection. 91 Defining the normal microbiota of URT is the first step to understand health 92 and disease development. Until recently, knowledge of the nasal bacteria was limited to 113 culture-dependent assays, and it is estimated that less than 1% of bacterial 114 species can be cultivated (Guclu et al., 2007;Smith-Vaughan et al., 2006). The  Broad-range PCR primers that anneal to highly conserved regions flanking the 121 variable regions of the gene allow amplification from the majority of known 122 bacteria, which has led to the identification of microbial diversity and has 123 provided compelling evidence for the existence of hitherto unknown bacteria.

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As a result of increasing challenge in modern farm associated with respiratory 125 disease, we tend to understand bacterial microbiota nasal cavity of modern 126 farming sow and its piglets, evaluate their relationship with potential disease 127 development. Summary the previous data of porcine upper respiratory tract as 128 well as our data, we provide a preliminary picture of the community structure of 129 the nasal bacteria ecosystem and discuss the potential pathogens, and their 130 impact on public health and food safety. were stored at -80°C until further processing.

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All biological specimens were first incubated in a preparation of enzymatic lysis 145 buffer (20 mM Tris at pH 8.0, 2 mM EDTA, 1.2% Triton X-100) and lysozyme 146 (20 mg/mL) for 30 min at 37°C, and samples were incubated overnight at 56°C 147 in Buffer AL and Proteinase K from the DNeasy DNA Extraction Kit (Qiagen).

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The standard protocol for the genomic DNA isolation was followed for steps     Estimations of species coverage, richness, evenness, and diversity were 255 calculated for the combined data set, as well as for three subsets of nasal 256 samples (As showed in Table 1). The Chao1 estimator of total species 257 richness was 439, which based on the distribution of singletons.   specific to human were also found. Among the summary in the table 3, most of 316 them were found specific in sow, while relative small number was in piglet.

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Interesting, we found specie Bacillus anthracis from sow sample, which can be 318 classified as biohazard level-3 pathogen. And host specific specie Shigella 319 flexneri was also found in nasal sample from sow. There were various swine specific pathogens found in swine nasal microbiome.

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Organisms from Pasteurellaceae family were common agents found in porcine 367 UTRs(Moller and Kilian, 1990). Haemophilus parasuis was the most common 368 nasal carriage in pigs, which was found throughout the world and organisms     calculates Shannon-Wiener diversity index; The index is one of several diversity indices used to measure diversity in categorical data. It is simply the Information entropy of the distribution, treating species as symbols and their relative population sizes as the probability; f The estimation of diversity coverage was calculated by Good's method, according to which the percentage of coverage was calculated with the formula (%) = [1-(n/N)] x 100, where n is the number of phylotypes represented by one clone only and N is the total number of sequences.
TABLE 3 Summary the potential pathogens associated with porcine and public health. a, the annotated sequence information was according to the seqmatch method described previously; b, the querying sample sequence identity is according to the N-BLAST identity to the preference sequence; c, the bioharzard level of each species was estimated as described in the methods; d, Zoonoses information of each species was determinated in the methods.