# data[].attributes.body
S. peroris is a facultative anaerobe, alpha-hemolytic, and Gram + bacteria.

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Phylum Firmicutes, Class Bacilli, Order Lactobacillales, Family Streptococcaceae, Genus Streptococcus, Streptococcus peroris
Kawamura et al. 1998.
Member of the 'Streptococcus mitis group'. Lancefield group was not determined.

Cells are nonsporulating, nonmotile, Gram-positive cocci, grouped in short chains.

Alpha-hemolytic, small (0.3-0.8 mm in diameter) colonies after 48 hour incubation.
Grows at 37 ºC. Facultatively anaerobic. Grows on Trypticase soy agar with 5% sheep
blood.

Isolated from human clinical samples - oral cavity (tooth surface) & pharynx.

1. Yoshiaki Kawamura, Xiao-Gang Hou, Yuko Todome, Ferdousi Sultana, Kenji Hirose, Sin-Ei Shu, Takayuki Ezaki & sp. nov., new members of the Streptococcus mitis group, Streptococcus peroris Streptococcus infantis isolated from human clinical specimens. Int. J. Syst. Bacteriol., 1998, 48, 921-927.

2. Robert A. Whiley and Jeremy M. Hardie, 2009. Genus I. Streptococcus Rosenbach 1884, 22AL. In: (Eds.) P.D. Vos, G. Garrity, Jones, N.R. Krieg, W. Ludwig, F.A. Rainey, K.-H. Schleifer, W.B. Whitman. Bergey's Manual of Systematic Bacteriology, The Firmicutes, Springer, 655-711.

Positive results for alanyl-phenylalanyl-proline arylamidase, acid production from: differential characters:
lactose, maltose & sucrose.

Negative results for alkaline phosphatase, alpha-galactosidase, arginine hydrolysis, catalase, esculin hydrolysis, hippurate

hydrolysis, beta-D-fucosidase, N-acetyl-beta-glucosaminidase, pyrrolidonyl arylamidase, beta-glucosidase, beta-mannosidase,
beta-glucuronidase, glycinetryptophan arylamidase, urease, Voges-Proskauer reaction, acid production from: amygdalin,
L-arabinose, D-arabinose, arabitol, arbutin, cyclodextrin, glycogen, inulin, mannitol, methyl beta-D-glucopyranoside, melibiose,
melezitose, pullulan, raffinose, ribose, sorbitol, tagatose & trehalose.

Variable results for alkaline phosphatase & beta-galactosidase.

Streptococcus infantis Streptococcus peroris

Legend: + more than 85% positive; - more than 85% negative; v variable (16-84% positive).

# data[].attributes.attachment_link.description
Phylum Firmicutes, Class Bacilli, Order Lactobacillales, Family Streptococcaceae, Genus Streptococcus, Streptococcus peroris Kawamura et al. 1998.Member of the 'Streptococcus mitis group'. Lancefield group was not determined. Cells are nonsporulating, nonmotile, Gram-positive cocci, grouped in short chains. Alpha-hemolytic, small (0.3-0.8 mm in diameter) colonies after 48 hour incubation. Grows at 37 ºC.

# data[].attributes.body
Kingdom: | Bacteria
Phylum: | Firmicutes
Class: | Bacilli
Order: | Lactobacillales
Family: | Streptococcaceae
Genus: | Streptococcus
Species: | S. peroris

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S. peroris is most closely related to pathogenic Streptococcus pneumoniae, that can result in several different human infections. S. peroris is less related to Neisseria pharyngis, another fairly pathogenic bacteria to animals.

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There isn't much information available for S. peroris, and the pathogenesis of the bacteria has yet to be determined. It can be found in human mouths and pharynx.

# data[].attributes.body
As mentioned, not much is known about S. peroris. But this study was conducted to observe the flora of an infants mouth from birth to 2 years old. S. peroris is found in the infants mouth until around 30-39 weeks old, where it rapidly decreases.

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ABSTRACT

Newborns are constantly exposed to various microbes from birth; hence, diverse commensal bacteria colonize the oral cavity. However, how or when these bacteria construct a complex and stable ecosystem remains unclear. This prospective cohort study examined the temporal changes in bacterial diversity and composition in tongue microbiota during infancy. We longitudinally collected a total of 464 tongue swab samples from 8 infants (age of <6 months at baseline) for approximately 2 years. We also collected samples from 32 children (aged 0 to 2 years) and 73 adults (aged 20 to 29 years) cross-sectionally as control groups. Bacterial diversities and compositions were determined by 16S rRNA gene sequencing. The tongue bacterial diversity in infancy, measured as the number of observed operational taxonomic units (OTUs), rapidly increased and nearly reached the same level as that in adults by around 80 weeks. The overall tongue bacterial composition in the transitional phase, 80 to 120 weeks, was more similar to that of adults than to that of the early exponential phase (EEP), 10 to 29 weeks, according to analysis of similarities. Dominant OTUs in the EEP corresponding to Streptococcus peroris and Streptococcus lactarius exponentially decreased immediately after EEP, around 30 to 49 weeks, whereas several OTUs corresponding to Granulicatella adiacens, Actinomyces odontolyticus, and Fusobacterium periodonticum reciprocally increased during the same period. These results suggest that a drastic compositional shift of tongue microbiota occurs before the age of 1 year, and then bacterial diversity and overall bacterial composition reach levels comparable to those in adults by the age of 2 years.

IMPORTANCE Evaluating the development of oral microbiota during infancy is important for understanding the subsequent colonization of bacterial species and the process of formation of mature microbiota in the oral cavity. We examined tongue microbiota longitudinally collected from 8 infants and found that drastic compositional shifts in tongue microbiota occur before the age of 1 year, and then bacterial diversity and overall bacterial composition reach levels comparable to those in adults by the age of 2 years. These results may be helpful for preventing the development of various diseases associated with oral microbiota throughout life.

INTRODUCTION

The oral cavity is one of the largest bacterial reservoirs in the human body, and numerous coexisting bacteria construct a complex and stable bacterial community ( 1-4). It is well known that mutans streptococci and the red complex, which includes Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola, are the most important bacteria associated with dental caries and periodontal disease, which are major oral diseases ( 5-9). Additionally, the existence of specific oral bacterial species related to systemic diseases such as obesity, cardiovascular diseases, and pneumonia have been reported in recent years ( 10-16). In contrast, oral dysbiosis (imbalance of overall bacterial composition including oral commensal bacteria and pathogenic bacteria) has attracted attention as an etiology of oral and systemic diseases ( 3, 4, 17-19).

After birth, the oral cavity of newborns is exposed to a wide variety of microbes, and the oral microbiota develops as newborns grow. This development begins with tolerated colonization of specific microbes in the human oral cavity. It is thought that facultative anaerobic bacteria such as Streptococcus and Actinomyces are pioneer colonizers in a newborn's oral cavity; particularly, Streptococcus salivarius is thought to be the most predominant pioneer colonizer ( 20). Although the phylum Firmicutes predominates in both oral and gut microbiota, the genus Streptococcus within the phylum Firmicutes is rarely detected in gut microbiota ( 1). Thus, the predominance of Streptococcus species is characteristic of the first step in forming oral microbiota. It has also been reported that formation of the oral indigenous microbiota begins within the first 6 weeks of life, and Streptococcus rapidly dominates the oral cavity during this stage ( 21).

