International Journal of Systematic and Evolutionary Microbiology (2013), 63, 2545–2552 DOI 10.1099/ijs.0.045237-0

Tonsilliphilus suis gen. nov., sp. nov., causing tonsil infections in pigs

Ryozo Azuma,1 Bak Ung-Bok,2 Satoshi Murakami,3 Hiroyuki Ishiwata,4 Makoto Osaki,5 Nobuaki Shimada,5 Yasuichiro Ito,6 Eichi Miyagawa,7 Takashi Makino,8 Takuji Kudo,9 Yoko Takahashi,10 Ikuya Yano,11 Ryo Murata3 and Eiji Yokoyama12

Correspondence 12-7-33 Higashi-tokura, Kokubunji-city, Tokyo 185-0002, Japan Satoshi Murakami 21002, 808-dong Suri-Apt, Sanbon-dong, Gunpo-city, Kyong-gi-do, Republic of Korea [email protected] 3Laboratory of Animal Health, Department of Animal Science, Tokyo University of Agriculture, 1737 Funako Atugi-city, 243-0034, Japan 4Technical Research Institute, Nishimatu Construction Co. Ltd Nakatu, Aikawa-machi Kanagawa-Pref., 243-0303, Japan 5Safety Research team, National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba-city 305-0856, Japan 61-3 Bangai, Tamiya, Ushiku-city, 300-1236, Japan 7Agricultural Microbiology, Faculty of Dairy Science, Rakuno Gakuen University, 582 Bunkyodai, Midorimachi, Ebetu, 069-8501, Japan 8Yakult Central Institute for Microbiological Research 1796 Yaho, Kunitachi-city, 186-8650, Japan 9Japan Collection of Microorganisms, RIKEN BioResource Center, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan 10Kitasato Life Science Institute, Kitasato University. 5-9-1 Shirogane, Minatoku, Tokyo 108-8641, Japan 11Japan BCG Laboratory, 3-1-5 Matuyama, Kiyose, Tokyo 204-0303, Japan 12Chiba Prefectural Institute of Public Health, 666-2 Nitona, Chuo, Chiba, 260-8715 Japan

A micro-organism resembling members of the genus Dermatophilus, strain W254T, which was isolated from the submandibular lymph node of a pig, and an additional 16 strains isolated from swine tonsils, were studied to establish their taxonomic status. Although all 17 strains were isolated

anaerobically under an atmosphere of 100 % CO2, all of them were aerotolerant anaerobes. The micro-organisms showed at least five cellular morphologies: (i) a radially protrusive thallus, which proliferated into tuber-like cells; (ii) segmentation in both tubers and thallus followed by multilocule formation, (iii) development of coccoid forms in the locules; (iv) a change from the coccoid forms to zoospores; (v) resting cells, which were able to develop into protrusive thalli again. The micro- organisms were positive for nitrate reduction, but negative for catalase, indole production, hydrolysis of urea and gelatin liquefaction. Milk was not decomposed and none of the strains was haemolytic. A total of 16 compounds, including glucose, were utilized as sole carbon sources and seven compounds, including L-arabinose, were not utilized. Three out of the 17 strains were subjected to further studies. The micro-organisms had meso-diaminopimelic acid in their peptidoglycan and galactose, glucose, madurose and a trace of mannose in their whole-cell sugar patterns. The major phospholipids were phosphatidylglycerol, diphosphatidylglycerol and phosphatidylinositol.

Abbreviations: DAP, diaminopimelic acid; SEM, scanning electron microscopy; TEM, transmission electron microscopy. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of Tonsilliphilus suis W254T, HT1-19 and HT5-225 are AB096084, AB096085 and AB602380, respectively. A supplementary figure and a supplementary table are available with the online version of this paper.

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Cellular fatty acids were C15 : 0 (35.7–23.1 %), C16 : 0 (5.9–2.4 %) C17 : 0 (62.9–39.5 %), C17 : 1

(24.4–0 %) and C18 : 0 (3–1.6 %). The predominant menaquinone was MK-8 (H4). The G+C content of the DNA was 69.6–71.8 mol%. Analysis of the 16S rRNA gene sequences showed that the strains clustered with the type strains of members of the family Dermatophilaceae. Based on the polyphasic taxonomic characterization carried out, all 17 strains are considered to belong to a novel species in a new genus, for which the name Tonsilliphilus suis gen. nov., sp. nov. is proposed. The type strain of the type species is W254T (5ATCC 35846T5CCM 3774T5DSM 21880T5JCM 15727T).

