Microbial communities in the tonsils of healthy pigs

doi:10.1016/j.vetmic.2010.06.025

Veterinary Microbiology

Volume 147, Issues 3–4, 27 January 2011, Pages 346-357

https://doi.org/10.1016/j.vetmic.2010.06.025 Get rights and content

Abstract

The tonsils of mammals such as humans and pigs are colonized with an extensive microbiota and are frequently the site for asymptomatic carriage of bacterial pathogens. The goal of this study was to determine the composition of the microbial community of the tonsils in healthy pigs. Tonsils were collected from eight pigs from two different healthy herds. Samples of the tonsils from each pig were used for culture dependent and culture independent identification of the microbial community. Aerobic cultivation identified Pasteurella multocida, Actinobacillus spp., Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus suis, Streptococcus dysgalactiae, and Escherichia coli from ≥50% of the pigs in both herds. For culture independent studies, microbial community members were identified by 16S rRNA sequences using the Ribosomal Database Project Pipeline programs developed at Michigan State University. Dominant genera identified by 16S rRNA analysis in pigs from both herds included Actinobacillus, Haemophilus, Pasteurella, Porphyromonas, Fusobacterium, Bacteroides, and Prevotella. These genera were detected in nearly every pig regardless of herd. In contrast, there was an asymmetric distribution of minor genera between the two herds, suggesting herd-specific differences in the microbial communities. In addition, we demonstrated primer bias between two frequently used forward primers when targeting the tonsillar community. Our results suggest that the major bacterial community members found in porcine tonsils are the same regardless of herd, while the minor species are unique to each herd. This is the first analysis using 16S rRNA sequence libraries of the composition of microbial communities in the porcine upper respiratory tract.

Introduction

The oral cavity, particularly the tonsils, plays an important role in host defense against pathogens. The palatine tonsils function as a secondary lymphoid organ and initiate an immune response to pathogens (Belz and Heath, 1996, Horter et al., 2003, Nave et al., 2001). The tonsils also act as a reservoir for both pathogenic and non-pathogenic bacteria, due mainly to the tubular crypts that extend deep within tonsillar tissue and provide a place for evasion of the immune system (Horter et al., 2003).

Porcine tonsils are a reservoir for some of the most virulent bacterial pathogens of pigs. Actinobacillus pleuropneumoniae, A. suis, Haemophilus parasuis, Pasteurella multocida, Mycoplasma hyopneumoniae, Salmonella spp., and Streptococcus suis are often carried asymptomatically in the tonsils of pigs (Arends et al., 1984, Chiers et al., 2002, Fedorka-Cray et al., 1995, Horter et al., 2003, Kamp et al., 1996, MacInnes et al., 2008, Marois et al., 2008). For many of these pathogens, the porcine upper respiratory tract is the only known reservoir. Other species frequently identified from porcine tonsils include commensal Pasteurellaceae such as A. minor, A. porcinus, A. indolicus and “A. porcitonsillarum” (MacInnes et al., 2008, Møller and Kilian, 1990, Tonpitak et al., 2007) and Gram-positive species such as Streptococcus dysgalactiae, S. porcinus, Staphylococcus aureus, Enterococcus faecalis, E. faecium, Arcanobacterium pyogenes, and Lactobacillus reuteri (Baele et al., 2001, Devriese et al., 1994).

Current knowledge of the microbial community in the porcine tonsils has focused mainly on isolation of specific genera and species rather than on identification of a wide range of microbes. There has not been a comprehensive study of the microbial community of the porcine tonsils. Our goal for this study was to utilize both standard culture dependent as well as new culture independent techniques to identify the tonsillar microbial community members from healthy pigs. This report represents the first culture-independent analysis of the microbial community of porcine tonsils.

Section snippets

Animals and specimen collection

Four 18–20 week old pigs from a high health status herd with no recent history of respiratory disease (herd 1) and four pigs of the same age from a currently healthy herd with a history of chronic but undefined respiratory problems (herd 2) were randomly selected for use in this study. Both herds are located in mid-Michigan. Herd 1 received no vaccinations or in-feed antibiotics. Herd 2 was vaccinated against PCV2, and received Tylan as an in-feed antibiotic. Additionally, herd 2 received

Gross and microscopic examination of porcine tonsils

Porcine palatine tonsils are large, flat, and located on the ventral surface of the soft palate. Large numbers of tonsillar crypts, recognizable as multiple crater-like indentations, identify the porcine palatine tonsils as follicular tonsils. There were no recognizable gross changes in the collected tonsils. Microscopically, the palatine tonsils were covered by non-keratinized, stratified squamous epithelium that extended into the tonsillar crypts. The lower and middle portions of the

Discussion

Until the recent development of culture independent techniques such as sequencing of the gene encoding 16S rRNA, the study of complex microbial communities was extremely difficult. Culture dependent analyses could only identify organisms for which there were well described culture media and incubation conditions. In many studies, it has become apparent that culture failed to identify a significant portion of the community (Amann et al., 1995, Hugenholtz et al., 1998b, Paster et al., 2006). In

Conclusion

Overall, our data agrees with many studies utilizing both culture dependent and independent methods, which suggest that microbial communities are both highly complex and very diverse (Aas et al., 2005, Leser et al., 2002, Paster et al., 2001). To our knowledge this is the first report using 16S rRNA analysis to identify the microbial community of the porcine tonsils. This research provides baseline data for studies of the tonsillar communities in pigs with disease due to known respiratory or

Acknowledgments

This research was supported by grants from the Michigan State University Center for Microbial Pathogenesis and the National Pork Board.

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Research articleMicrobial communities in the tonsils of healthy pigs

Abstract

Introduction

Section snippets

Animals and specimen collection

Gross and microscopic examination of porcine tonsils

Discussion

Conclusion

Acknowledgments

Vet Microbiol

Vet Microbiol

Vet Microbiol

Curr Opin Microbiol

Defining the normal bacterial flora of the oral cavity

J Clin Microbiol

Phylogenetic identification and in situ detection of individual microbial cells without cultivation

Microbiol Rev

Carrier rate of Streptococcus suis capsular type 2 in palatine tonsils of slaughtered pigs

J Clin Microbiol

The Gram-positive tonsillar and nasal flora of piglets before and after weaning

J Appl Microbiol

Tonsils of the soft palate of young pigs: crypt structure and lymphoepithelium

Anat Rec

Effect of pre-operative antibiotic treatment on the bacterial content of the tonsil

Clin Otolaryngol Allied Sci

The Ribosomal Database Project (RDP-II): sequences and tools for high-throughput rRNA analysis

Nucleic Acids Res

Identification and composition of the streptococcal and enterococcal flora of tonsils, intestines and faeces of pigs

J Appl Bacteriol

Alternate routes of invasion may affect pathogenesis of Salmonella typhimurium in swine

Infect Immun

A review of porcine tonsils in immunity and disease

Anim Health Res Rev

Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity

J Bacteriol

Novel division level bacterial diversity in a Yellowstone hot spring

J Bacteriol

A specific and sensitive PCR assay suitable for large-scale detection of toxigenic Pasteurella multocida in nasal and tonsillar swabs specimens of pigs

J Vet Diagn Invest

Research article
Microbial communities in the tonsils of healthy pigs