A recent cross-sectional study investigated subjects with different dentition states, including the predentate stage (age, 0 years) and primary dentition stage (age, 0 to 5 years). The results revealed a drastic expansion of bacterial diversity after tooth eruption ( 22). The authors also suggested that certain bacterial species were acquired in the predentate stage as consistent core species of human oral microbiota. Given that these early bacterial communities, including core species and species that immediately colonize after primary tooth eruption, lead to subsequent colonization as a foundation and affect the formation of adult oral microbiota related to various diseases in later life, it is important to understand bacterial acquisition during this period. However, the detailed characteristics of the acquisition process and temporal shift of oral microbiota composition and diversity during this period remain unclear.

In this prospective cohort study, we focused on the development of tongue microbiota during infancy. We analyzed tongue microbiota collected from 8 infants at short intervals by 16S rRNA gene amplicon deep sequencing and followed the infants' transitions from 6 months to approximately 2 years of age. The aim of this study was to clarify temporal changes in infant tongue microbiota in detail and to reveal the characteristics of tongue microbiota in infancy compared to those in adults.

RESULTS

Study subjects and sequence.Tongue swab samples were collected longitudinally from 8 infants, aged 13 to 26 weeks at baseline, for 82 to 98 weeks. A total of 464 samples (average, 58.0 samples per subject; range, 33 to 88 samples) were obtained. The characteristics and details of follow-up in each subject are presented in Table 1. We also collected cross-sectional tongue swab samples from 32 children and 73 adults. We analyzed these samples by 16S rRNA gene amplicon analysis, resulting in 10,175,374 high-quality reads (17,883 ± 11,918 reads per sample) to determine their bacterial compositions and diversities. The data were further analyzed to characterize the development of tongue microbiota in infants.

Transition of bacterial diversity in tongue microbiota.We first investigated the number of observed operational taxonomic units (OTUs) in tongue microbiota to evaluate the maturity of tongue bacterial populations. The number of OTUs detected from tongue microbiota rapidly increased with age until 80 weeks ( Fig. 1). In the cross-sectional samples from children, a similar increasing tendency in the number of OTUs was observed until approximately 80 weeks of age ( Fig. 2). We defined 10 to 29 weeks as the early exponential phase (EEP), corresponding to the initial stage of increase, and 80 to 120 weeks as the transitional phase (TP), corresponding to the phase between exponential and stationary stages in infancy. When bacterial alpha diversity was compared between the EEP and TP, the number of OTUs in the TP was significantly higher than that in the EEP in all subjects (Mann-Whitney U test) ( Table 2). Interestingly, the number of OTUs in the TP was either not significantly different or significantly higher than that of adult samples in all subjects except for subject 3. This result suggests that the bacterial alpha diversity of tongue coating in the TP had reached the same level as that in the adult phase.

Similarities of bacterial compositions between phases. Figure 3A shows a principal-coordinate analysis (PCoA) plot of the tongue bacterial compositions of all samples belonging to the EEP, TP, and adult phase. Plots of the EEP clearly differed from those of the TP and adult samples. Furthermore, we statistically estimated the similarities (dissimilarities) of bacterial compositions between the EEP, TP, and adult samples using analysis of similarities (ANOSIM) based on the weighted UniFrac distance. ANOSIM can statistically reveal significant differences in bacterial compositions between groups, and an R value (ranging from −1.0 to 1.0) close to 1.0 suggests maximum dissimilarity. ANOSIM showed that the overall bacterial composition of the TP was more similar to that of the adult samples (analysis of similarity R = 0.326) than that of the EEP ( R = 0.489). Moreover, when samples were compared individually as shown in Fig. 3B, the similarities between the TP and adult samples were significantly higher ( R value range, 0.14 to 0.64) than those between the TP and EEP samples ( R value range, 0.57 to 0.92) (Wilcoxon's signed-rank test, P  = 0.016) although the former comparison used data from a single individual while the latter used data of different individuals. These results indicate that the bacterial composition drastically changed between the EEP and TP and that the bacterial composition of the TP was already more similar to that of the adult phase than to that of the EEP.

Dominant OTUs and transition of their abundances.We examined the temporal changes in bacterial composition in tongue microbiota at the species level to clarify the details of bacterial colonization and substitution during the early developmental stage. First, we identified dominant OTUs with a ≥1% mean relative abundance in the EEP, TP, and adult phase, and summarized their distributions among these phases using a Venn diagram ( Fig. 4). Seven OTUs corresponding to bacterial species such as Neisseria flavescens HOT-610 (human oral taxon numbers in Human Oral Microbiome Database [HOMD]), Prevotella melaninogenica HOT-469, and Rothia mucilaginosa HOT-681 were commonly dominant in all phases (core OTUs). In addition, 10 OTUs corresponding to bacterial species such as Porphyromonas pasteri HOT-279, Granulicatella adiacens HOT-534, and Fusobacterium periodonticum HOT-201 were commonly dominant in both the TP and adult phase (TP-adult common OTUs). No common OTUs were observed in the EEP and TP except for core OTUs. This finding is consistent with the above result of ANOSIM showing that the bacterial composition in the TP was more similar to that in the adult phase than to that in the EEP.

Thereafter, we studied the temporal shift in the total relative abundance of dominant OTUs according to Venn diagram analysis. Core OTUs showed a high relative abundance in the range of 28.89 to 42.29% in all phases ( Fig. 5). Eight OTUs that were dominant only in the EEP (EEP-OTUs) consisted of OTUs corresponding to bacterial species such as Streptococcus peroris HOT-728 and Streptococcus lactarius HOT-948, and their total relative abundance exponentially decreased immediately after the EEP, particularly around 30 to 49 weeks, from approximately 60% to below 3%. Additionally, TP-adult common OTUs rapidly increased during this phase, accounting for approximately 20% of tongue microbiota after that, even in the adult phase. These results suggest that a drastic change occurs in the bacterial composition before the age of 1 year.

DISCUSSION

This prospective cohort study revealed the early development of bacterial diversity and composition of tongue microbiota. The drastic compositional shift in tongue microbiota occurred in an early stage of infancy, around 30 to 49 weeks, and then the bacterial diversity and overall bacterial composition in the TP reached the same levels as those in adults and were less comparable to those in the EEP. The result is particularly interesting considering that the TP was only approximately 1 year later than the EEP and that the comparison between the TP and EEP was carried out within the same individual although the comparison between the TP and the adult phase was conducted among different individuals.

A recent cross-sectional study suggested that oral bacterial diversity in the primary dentition period is significantly higher than that in the predentate stage ( 22). Although we did not consider the effect of primary dentition because of a lack of sufficient edentulous samples, this prospective cohort study revealed that expansion of the bacterial diversity in the oral cavity may begin during the earliest phase of the primary dentition period. These results suggest that tongue microbiota drastically develop and approach maturity immediately after eruption of the primary teeth. Additionally, dominant OTUs in adults, such as N. flavescens, P. melaninogenica, and R. mucilaginosa, were already detected and colonized in an early stage of infancy as core bacterial species. These results suggest that the foundation of tongue microbiota is established in infancy.

Although we frequently collected samples at short intervals and obtained information from several events to identify factors affecting the development of tongue microbiota, an obvious factor affecting the drastic increase of bacterial alpha diversity and compositional changes in bacterial species was difficult to identify because of the small number of subjects. Additionally, samples from patients that would contribute to understanding the origins of oral bacteria in infants were not collected. Further studies involving larger sample sizes focusing on early infancy are required to identify the factors that critically affect the development of oral microbiota.