In 1971, a micro-organism resembling members of the double fixation was used as for SEM and the specimens genus Dermatophilus, strain W254T, was isolated from an were embedded in Epon 812. The resulting blocks were infected submandibular lymph node of a pig in Japan sectioned and the sections were stained with uranyl acetate (Azuma & Bak 1980). In 1978 and 1979, we isolated 10 and lead acetate. The specimens were examined under a strains from infected swine tonsils in Korea and their Hitachi EU-12 electron microscope. similarity to strain W254T was inferred from their Five per cent sheep blood BHI agar for haemolysis; a morphological resemblance to this strain (Bak & Azuma, TMVL or BHI culture for catalase; 0.1 % KNO BHI broth 1980, 1991). Six more strains were isolated from swine 3 for nitrate reduction; 1 % yeast extract Trypticase (BBL) tonsils in Japan (Azuma et al., 1989). Morphological and broth for indole production and SSR (Stuart, van Stratum physiological studies were performed on all of the isolates & Rustigan) medium for urea hydrolysis, were used and strains W254T, HT1-19 and HT5-225 were also (Cowan, 1974). Gelatin liquefaction was performed using subjected to chemotaxonomic and genetic characterization. a denatured gelatin block (Kohn, 1953). Skim-milk (10 %) In parallel with the microbiological characterization, added to BHI was used for the milk decomposition test. pathology studies on natural and experimentally induced For sugar utilization, basal semi-solid medium consisting lesions have been conducted (Murakami et al., 1991 and of polypeptone (Daigo) 1 %, yeast extract (Difco) 0.2 %, 1992). In this paper, we report the taxonomic character- NaCl 0.5 %, Andrade’s indicator (1 ml) and bactoagar ization of the isolates. (Difco) 0.2 % was used. Each sugar (10 % w/v) was All of the strains were isolated using the gas jet method with sterilized by autoclaving with the exception of L-arabinose, MVL medium (Azuma & Suto, 1970; Azuma & Bak, 1980; xylose and ribose which were filter-sterilized. All carbon Azuma & Ito, 1987; Cote, 1992). In some cases a selective sources were added to the basal medium at a final medium, MVL containing vancomycin (5 mgml21), concentration of 1 % (w/v). Sugars used are indicated in kanamycin (50 mgml21), nalidixic acid (250 mgml21)and Table S1 (available in IJSEM Online). cephaloridine (3.2 mgml21) (Azuma et al., 1992), was used. The isomer of diaminopimelic acid (DAP) in the Maintenance cultures of the isolates for inoculating test peptidoglycan was determined as described by Azuma media were grown on slants of MVL agar for 3–5 days, at et al. (2009). A standard amino acid mixture solution 37 uC. For growth under aerobic conditions, brain heart (Type H, Wako Pure Chemical Industries) and a mixture infusion (BHI) or TS (Tripto-soy agar; Eiken) were used. prepared from DAP, ornithine–HCl and N-acetyl muramic Growth under an atmosphere of 10 % CO was also tested 2 acid (all from Wako Pure Chemical Industries) were used on BHI and TS plates. As indicated above, strain W254T was as standards. Whole cell sugars were determined by the isolated from an infected swine submandibular lymph node method of Staneck & Roberts (1974) using descending in Japan in 1971 and the other strains were isolated from paper chromatography. Phospholipids were determined swine tonsils in Korea and Japan from 1978 to 1988. by the method of Folch et al. (1957). For detection of Dermatophilus congolensis ATCC 14637T was used as a phosphorus, amino groups and reducing sugars, the reference strain. TMVL medium, in which 0.1 % Tween 80 is Dittmer, ninhydrin and diphenylamine reagents were used, added, was used in place of MVL medium. respectively. As standards, diphosphatidylglycerol (A42, Strain W254T and selected other strains were examined by Funakoshi), phosphatidylglycerol (A431, Funakoshi) and scanning electron microscopy (SEM) and transmission phosphatidylinositol (A39, Funakoshi) were applied to the electron microscopy (TEM) of ultrathin sections. The same plate. Cellular fatty acids were determined as isolates were also examined by Gram-, Loffler methylene described by Azuma et al. (1992) for strains W254T, blue- (Cowan, 1974), lactophenol- (anonymous 1989) and HT1-19, HT5-225 and D. congolensis ATCC 14637T grown flagellar-staining (Research Group 1951). For SEM, the in TS broth (Rohde, 1973) at 37 uC for 5 days. The fatty specimens were fixed with both 2 % glutaraldehyde and acids were analysed using a model 163 gas chromatograph 1 % osmium oxide, then dehydrated, critical point dried, (Hitachi) equipped with a PEG-HT column, (0.25 mm coated with Palladium–Platinum and viewed under a internal diameter 6 50 m; GL Science). A M-80B double Hitachi S-450 electron microscope. For TEM, the same focusing mass spectrometer (Hitachi) was also used for the