A set of twins (subjects 4 and 5) among our subjects showed interesting results. Their parents began the feeding of baby food starting at 7 months; while one (subject 4) child quickly accepted the baby food, the other (subject 5) did not accept this food until it was approximately 1 year old. However, there was no drastic difference in the development of bacterial diversity and compositional shift between the twins. These results suggest that the change in nutrition from lactation to weaning food did not greatly affect the composition of tongue microbiota and that environmental factors other than eruption of primary teeth or genetic factors may have stronger effects on temporal changes in tongue microbiota from the EEP to TP.

Our study revealed that the bacterial composition of the TP was more similar to that of the adult phase than to that of the EEP according to ANOSIM. ANOSIM also revealed a significant difference in bacterial compositions between the TP and adult phase in most subjects (data not shown). Indeed, seven OTUs corresponding to bacterial species such as Neisseria mucosa HOT-682 and Streptococcus cristatus HOT-578 were dominant in only the TP (TP-OTUs), whereas six OTUs corresponding to bacterial species such as Alloprevotella species strain HOT-308 and Prevotella histicola HOT-298 were inversely dominant in only adult samples (adult-OTUs). These OTUs were thought to be discriminative OTUs between the TP and adult phase. More interesting is that Streptococcus cristatus and Lautropia mirabilis HOT-022, which are predominant in supragingival plaque compared to their presence at other oral sites in the adult phase ( 23, 24), were dominant in the TP, suggesting that the emergence of tooth surfaces contributes to colonization of these microbes in the oral cavity. These microbes disappeared from the tongue coating of adults, even though there are fewer teeth in the TP than in adults. Although we could not confirm whether these microbes were present in other niches because we examined only tongue swabs in this study, the reduction in the populations of S. cristatus and L. mirabilis in the tongue coating may not mean that they are absent from the oral cavity; these microbes tightly colonize tooth surfaces by coaggregating with new inhabitants in the adult phase. Alternatively, unknown factors emerging during growth enhance the tight colonization of these microbes in supragingival plaque, which results in the reduction of the relative abundances of these microbes in the tongue coating and saliva. If the latter hypothesis is true, identification of such factors would reveal a new mechanism by which colonization of oral bacteria and formation of dental plaque occur.

Streptococcus is among the most dominant genera in the human oral cavity, and previous reports of oral microbiota of infants demonstrated that S. salivarius is frequently detected in the infant oral cavity ( 20, 22). In fact, S. salivarius belonged to the core OTUs and showed a high relative abundance staring at the EEP ( Fig. 4 and Fig. S1 in the supplemental material). In contrast, S. peroris, S. lactarius, and other Streptococcus species belonging to the EEP-OTUs exponentially decreased and disappeared by the TP. This result suggests that substitution of bacterial species, even in the same genus, occurred in tongue microbiota during this period. Interestingly, an OTU that nearly corresponded to S. salivarius (97.2% identity against the HOMD) was dominant in the EEP of some subjects but disappeared from the oral cavity in the TP and adult phase. This suggests that there are two types of S. salivarius with slightly different base sequences and different behaviors residing in or leaving the oral cavity. Further studies of these bacterial species using comparative genomics may reveal the detailed colonization mechanisms of oral commensal bacteria.

FIG S1
Copyright © 2019 Kageyama et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

While 16S rRNA gene sequencing may not be suitable for detecting specific bacteria present at low proportions, we examined abundances of Porphyromonas gingivalis and Streptococcus mutans, which are the major pathogens of periodontitis and dental caries, in each subject ( Fig. S2). As a result, we detected P. gingivalis in the tongue coating of most subjects from a very early period, even when the subjects did not have periodontitis or teeth. Additionally, P. gingivalis disappeared by the TP in most subjects, but subjects 1 and 8 had evidence of P. gingivalis until the TP. These results suggest that the tongue dorsum harbors periodontal pathogens as a reservoir starting in early infancy. In contrast, the detection of S. mutans was transient and unstable compared to that of P. gingivalis.

FIG S2
Copyright © 2019 Kageyama et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

Although the number of subjects was small in the present study, no previous studies have evaluated an average of 58 tongue samples per individual, collected consistently for approximately 2 years, based on next-generation sequencing. To compensate for the small sample size of our study, we validated our results by utilizing cross-sectional control samples. Therefore, the results obtained in the present study appear to be generalized to the development of tongue microbiota in infancy in Japanese infants.

In conclusion, the present longitudinal study demonstrated that the bacterial diversity and composition of tongue microbiota rapidly develop in an early stage of infancy. To prevent the development of oral and systemic diseases associated with oral microbiota, oral care based on a bacteriological basis, e.g., changing the oral microbiota from a dysbiotic state to a healthy state or maintaining a healthy state, is indispensable in future preventive health care. Although little is known about how to change the oral microbiota, the monitoring and intervention of infant oral microbiota, particularly until the first year of life, may help to prevent the development of various diseases later in life.

MATERIALS AND METHODS

Study subjects.We enrolled 8 infants (2 male and 6 female), aged 13 to 26 months, living in Fukuoka City, Fukuoka Prefecture, Japan, for a longitudinal study spanning approximately 2 years. To confirm the results of the longitudinal study, 32 healthy children (18 male and 14 female; age, 0 to 2 years old) who had not used antibiotics within a month preceding sampling were also enrolled as cross-sectional controls from our previous project of disease in children. As a reference adult control, 73 healthy participants (36 men and 37 women; age, 20 to 29 years), consisting of dental students at Kyusyu University who had not used antibiotics within a month preceding sampling, were also enrolled from our previous project of dental plaque. Written informed consent was obtained from all subjects or their parents. The ethics committee of Kyushu University approved this study and the informed consent procedure (approval numbers 22-146, 25-110, and 28-127).

Sample of microbiota and data collection.Tongue swab samples were collected from all children by scraping the tongue dorsum with a cotton swab. Longitudinal samples were collected by their parents and immediately stored in the home freezer. Samples were transported to our laboratory and stored at −30°C until further analysis. Feeding information (breast milk, artificial milk, or baby food), eruption of teeth, start of outside play, and antibiotic administration were recorded by the parents. Tongue swab samples from adults were collected using a circular bonded-fiber fabric with a diameter of 15 mm as described previously ( 4). These samples were immediately stored at −30°C until further analysis.

16S rRNA gene amplicon sequencing analysis of tongue swab samples.Tongue swab samples were examined by 16S rRNA gene next-generation sequencing using an Ion PGM system (Thermo Fisher Scientific, Waltham, MA, USA). DNA was extracted from the samples collected from children using the bead-beating method ( 25), and DNA was extracted from samples collected from adults using lysozyme and achromopeptidase ( 26), as described previously. The V1-V2 regions of the 16S rRNA gene were amplified using the following primers: 8F (5′-AGA GTT TGA TYM TGG CTC AG-3′) with the Ion Torrent adaptor A and a sample-specific 8-base tag sequence and 338R (5′-TGC TGC CTC CCG TAG GAG T-3′) with the Ion Torrent trP1 adaptor sequence. PCR amplification, purification, and quantification of each PCR amplicon were performed as previously described ( 3). Equal amounts of the purified PCR amplicons were pooled, and gel purification was carried out using a Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA). The DNA concentration was determined using a KAPA Library Quantification kit (KAPA Biosystems, Wilmington, MA, USA), and DNA was diluted to 8 pM for use as the template DNA for emulsion PCR. Emulsion PCR and enrichment of template-positive particles were performed using an Ion PGM Template OT2 400 kit and Hi-Q View OT2 kit (Thermo Fisher Scientific) in an Ion One Touch 2 system (Thermo Fisher Scientific). The enriched particle was loaded onto 10 Ion 318 v2 chips (Thermo Fisher Scientific), and sequencing was performed on the Ion PGM (Thermo Fisher Scientific) using an Ion PGM Hi-Q sequencing kit and Hi-Q view sequencing kit (Thermo Fisher Scientific).

Data analysis and taxonomy assignment.We conducted quality filtering of the raw sequence reads using a script written in R (version 3.5.1). The reads were excluded from the analysis when they were ≤200 bases, had an average quality score of ≤25, did not include the correct forward primer sequence, did not include the correct reverse primer sequence (one mismatch was allowed), or had a homopolymer run of >6 nucleotides (nt). The quality-checked reads (including singleton reads) were assigned to the appropriate sample by examining the tag sequence, and then forward and reverse primer sequences were trimmed. OTUs were constructed by clustering quality-checked reads, excluding singleton reads, with a minimum pairwise identity of 97% using UPARSE ( 27) as described previously ( 3). All quality-checked reads (including singleton reads) were mapped to each OTU with an identity of ≥97% using UPARSE ( 3). The taxonomy of representative sequences was determined using BLAST against 889 oral bacterial 16S rRNA gene sequences (HOMD 16S rRNA RefSeq, version 14.51) in the Human Oral Microbiome Database ( 28). Nearest-neighbor species with ≥98.5% identity were selected as candidates for each representative OTU. The taxonomy of sequences without hits was further determined using the Ribosomal Database Project (RDP) classifier with a minimum support threshold of 80%. The number of OTUs and UniFrac distance ( 29) were calculated following rarefaction to 4,000 reads per sample using R.

Statistical analysis.A logistic curve was fitted to the transition data of the bacterial diversities (number of OTUs) in tongue microbiota using the nonlinear least squares (NLS) function, based on the self-start logistic (SSlogis) model that can avoid the step of determining an initial value in the R package ( 30). The 95% confidence interval of the logistic curve was estimated using the predictNLS function in the propagate library of R. We compared the number of OTUs between different phases using a Mann-Whitney U test. The UniFrac metric was used to determine the dissimilarity between any pair of bacterial compositions. Principal-coordinate analysis (PCoA) was performed based on the weighted UniFrac distance. We estimated the R value between phases by analysis of similarities (ANOSIM) with 999 permutations based on the weighted UniFrac distance in each subject and compared the R values using Wilcoxon's signed-rank test.

Data availability.The sequence data have been deposited in DDBJ/NCBI Sequence Read Archive under accession number DRA008297.

ACKNOWLEDGMENTS

This work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI grants JP25463249, JP16H02692, JP16H05850, JP16H05557, and JP18H06296 and an Institute for Fermentation, Osaka (IFO) grant (to T.T., 2014 to 2016).

We declare that we have no conflicts of interest.

FOOTNOTES

# data[].attributes.attachment_link.title
Transition of Bacterial Diversity and Composition in Tongue Microbiota during the First Two Years of Life

# data[].attributes.attachment_link.description
Newborns are constantly exposed to various microbes from birth; hence, diverse commensal bacteria colonize the oral cavity. However, how or when these bacteria construct a complex and stable ecosystem remains unclear. This prospective cohort study examined the temporal changes in bacterial diversity and composition in tongue microbiota during infancy.

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E. tonsillarum is a Gram positive, rod that can come as single rods, V-shaped pairs, or chains.

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Erysipelothrix is a genus of bacteria containing four described species, Erysipelothrix rhusiopathiae, Erysipelothrix tonsillarum, Erysipelothrix inopinata and Erysipelothrix larvae. Additional species have been proposed based on DNA-DNA hybridization studies "The hallmark of Erysipelothrix is the presence of a type B cell wall, in which the peptide bridge is formed between amino acids at positions 2 and 4 of adjacent peptide side-chains and not, as in the vast majority of bacteria, between amino acids at positions 3 and 4."

The best known species within the genus is Erysipelothrix rhusiopathiae, which is the cause of erysipelas of domestic pigs, poultry and other animal species. In humans, E. rhusiopathiae infections are termed erysipeloid. Erysipelothrix tonsillarum has been described as a pathogen for dogs and has been isolated from the tonsils of healthy pigs and cattle. Disease caused by E. inopinata or E. larvae have not been described.

Bacteria of genus Erysipelothrix are straight, or slightly curved, slender, nonmotile rods which may exist singly, in V-shaped pairs, or in short chains. Some strains have a tendency to form long filaments. The bacteria are Gram-positive but can be mistaken for Gram-negative bacteria during analysis because they lose their staining easily. They are aerobic to facultatively anaerobic but not acid-fast.

# data[].attributes.attachment_link.description
Erysipelothrix is a genus of bacteria containing four described species, Erysipelothrix rhusiopathiae , Erysipelothrix tonsillarum, Erysipelothrix inopinata and Erysipelothrix larvae. Additional species have been proposed based on DNA-DNA hybridization studies "The hallmark of Erysipelothrix is the presence of a type B cell wall, in which the peptide bridge is formed between amino acids at positions 2 and 4 of adjacent peptide side-chains and not, as in the vast majority of bacteria, between amino acids at positions 3 and 4."

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E. tonsillarum isn't very much of a risk for humans, but is a risk to common farm animals. This risk affects humans economically. E. tonsillarum can affect pigs, lambs, turkey, and calves by causing septicemia.

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Since E. tonsillarum is very infectious to livestock animals, it's important to find a way to keep the animals healthy in order to not lose money. This new research is continuing to examine the efficacy of current vaccines used for Erysipelothrix species.

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ABSTRACT

The aim of this study was to characterize Erysipelothrix sp. isolates from clinically affected pigs and their environment and compare them to the Erysipelothrix sp. vaccines used at the sites. Samples were collected during swine erysipelas outbreaks in vaccinated pigs in six Midwest United States swine operations from 2007 to 2009. Pig tissue samples were collected from 1 to 3 pigs from each site. Environmental samples (manure, feed, central-line water, oral fluids, and swabs collected from walls, feed lines, air inlets, exhaust fans, and nipple drinkers) and live vaccine samples were collected following the isolation of Erysipelothrix spp. from clinically affected pigs. All Erysipelothrix sp. isolates obtained were further characterized by serotyping. Selected isolates were further characterized by PCR assays for genotype ( E. rhusiopathiae, E. tonsillarum, Erysipelothrix sp. strain 1, and Erysipelothrix sp. strain 2) and surface protective antigen ( spa) type (A, B1, B2, and C). All 26 isolates obtained from affected pigs were E. rhusiopathiae, specifically, serotypes 1a, 1b, 2, and 21. From environmental samples, 56 isolates were obtained and 52/56 were E. rhusiopathiae (serotypes 1a, 1b, 2, 6, 9, 12, and 21), 3/56 were Erysipelothrix sp. strain 1 (serotypes 13 and untypeable), and one was a novel species designated Erysipelothrix sp. strain 3 (serotype untypeable). Four of six vaccines used at the sites were commercially available products and contained live E. rhusiopathiae serotype 1a. Of the remaining two vaccines, one was an autogenous live vaccine and contained live E. rhusiopathiae serotype 2 and one was a commercially produced inactivated vaccine and was described by the manufacturer to contain serotype 2 antigen. All E. rhusiopathiae isolates were positive for spaA. All Erysipelothrix sp. strain 1 isolates and the novel Erysipelothrix sp. strain 3 isolate were negative for all currently known spa types (A, B1, B2, and C). These results indicate that Erysipelothrix spp. can be isolated from the environment of clinically affected pigs; however, the identified serotypes in pigs differ from those in the environment at the selected sites. At one of the six affected sites, the vaccine strain and the isolates from clinically affected pigs were of homologous serotype; however, vaccinal and clinical isolates were of heterologous serotype at the remaining five sites, suggesting that reevaluation of vaccine efficacy using recent field strains may be warranted.