Downloaded from www.microbiologyresearch.org by 2546 International Journal of Systematic and Evolutionary Microbiology 63 IP: 54.70.40.11 On: Sun, 25 Nov 2018 09:23:34 Tonsilliphilus suis gen. nov., sp. nov. confirmation of some fatty acids. Menaquinones were according to the manufacturer’s directions. Sequence determined by the method of Collins et al. (1977) using an analysis was outsourced to Greiner Japan (Tokyo). HPLC (LC 6A; Shimadzu) with an Ultron S-C column 18 The 16S rRNA gene sequences of strains W254T, HT1-19 (Chromato Pack) and a mass spectrometer (QP-1000). The and HT5-225 were investigated with those of other bacteria G+C content of the DNA was determined according to the of the suborder Micrococcineae (Schumann et al., 2009) method of Ezaki et al. (1990). using MEGA software version 5 (Tamura et al., 2011). All the DNA–DNA hybridization experiments were performed sequences registered in GenBank were retrieved from according to the method of Tamura et al. (2000). Strains positions equivalent to positions 41 to 1475 of the sequence W254T, HT1-19 and HT5-225 were cultured at 37 uC for of Escherichia coli (accession number J01695) and subjected 1 week in TMVL broth and their DNAs were extracted. to analysis. Alignment of these sequences was carried out Calf thymus DNA was used as a negative control. using CLUSTAL W and phylogenetic trees were reconstructed by maximum-likelihood and maximum-parsimony meth- Genomic DNA was used for amplification of the 16S rRNA ods using MEGA version 5. Bootstrap values were deter- gene as described by Saiki et al. (1988). The amplification mined based on 1000 replications. was carried out with a TaKaRa PCR amplification kit. For cloning, the PCR products were ligated into the pGEM-T The 17 strains examined were isolated anaerobically under vector (Promega) using the TaKaRa ligation kit ver. 2. 100 % CO2. Cultures on BHI under 100 % CO2 grew better Plasmid purification from Escherichia coli strain JM109 was than those incubated under aerobic conditions. The growth performed with a Qiagen plasmid mini kit. Five clones of cultures of D. congolensis ATCC 14637T incubated under were pooled for the sequencing reaction in order to reduce 100 % CO2 was distinctly poorer than that of those possible errors due to the Taq DNA polymerase. The DNA incubated under aerobic conditions. Incubation of the T sequences of cloned fragments from strains W254 and isolates on agar slants under 100 % CO2 resulted in dense HT1-19 and HT5-225 were determined by using an and granular colonies or irregular, rough colonies of Applied Biosystems model 377 DNA sequencer and big various sizes. However, under aerobic conditions the dye primer cycle sequencing kit (Applied Biosystems) colonies were larger, harder and had various shapes (e.g.

(a) (b) (c)

(d) (e)

Fig. 1. (a) Tonsilliphilus suis sp. nov. strain 79, 12-1 grown on BHI agar in 10 % CO2 for 11 days at 37 6C. Prepared by the lactophenol method. A radiating segmented thallus in an early stage of growth. Bar, 10 mm. (b) Strain 79, 12-1 grown on BHI agar

in 10 % CO2 for 11 days at 37 6C. Prepared by the lactophenol method. A chlamydospore on a mature thallus is shown (indicated with an arrow). Bar, 10 mm. (c) Light micrograph of Tonsilliphilus suis sp. nov. strain 79, 3-1 grown on BHI agar in 10 % CO2 for 8 days at 37 6C. Prepared using Loffler’s methylene blue staining method. A thallus is shown in which segmentation is visible as well as coccoid forms in locules. Inset: a chlamydospore (arrow) is shown. Bar, 500 nm. (d) Transmission electron micrograph of T strain W254 grown on a MVL slant in 100 % CO2 for 5 days at 37 6C showing a cross section through a tuber. The development of coccoid forms in multilocules can be seen. Bar, 10 mm. (e) Strain 79, 12-1 grown on TS agar in 10 % CO2 for 3 days at 37 6C showing zoospores with flagella. Prepared by the flagellar staining method. Bar, 10 mm.

Downloaded from www.microbiologyresearch.org by http://ijs.sgmjournals.org 2547 IP: 54.70.40.11 On: Sun, 25 Nov 2018 09:23:34 R. Azuma and others umbonate and molar tooth colonies). On occasion, whole Chemotaxonomic characteristics are shown in Table 1. The colonies were covered with a slimy substance. isomer of DAP, the phospholipid pattern (Azuma et al., 1989) and the major menaquinone of the novel strains The dominant cellular morphology was short radial were the same as those of D. congolensis ATCC 14637T. protrusions or thalli (Fig. 1a). The thalli developed into However, the whole cell sugar pattern of the novel strains tubers, by growing wider (Fig. 1b). Horizontal and vertical contained four sugars, including madurose (Azuma et al., segmentation occurred in the tubers and thalli, which led 1989), whereas D. congolensis ATCC 14637T contains only to formation of multi-loculi (Fig. 1c) in which coccoid cells madurose. later appeared (Fig. 1d). A single flagellated zoospore (Fig. 1e) emerged from each mature locule. Each zoospore The cellular fatty acid profiles of the novel strains and D. germinated to become a resting cell. Budding of the resting congolensis ATCC 14637T were different, with those of the cells led to the development of a young thallus. Sometimes novel strains containing fewer fatty acids. The difference in chlamydospores were seen (Figs 1b, d). the DNA G+C content of the novel strains and D. congolensis ATCC 14637T was marked. All of the strains were positive for nitrate reduction, but negative for catalase, indole, urea hydrolysis, gelatin The homologous reassociation values between labelled liquefaction, milk decomposition and haemolysis. D. DNA of strain W254T and unlabelled DNA of strains congolensis ATCC 14637T produced the opposite result HT5-225 and HT1-19 were 82.9 % and 86.4 %, respect- for each of these tests (except indole production). The pig ively. The DNA relatedness values between labelled DNA isolates utilized 16 of 26 compounds tested as sole carbon of strain HT5-225 and unlabelled DNA of strains W254T sources. Seven compounds were not utilized and the results and HT1-19 were 85.2 % and 87.5 %, respectively. The for ribose, cellobiose and soluble starch varied between the DNA relatedness values between labelled DNA of strain strains. D. congolensis ATCC 14637T utilized only four HT1-19 and unlabelled DNA of strains W254T and HT5- sugars (Table S1). Thus the pig isolates are saccharolytic, 225 were 88.3 % and 89.4 %, respectively. The levels of whereas D. congolensis ATCC 14637T is proteolytic. DNA relatedness for the three strains did not fall below

Table 1. Chemotaxonomic characteristics that differentiate strains W254T, HT1-19 and HT5-225 from their close relatives

Strains: 1, W254T; 2, HT1-19; 3, HT5-225; 4, D. congolensis ATCC 14637T;5,Piscicoccus intestinalis Ngc37-23T. For strain W254T: MVL glucose (MVLG) was used for amino acids and whole cell sugars; MVLG broth was used for cellular fatty acids, menaquinone and DNA G+C content; BHI broth was used for phospholipids. For D. congolensis ATCC 14637T: BHI glucose (BHIG) was used for amino acids and whole cell sugars; BHIG broth was used for cellular fatty acids, menaquinones and DNA G+C content. BHI broth was used for phospholipids. All strains contain meso-DAP.