Organisms of the genus Erysipelothrix are facultative anaerobic small, slender, Gram-positive rods and are distributed worldwide. Erysipelothrix spp. have been isolated from domestic and wild species of both birds and mammals and have been identified as the causative agent of the clinical disease known as "erysipelas" in animals and "erysipeloid" in humans ( 2). The genus Erysipelothrix consists of four species and 25 associated serotypes: E. rhusiopathiae (serotypes 1a, 1b, 2, 4, 5, 6, 8, 9, 11, 12, 15, 16, 17, 19, 21, N), E. tonsillarum (serotypes 3, 7, 10, 14, 20, 22, 23), Erysipelothrix sp. strain 1 (serotype 13), and Erysipelothrix sp. strain 2 (serotype 18) ( 27, 28). Among the four species, E. rhusiopathiae causes the greatest economic loss, primarily to the swine and turkey industries ( 34, 36).

Three clinical presentations of swine erysipelas are recognized, i.e., acute, subacute, and chronic, and serotypes 1a, 1b, and 2 are frequently isolated from all disease stages ( 36). The additional serotypes (3 to 23 and N) have little clinical significance in swine. It is estimated that 30 to 50% of healthy pigs harbor E. rhusiopathiae in tonsils and lymphatic tissue. Subclinically affected pigs are thought to be the source for acute erysipelas outbreaks due to shedding of the organism in urine, feces, saliva, and nasal secretions ( 36).

Economic losses due to swine erysipelas continue to occur worldwide. For this reason, accurate, reliable, and timely diagnostic strategies are important ( 4). Immunohistochemistry techniques have been shown to be highly sensitive and specific, especially when diagnostic specimens include lesions from antimicrobial-treated pigs or chronically affected pigs ( 16). Our previous studies confirmed that an Erysipelothrix species-selective broth technique is more sensitive than traditional direct plating of regular and contaminated specimens ( 1). Although the enrichment technique has been used by other countries for a number of years, it has only recently been adopted by diagnostic laboratories within the Midwestern United States ( 1). PCR technology is also being employed to complement traditional detection methods ( 10, 19, 28, 39). In addition to improved diagnostic assays, methods to further characterize and differentiate Erysipelothrix spp. through the use of randomly amplified DNA, pulsed-field gel electrophoresis, and ribotyping have been shown to be useful and credible ( 13- 15, 17). Recent investigations have focused on antibodies against the cell surface components of E. rhusiopathiae and their protective role. Genes encoding surface protective antigens (Spa) have been cloned, and nucleotide sequences have been determined ( 11, 22). Spa-related genes of all E. rhusiopathiae serotypes and Erysipelothrix sp. strain 2 (serotype 18) were analyzed, and Spa proteins can be classified into three molecular species, SpaA, SpaB, and SpaC ( 29). The SpaA protein was identified in E. rhusiopathiae serotypes 1a, 1b, 2, 5, 8, 9, 12, 15, 16, 17, and N, the SpaB protein was identified in E. rhusiopathiae serotypes 4, 6, 11, 19, and 21, and the SpaC protein was identified only in serotype 18 ( 29). Additional work further differentiated SpaB into subtypes SpaB1 (serotypes 4, 6, 8, 19, 21) and SpaB2 (serotype 11) ( 21).

Previous characterization of Erysipelothrix sp. isolates from affected pigs or isolates from the environment of U.S. swine sites dates back to the 1970s. The objective of this study was to identify, characterize, and compare Erysipelothrix sp. isolates from affected pigs and the environment from erysipelas outbreaks in six swine operations and to compare those isolates to the vaccine strains routinely used for vaccination in those same six operations.

MATERIALS AND METHODS

Site selection.Submissions to the Iowa State University Veterinary Diagnostic Laboratory (ISU-VDL) between December 2007 and February 2009 from pigs with a clinical history consistent with acute septicemia (fever, lethargy, skin lesions, decreased feed intake) or chronic changes (swollen joints, lameness) suggestive of swine erysipelas were cultured for Erysipelothrix spp. Following a positive isolation, the submitting veterinarian was contacted to determine the vaccination status of the herd. Six sites with a vaccination program against E. rhusiopathiae were identified for environmental sample collection. The six sites were located in Iowa (sites A to D), Indiana (site E), and Illinois (site F) and environmental sample collections were done on these sites from 2007 to 2009 within 7 to 14 days of a positive isolation. Samples were collected from the same barn in pens with additional clinically affected pigs or pens where clinically affected pigs had been housed recently. The site structures, types, and clinical signs present are summarized in Table 1. All sites housed pigs in confinement equipped with automatic feeders and waters.

Pig samples.Sections of spleen, liver, lung, tonsil, kidney, and skin were collected from clinically affected pigs. A total of 31 samples from 1 to 3 pigs from each of the six sites were cultured. All isolates obtained were frozen at −80°C for future evaluation.

Environmental samples.Environmental samples were collected using swabs (Culturette, Becton Dickinson, Sparks, MD) from the water supply area and nipple drinkers, wall surfaces, feed lines, air inlets, and exhaust fans. Feed, manure, and central-line water samples were also collected in sterile 50-ml tubes (Thermo Fisher Scientific Remel, Lenexa, KS). For purposes of this study, oral fluid samples were also collected and classified as environmental samples, as they were from pigs on-site and not from the same pigs from which tissue specimens were collected. Oral fluids (mainly saliva) were collected only from sites C and D. In brief, a 3-strand, 1.27-cm-diameter cotton rope was placed in pens with 4 to 6 pigs. The rope was hung at approximately the height of the pigs' shoulders and left in place for 20 to 30 min. Oral fluids were then collected by mechanically compressing the rope and collecting the fluid in sterile 5-ml snap cap tubes ( 20). All environmental samples were immediately placed on ice following collection and stored at −20°C until microbiologic evaluation. Evaluation was done within 3 months of collection.

Vaccine strains.Four attenuated live vaccine strains (sites A, B, D, and E), one attenuated live autogenous vaccine strain (site F), and one inactivated vaccine strain (site C) were collected from the respective sites. Attenuated live vaccines included ERY VAC 100 (Arko Laboratories Limited, Jewell, IA), which was used at sites A and B; Suvaxyn E-oral (Fort Dodge Animal Health, Inc., Fort Dodge, IA), which was used at site D; and Ingelvac ERY-ALC (Boehringer Ingelheim Vetmedica, Inc., St. Joseph, MO), which was used at site E. An attenuated live autogenous vaccine produced by using a site-specific Erysipelothrix sp. isolate (Newport Laboratories, Worthington, MN) was used at site F. An inactivated vaccine (Suvaxyn Parvo/E, Fort Dodge Animal Health, Inc., Fort Dodge, IA), which was serotype 2 based on the manufacturers' information, was used at site C. The five attenuated live vaccine strains were immediately placed on ice following collection and stored at −20°C until evaluation. Evaluation was done within 3 months of collection.