Characteristic 1 2 3 4 5*

Whole-cell Galactose, glucose, Galactose, glucose, Galactose, glucose, Madurose Ribose, Mannose sugars mannose, madurose mannose, madurose mannose, madurose Phospholipids Phosphatidylinositol Phosphatidylinositol Phosphatidylinositol Phosphatidylinositol Phosphatidylglycerol, phosphatidylglycerol, phosphatidylglycerol, phosphatidylglycerol, phosphatidylglycerol, Phosphatidylinositol piphosphatidylglycerol diphosphatidylglycerol diphosphatidylglycerol diphosphatidylglycerol lyso-Phosphatidyle- thanolamine Cellular fatty acids (%)D

C13 : 0 2.6 Un. 1.2

C14 : 0 9.4 1.3 C15 : 0 23.3 23.1 35.7 30.0 7.4 Un. 1.4

C16 : 0 5.9 2.9 2.4 16.7 7.4 C17 : 0 39.5 62.9 45.6 12.3 6.0 C17 : 1 24.4 16.7 23.9 C18 : 0 3.0 2.3 1.6 4.1 Menaquinone MK-8(H4) MK-8(H4) MK-8(H4) MK-8(H4) MK-8(H4) DNA G+C 72.2 67.2 71.5 58.99 71.5 content (mol%)

T *P. intestinalis Ngc37-23 also contains major amounts (.10 %) of iso-C14 : 0 and iso-C16 : 0. DData taken from Hamada et al. (2010).

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Tonsilliphilus suis HT1-19 (AB096085) 100 0.05 Tonsilliphilus suis HT5-225 (AB602380) 90 Tonsilliphilus suis W254T (AB096084) 40 Dermatophilus congolensis DSM 44180T (AJ243918) 48 Piscicoccus intestinalis Ngc37-23T (AB550799) 61 Kineosphaera limosa Lpha5T (AF109792)

T 82 Mobilicoccus pelagius Aji5-31 (AB550798) 47 Austwickia choelonae DSM 44178T (AJ243919) 68 Dermacoccus nishinomiyaensis DSM 20448T (X87757) 23 Intrasporangium calvum DSM 43043T (CP002343) Brevibacterium linens DSM 20425T (X77451) Dermabacter hominis DSM 7083T (X91034) 26 T 57 20 Micrococcus luteus DSM 20030 (AJ36198) 88 Yaniella halotolerans YIM 70085T (AY228479)

48 Bogoriella caseilytica HKI 0088T (Y09911) Beutenbergia cavernae DSM 12333T (Y18378)

11 Ruania albidiflava 3-6T (DQ343153) 8 19 Promicromonospora citrea DSM 43110T (X83808)

9 Jonesia denitrificans DSM 20603T (X83811) 13 Rarobacter faecitabidus DSM 4813T (Y17870) 3 Sanguibacter keddieii ST-74T (X79450)

20 Demequina aestuarii JC2054T (DQ10160) 50 28 Actinotalea fermentans DSM 3133T (X83805) Microbacterium lacticum DSM 20427T (X77441) Cellulomonas flavigena DSM 20109T (X83799) Tropheryma whipplei Twist-MarseilleT (AF251035) Escherichia coli (J01695)

Fig. 2. Maximum-likelihood phylogenetic tree based on 16S rRNA gene sequences (1435 nt). The sequence of Escherichia coli (J01695) was used as an outgroup. Numbers at nodes indicate percentage bootstrap support based on a maximum- likelihood analysis of 1000 resampled datasets. Bar, 5 nt substitutions per 100 nt.

80 % for any pair. By convention, strains which exhibit in the family Dermatophilaceae, indicating that these three more than 70 % DNA relatedness are considered members strains belong to this family. of the same genomic species (Wayne et al., 1987). Strain W254T was sent to Dr H.A. Lechevalier in 1984. He When the common regions of the 16S rRNA gene informed us of the presence of meso-DAP and madurose sequences of strains W254T (1470 bp), HT1-19 (1517 bp) (Personal communication, 1984). Tonsillophilus suis was and HT5-225 (1480 bp) were analysed, only one base was subsequently included in Bergey’s manual (Gordon, 1989). different in HT1-19, indicating that the similarity between Although strain W254T was isolated from a submandibular these three strains was more than 99 %. Comparative lymph node, the other 16 strains were all isolated from sequence analysis showed that the 16S rRNA gene sequence swine tonsils (Azuma et al., 1990). From this, it is possible similarity between strain W254T and D. congolensis ATCC to suggest that the habitat of the isolates might be infected 14637T was 94.7 %. The maximum-likelihood phylogenic swine tonsils, and by chance the organisms invade tree (Fig. 2) and maximum-parsimony phylogenetic tree neighbouring lymph nodes. (Fig. S1) clearly revealed that strains W254T, HT1-19 and By cooperative pathological research, it became clear that HT5-225 formed a cluster with the type strains of bacteria the micro-organisms might preferably infect the swine

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tonsillar crypt (Bak & Azuma, 1991; Murakami et al., is MK-8(H4). The genus belongs to the family Dermatophi- 1991). Also, it was revealed that the incidence of tonsil laceae. The type species is Tonsilliphilus suis. infection reached 10.5–57.1 % in fattening pigs (Bak & Azuma, 1980; Murakami et al., 1991) and breeding sows (Shiozawa et al., 1991). With regard to the infection of the Description of Tonsilliphilus suis sp. nov. swine tonsillar crypt, Johne (1881, 1882) described the Tonsilliphilus suis (su9is. L. gen. n. suis of a pig). involvement of a fungus of the genus Echinobotryum. The organism sketched by him appears to be similar to one of General chemotaxonomic characteristics are as described the novel isolates. Gilka (1983) observed club-forming in the genus description. Cells show stronger growth under micro-organisms in swine tonsillar crypts by electron 100 % CO2 than under aerobic conditions. Cells develop microscopy. Experimental infection using the novel isolates into a short radial protrusion or thallus, which then in mice (Momotani et al., 1983), rats, guinea pigs develops into tubers. In both thalli and tubers, horizontal and vertical segmentation occurs to form multi-loculi. (Murakami et al., 1992) and sheep (Momotani et al., Thereafter, coccoid cells develop in the multi-loculi. A 1985) were performed, in which some pathogenic potential single flagellated zoospore emerges from each mature of the organisms was shown. locule. The zoospore germinates to become a resting cell. Comparative infectivity of the novel isolates and that of D. Budding of the resting cells leads to the development of a congolensis (Gordon, 1964) should be mentioned. The new thallus. Globose bodies or chlamydospores are novel isolates were associated with swine tonsillar crypts, sometimes formed. Colonies are dense and granular or while D. congolensis is associated with bovine hair follicles irregular and rough on the surface after growth on TMVL (Abu-Samra et al., 1976; Kinjo et al., 1981), which are slants in 100 % CO2 after 3–5 days at 37 uC. Positive for anatomically similar to tonsillar crypts. In both species, nitrate reduction but negative for catalase, indole produc- adaptive features of the micro-organisms to host animals, tion, urease, gelatin liquefaction and milk decomposition. that is, microbial elements, histological constituents and Not haemolytic. Aesculin, fructose, galactose, glucose, the process of lesion development seem very similar. The inositol, inulin, lactose, maltose, mannitol, mannose, novel isolates seem to have weak infectivity, because lesions melibiose, raffinose, salicin, sorbitol, sucrose and trehalose are confined to the tonsil itself, although spread from one are utilized as sole carbon sources, but adonitol, L- crypt to a neighbouring crypt or those of another host are arabinose, dulcitol, glycerin, melezitose, rhamnose and suspected. D. congolensis has a similar infection pattern and xylose are not used. The utilization of cellobiose, ribose and hair follicles in the skin of cattle are infected. The described starch varies between strains. The G+C content of the cases give the impression of serious illness, although some DNA is 69.6–71.8 mol%. Most strains have been isolated bad climatic or feeding conditions might also have been from inflamed swine tonsillar crypts. involved (Hyslop, 1979). One reason is the fact that D. The type strain, W254T (5ATCC 35846T5CCM congolensis has proteolytic and haemolytic ability. From the 3774T5DSM 21880T5JCM 15727T), was isolated from above consideration, the virulence of D. congolensis might an infected swine submandibular lymph node. be higher than that of the novel isolates. Other actinobacteria have been reported from swine tonsils. The presence of species of the genus Actinomyces Acknowledgements in swine tonsils was reported by Johne (1881), Davis (1923) The authors express sincere gratitude to Y .Okada, F. Yoshikawa, and Ogura (1925), and ‘Actinomyces suis’ was isolated from S. Ikemoto M. Shiozawa, Dr E. Momotani, Dr H. Fujiwara, Dr H. swine tonsils/the swine oral cavity (Oomi et al., 1994). Oomi, Dr T. Watanabe, Dr S. Suzuki, the late Dr N. Hara, the late Subsequently, the presence of the bacteria in swine tonsils Dr S. Inui and the late T. 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