Bacterial isolation.An Erysipelothrix species-selective broth protocol as previously described was utilized for bacterial isolation ( 1). The selective broth was prepared as follows: 25 g heart infusion broth (Becton Dickinson) was dissolved in 1 liter of 0.1 phosphate buffer solution (12.02 g of Na ₂HPO ₄ and 2.09 g of KH ₂PO ₄ per liter of distilled water) and autoclaved. Sterile fetal bovine serum (5%; Sigma-Aldrich, St. Louis, MO), kanamycin (400 mg/ml; Sigma-Aldrich), and neomycin (50 mg/ml; Sigma-Aldrich) were added to the broth ( 31). Specimens were cultured on an Erysipelothrix species-selective agar as previously described ( 18).

Sample preparation. (i) Tissue specimens.Samples were cut into 2- by 3-cm sections, added to 2 ml of 0.85% physiologic saline solution, and homogenized using a stomacher (Seward, Bohemia, NY), and 300 μl of the resulting tissue homogenate was added to 3 ml of Erysipelothrix species-selective broth and incubated at 35°C for 24 to 48 h. At 24 h and again at 48 h, a 100-μl subculture from the Erysipelothrix species-selective broth was put onto a Trypticase soy agar plate containing 5% sheep blood, a colistin-nalidixic acid (Becton Dickinson) agar plate containing 5% sheep blood, and an Erysipelothrix-selective plate as described previously ( 1). Colonies were subcultured on sheep blood agar plates, incubated for 24 h, and then biochemically confirmed using standard laboratory methods ( 27, 31).

(ii) Water, oral fluids, and vaccines.Each specimen (300 μl) was added to 3 ml of Erysipelothrix species-selective broth and incubated.

(iii) Swabs.Culturette swabs were placed directly into the Erysipelothrix species-selective broth.

(iv) Manure and feed samples.A portion of the sample (75 to 100 g) was placed into sterile flasks, and 0.1 M phosphate buffer solution (pH 7.231) was added for a total volume of approximately 230 ml. The homogenate was mixed for 10 min with magnetic metal stir bars ( 37) and then transferred to centrifuge tubes and centrifuged for 10 min at 1,000 rpm. The supernatant (approximately 2 to 5 ml) was transferred to a flask to which 250 ml of the Erysipelothrix species-selective broth was added. Each flask was thoroughly mixed, incubated at 35°C for 24 h, and then subcultured to media in a manner similar to that used for tissue and liquid specimens ( 37).

Further characterization of the Erysipelothrix sp. isolates. (i) Serotyping.A pure culture was grown at 37°C for 36 h in 30 ml of heart infusion broth (Becton Dickinson) supplemented with 10% equine serum (Sigma-Aldrich) ( 35). The culture was then killed by adding 1% formalin (Sigma-Aldrich), held at room temperature for 12 h, harvested by centrifugation, and washed twice in 0.85% NaCl solution containing 0.5% formalin. Washed cells were suspended in 1.5 ml of distilled water and autoclaved at 121°C for 1 h ( 35). The supernatant was collected and used for the agar gel precipitation test ( 12). Homologous positive controls were used with each test run. Reactions were recorded after 24 h ( 35).

(ii) DNA extraction for PCR assays.For DNA extraction, bacterial colonies were suspended in 200 μl of sterile water by vigorous stirring. The suspension was then used for DNA extraction using the QIAmp DNA blood minikit (Qiagen, Valencia, CA) following the manufacturer's instruction, and the extracted DNA was kept at −20°C until use.

(iii) Genotype multiplex PCR assay.Further characterization was done on randomly selected representative isolates of each serotype collected at each site. A multiplex real-time PCR assay previously described was used to confirm the presence of E. rhusiopathiae, E. tonsillarum, or Erysipelothrix sp. strain 2 ( 19). A modified Erysipelothrix sp. strain 2 primer was utilized to increase the sensitivity of the assay described by Shen et al. ( 21).

(iv) Identification of Erysipelothrix sp. strain 1 by conventional PCR assay.A pair of specific primers, Sp11508F (5′-AGACGAAAGCGGCGATTACT-3′) and Sp12362R (5′-CCCCTACCACTTGCATTTAATGC-3′), were designed from the 16S rRNA gene of Erysipelothrix sp. strain 1 (GenBank accession no. AB019249). The PCR was performed in a model 9700 GeneAmp PCR system (Applied Biosystems, Foster City, CA) in 25-μl mixtures containing 1.25 U (0.25 μl) Taq DNA polymerase (Invitrogen, Carlsbad, CA), 0.2 mM deoxynucleoside triphosphate (dNTP), 0.4 μM each of the primers, 1.5 mM MgCl ₂, and 4 μl DNA extract. The cycling conditions were 5 min at 95°C followed by 35 cycles of 40 s at 95°C, 40 s at 55°C, and 1 min at 72°C and a final extension at 72°C for 7 min. The amplified PCR products were separated by gel electrophoresis on a 1% agarose (Amresco, Solon, OH) gel and visualized by UV irradiation after ethidium bromide staining of the gel. The specificity of the Erysipelothrix sp. strain 1 conventional PCR assay was investigated by testing E. rhusiopathiae reference strain Tuzok (serotype 6), E. tonsillarum reference strain Lengyel-P (serotype 10), Erysipelothrix sp. strain 2 reference strain 715 (serotype 18), and Erysipelothrix sp. strain 1 reference strain Pécs 18 (serotype 13). A specific product of 855 bp was amplified from the Erysipelothrix sp. strain 1 reference strain, whereas no PCR products were amplified from the other isolates used. The sensitivity of this conventional PCR was determined to be 1 × 10 ⁴ CFU per reaction (data not shown).

(v) Spa multiplex real-time PCR.A multiplex real-time PCR assay was utilized for identification of the Spa type present ( spaA, spaB1, spaB2, and spaC) on the same isolates used for genotyping ( 21).

(vi) DNA sequencing.From selected samples (of undetermined serotype), the 16S rRNA gene was amplified as described previously ( 5). Amplified products were sequenced at the DNA facility of Iowa State University, Ames, IA. The consensus 16S rRNA sequence was evaluated by comparison with those available in GenBank using the Basic Local Alignment Search Tool (BLAST).

Nucleotide sequence accession number. Erysipelothrix sp. strain 3 was deposited in GenBank under accession no. HM216182.

RESULTS

Isolation and further characterization of isolates (genotype, serotype, and spa type). (i) Clinically affected pigs.Among the pig tissue samples examined, 83.9% (26/31) were found to be culture positive for Erysipelothrix spp. (see Table 3). Skin samples were received from all six sites, and 100% (11/11) were culture positive. Erysipelothrix spp. were also isolated from all tonsil (5/5) and kidney (3/3) samples obtained; however, these sample types were not submitted from every site. Table 3 describes the distribution of Erysipelothrix sp. isolates in tissues submitted from each site. The isolation success was 50% for heart (1/2) and liver (2/4) tissues and 66.7% for spleen tissues (4/6). Based on serotyping and multiplex PCR, all 26 isolates were found to be E. rhusiopathiae. The E. rhusiopathiae isolates recovered from each site were found to belong to the same serotype. The more common serotypes in affected pigs were serotypes 1a (sites D and F) and 2 (sites A and B). In addition, serotype 21 was present in pigs from site C, and serotype 1b was identified in pigs from site E. All 26 E. rhusiopathiae isolates recovered from pig tissues were positive for spaA.

(ii) Environmental samples.Of 142 environmental samples examined, 39.4% (56/142) were found to be culture positive for Erysipelothrix spp. (Table 2). Genotyping and serotyping revealed that 92.9% (52/56) of the environmental isolates belonged to E. rhusiopathiae and 5.4% (3/56) belonged to Erysipelothrix sp. strain 1, while in one isolate (1.8%) the genotype was novel and not previously described. For the purpose of this study, this genotype was designated Erysipelothrix sp. strain 3 (GenBank accession no. HM216182). The Erysipelothrix sp. strain 3 isolate was PCR negative for all known genotypes; however, its 16S rRNA region sequence was identical to those of GenBank accession no. AB055910.1 and AB055909 ( E. rhusiopathiae strain KG-BB2), confirming its Erysipelothrix sp. origin. The most commonly identified serotype was 1a, which was identified in 37.5% (21/56) of the isolates, followed by serotype 2 (33.9% [19/56 isolates]), serotype 1b (10.7% [6/56 isolates]), serotypes 6 and 21 (each 3.6% [2/56 isolates]), and serotypes 9, 12, and 13 (each 1.8% [1/56 isolates]). The serotype of 5.4% (3/56) of the isolates was not determinable. All environmental isolates identified as E. rhusiopathiae were found to be positive for spaA, and all isolates identified as Erysipelothrix sp. strain 1 or with an undeterminable genotype were negative for spaA, -B1, -B2, and -C. The frequency of detection of Erysipelothrix spp. in environmental samples is summarized in Table 2, with nipple drinkers and feed as the sources where samples had the highest positive isolation rate. Serotypes that were detected in clinically affected pigs were also found in manure, feed, wall swabs, central-line water, nipple drinkers, and fans for serotype 1a; central-line water and nipple drinkers for serotype 1b; manure, feed, wall swabs, central-line water, and fans for serotype 2; and oral fluid for serotype 21.

(iii) Vaccine strains.All four commercially available attenuated live vaccine strains (sites A, B, D, E) were identified as E. rhusiopathiae serotype 1a, and the autogenous vaccine strain used on site F was identified as E. rhusiopathiae serotype 2. All five vaccine strains were positive for spaA.

Isolation success and distribution of isolates from the different sites. (i) Site A.A total of 7 tissue samples from affected pigs were obtained and 43 environmental samples were collected (Table 3). E. rhusiopathiae serotype 2 was isolated from 5/7 tissues and from 18/43 environmental samples (nipple drinker swabs, manure, wall swabs, feed, and fan swabs). In addition, E. rhusiopathiae serotype 1a was identified in 2/5 feed samples.

(ii) Site B.A total of five tissue samples from affected pigs were obtained and 39 environmental samples were collected (Table 3). E. rhusiopathiae serotype 2 was isolated from all tissue samples and from 1 nipple drinker swab. E. rhusiopathiae serotype 1a was isolated from 8 environmental samples (central-line water, manure, wall swab, and feed). In addition, E. rhusiopathiae serotype 6 was isolated from a central-line water sample and a nipple drinker swab, and serotype 13 ( Erysipelothrix sp. strain 1) was isolated from a central-line water sample.

(iii) Site C.A total of three tissue samples from affected pigs were obtained and 31 environmental samples were collected (Table 3). E. rhusiopathiae serotype 21 was isolated from both tissue samples and from two oral fluid samples.

(iv) Site D.A total of two tissue samples from affected pigs were obtained and 12 environmental samples were collected (Table 3). E. rhusiopathiae serotype 1a was isolated from both tissue samples but was not identified in any of the environmental samples collected. Instead, E. rhusiopathiae serotypes 9 and 12 were isolated from oral fluids and Erysipelothrix sp. strain 1 was isolated from oral fluid and manure (serotype undeterminable). One additional isolate (novel Erysipelothrix sp. strain 3; serotype undeterminable) was isolated from manure.

(v) Site E.A total of six tissue samples from affected pigs were obtained and 6 environmental samples were collected (Table 3). E. rhusiopathiae serotype 1b was isolated from all tissue and environmental samples.

(vi) Site F.A total of eight tissue samples from affected pigs were obtained and 11 environmental samples were collected (Table 3). E. rhusiopathiae serotype 1a was isolated from all tissue and environmental samples.

DISCUSSION

Results from this investigation indicate that during a clinical outbreak of swine erysipelas, Erysipelothrix spp. can be isolated from a variety of environmental samples. Earlier investigations on swine erysipelas conducted in the United States have reported similar results in identifying Erysipelothrix spp. from swine and swine production premises; however, studies have not been conducted on sites with acute swine erysipelas outbreaks ( 35, 37). Interestingly, in this study we identified three Erysipelothrix sp. strain 1 isolates (1/3 was serotype 13 and 2/3 were untypeable), indicating the possibility of one or more new serotypes within Erysipelothrix sp. strain 1 in addition to serotype 13. Moreover, a new genotype of Erysipelothrix designated Erysipelothrix sp. strain 3, whose serotype and spa type were also unknown, was identified in an environmental sample, indicating the possibility of a new serotype within the species.

When tissues from affected pigs were investigated, it was found that skin specimens with visible rhomboid lesions were consistently culture positive, implicating skin as the tissue of choice for isolation of Erysipelothrix spp., which is in agreement with previous work ( 1). In addition, all tested tonsil samples (5/5) were found to be culture positive. The existence of Erysipelothrix spp. in the tonsils of healthy pigs is suspected ( 24); however, in the current study, all selected isolates obtained from tonsils had the same serotype as isolates recovered from other organs of affected pigs. E. rhusiopathiae serotypes 1a, 1b, and 2 are commonly associated with clinical disease in pigs and were associated with clinically affected pigs in five of the six sites. Interestingly, serotype 21 was found in affected pigs from one of the six sites. While uncommon, this has been reported previously in a larger study that characterized 1,046 isolates recovered from pigs with swine erysipelas in Japan and found that 1.1% of Erysipelothrix sp. isolates belonged to serotype 21 ( 26). In addition, isolates with reactivity to both serotypes 1b and 21 (termed 1b × 21) have been isolated on four occasions from affected pigs in three Australian herds with clinical erysipelas ( 3).

Among the environmental samples, Erysipelothrix spp. were most frequently isolated in feed and nipple drinkers (both 9/17 samples). This was followed by isolation from wall swabs (11/23 samples) and manure (13/28 samples). While isolation of Erysipelothrix spp. has been described from manure and soil ( 31), to our knowledge this is the first description of isolation of Erysipelothrix spp. from nipple drinkers, walls, and ventilation fans. Although clinical disease was present at all sites, relatively high percentages of environmental samples were culture negative (53.5% at site A, 69.2% at site B, 93.5% at site C, and 41.7% at site D). This may have to do with the total number of samples collected, the sample types, storage, or a delay between the original case submission and follow-up site visit or may be related to shedding mechanisms of Erysipelothrix spp. Previous studies demonstrated no evidence of growth or maintenance of Erysipelothrix spp. in soil or manure samples from swine pens ( 32), but the role of soil, manure, and pit slurry as reservoirs or sources of infection is not completely understood ( 34).

The attenuated live vaccine strains utilized on the different sites were also collected, cultured, and characterized. All sites in this investigation utilized a vaccine as part of an erysipelas control plan. Four of six sites (sites A, B, D, and E) utilized attenuated live vaccines, produced by three manufacturers. All three of these commercially available vaccines were found to be positive for E. rhusiopathiae serotype 1a. Interestingly, two of the four sites using these vaccines had clinical infections with serotype 2, one of the four sites had infections with serotype 1b, and one site had infections with serotype 1a based on the isolation of these serotypes from affected pigs. One site (F) used an autogenous attenuated live vaccine based on a site-specific isolate. The isolate recovered from the autogenous vaccine was identified as serotype 2; however, E. rhusiopathiae serotype 1a was isolated from affected pigs from this site and was also the only serotype present in the environment. Many factors affect the ability of a live vaccine to elicit protection, including but not limited to vaccine storage, route and dose of administration, age, maternal immunity, antimicrobial therapy, and the vaccine strains used. Based on cross-protection studies done in the 1980s, it was found that live serotype 1a protected against serotypes 1b and 2 ( 25). At site D, we found that the serotype of the live vaccine used was identical to that isolated from the pig tissues. In Japan in 1932, an acriflavine-resistant attenuated live vaccine was developed; this vaccine has been used intensively since the erysipelas outbreaks in 1966 and 1967. However, since the 1990s, approximately 2,000 pigs annually have been shown to have acute and subacute septicemia. It is believed that the live vaccine was the causal agent for this high number of pigs with swine erysipelas ( 6), and this could also be a possibility in herd D. Earlier studies have shown that serotype 2 bacterins can protect against serotype 1 challenge ( 33, 38). Cross-protection was apparently not sufficient to prevent clinical disease in this case, providing evidence of the need to further evaluate cross-protection in the swine model using recent field isolates.

In this study, Erysipelothrix spp. were isolated from the environments of clinically affected swine. In addition, the Erysipelothrix sp. isolates recovered from clinically affected pigs and the majority of the Erysipelothrix sp. isolates recovered from the pigs' environments were found to be identical in four of the six sites (site A, serotype 2; site C, serotype 21; site E, serotype 1b; and site F, serotype 1a; Table 3). However, dissimilarities between isolates in pigs and isolates from their environments were identified in two of the six sites investigated. While pigs in these two sites were infected with very common serotypes (serotypes 1a and 2), a variety of serotypes were identified in the environment (serotypes 1a, 2, 6, 9, 12, 13, and untypeable) (Table 3). It has been determined that E. rhusiopathiae serotypes remain stable through swine serial passage or under different storage conditions ( 23, 30). In this investigation, serotypes identified in the environment were both identical to (4/6 sites) and different from (2/6 sites) what was isolated from clinically affected pigs from these sites; however, affected sites were visited only once. To fully understand the interactions and relations between isolates associated with disease in pigs and isolates present in the environment, repeated collection over time of samples would provide more complete information.

In this study, all recovered isolates were also tested for their dominant spa type. The Spa protein of E. rhusiopathiae has been shown to be attached at the cell surface level of the bacteria and also to be a major protective antigen against infection by E. rhusiopathiae ( 7, 8, 11, 29). All E. rhusiopathiae isolates obtained from affected pig tissues (serotypes 1a, 2, and 21), environmental samples (serotypes 1a, 1b, 2, 6, 9, 12, and 21), and vaccine strains (serotypes 1a and 2) were found to contain a single spa type, spaA. The identification of spaA in serotype 21 is in contrast to a previous report where spaB was identified ( 29) but in agreement with another study which reported serotype 21 as expressing SpaA ( 9). Moreover, the findings of this study support previous results that spaA is highly conserved in serotypes most often associated with clinical swine erysipelas ( 9). All five vaccine strains characterized were found to be positive for spaA, and based on the current state of knowledge, cross-protection should have occurred ( 9, 29). However, the role of vaccine handling, administration, and timing should not be overlooked and can be considered a potential explanation for the lack of protection. In addition, vaccine efficacy testing of vaccine batches may be unreliable, as testing is often done in mice, in which vaccine efficacy may or may not mimic what occurs in pigs. All three Erysipelothrix sp. strain 1 isolates and the Erysipelothrix sp. strain 3 isolate were found to be negative for all spa types investigated (A, B1, B2, C). These isolates were identified in two sites and were present in central-line water, oral fluid, and manure. No recent reports have implicated Erysipelothrix sp. strain 1 to be pathogenic. Moreover, as this strain was not isolated from pig tissue based on present evidence, it is unlikely that Erysipelothrix sp. strain 1 is an important pathogen.

Much interest has recently been generated regarding spa types. Results of this study agree with previous work and indicate that spa types are likely to be quite conserved among swine isolates of Erysipelothrix spp. associated with disease. However, all of the isolates obtained from clinically affected pigs were E. rhusiopathiae strains, which are known to contain only either spaA or spaB. In this study, spaA was the only type found, which is consistent with findings of others who have reported that serotypes 1a, 1b, and 2 (all spaA) predominate in clinically affected pigs. What is interesting is that the serotype 21 isolate from pig tissue from site C contained spaA and not spaB as previously described ( 29). In one investigation in Australia, four farms were found to have isolates that were reactive to both 1b and 21 ( 3), which could indicate a possible reason for why spaA was found in this case.

Data from acute swine erysipelas outbreaks investigated in this study indicate that during an acute outbreak situation, Erysipelothrix spp. can be isolated from both clinically affected pigs and their environments. Characterization of the Erysipelothrix sp. isolates using serotyping and genotyping assays indicated that isolates from affected pigs and the environment are not necessarily the same on individual sites.

ACKNOWLEDGMENTS

We thank the National Pork Board for checkoff dollars for funding of this study.

View Abstract

# data[].attributes.attachment_link.title
Characterization of Erysipelothrix Species Isolates from Clinically Affected Pigs, Environmental Samples, and Vaccine Strains from Six Recent Swine Erysipelas Outbreaks in the United States

# data[].attributes.attachment_link.description
The aim of this study was to characterize Erysipelothrix sp. isolates from clinically affected pigs and their environment and compare them to the Erysipelothrix sp. vaccines used at the sites. Samples were collected during swine erysipelas outbreaks in vaccinated pigs in six Midwest United States swine operations from 2007 to 2009.

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It is extremely important to learn how to use the 16S rRNA and phylogenetic trees for unknown organisms. In this project, we were given the sequences to begin with and didn't have to worry about sequencing our organisms. However, if in the future we get to research where we need to identify an organism by sequencing them, this process is how it would be done. It's important to understand how 16S rRNA sequences can also be used to show the relationship of the organism being researched, and what some similar organisms are. This is helpful in trying to identify potentially new characteristics of an organism. It is extremely important to continue researching the microbiome because the more we know about the microbes that are around us, the more we can help people that they may affect. Two projects that really stood out to me and were done very well were Kayla Smith's and Gabby Fricker's.