Amycolatopsis kentuckyensis sp. nov., Amycolatopsis lexingtonensis sp. nov. and Amycolatopsis pretoriensis sp. nov., isolated from equine placentas

Over the past decade, actinomycetes have been reported to be a significant emergent cause of placentitis and abortion in horses in Kentucky (Giles et al., 1993; Hong et al., 1993; Donahue & Williams, 2000). The term nocardioform placentitis has been used to describe this distinct type of placentitis in horses, in which lesions are observed on the chorionic surface of the placenta and Gram-positive branching micro-organisms are recovered upon culture. The condition was first diagnosed in 1986 at the University of Kentucky Livestock Disease Diagnostic Center, but this type of placentitis has only recently been confirmed to occur outside Kentucky. In Kentucky, at least three different groups of bacteria (all Gram-positive branching bacilli) have been associated with nocardioform placentitis. Most of the severe infections were caused by the recently described actinomycete Crossiella equi (Donahue et al., 2002). A small number of strains isolated from placental lesions appear to be members of the genus Streptomyces, whereas additional strains identified as members of the genus Amycolatopsis have been isolated from placental lesions since the late 1980s. Two of these strains, isolated from lesions on placentas from mares in Kentucky during 1999, along with a strain isolated from an equine placenta in Pretoria, South Africa, during 2000 (Volkmann et al., 2001), were characterized in a polyphasic taxonomic study. Primary storage of strains was as lyophilized ampules of mycelial and spore suspensions in sterile beef serum held at 4 °C. Working stock cultures were maintained on slants of ATCC medium no. 172 (Cote et al., 1984) and stored at 4 °C until needed. Gross morphological observations of colonial characteristics were made after 14 days on ATCC medium no. 172 (Cote et al., 1984) and those described by Shirling & Gottlieb (1966) for the International Streptomyces Project. Biomass for extraction of DNA was grown as 7-day streak cultures on ATCC medium no. 172 agar plates. Chemotaxonomic analysis of strains for fatty acids, cell-wall diamino acid and whole-cell sugars was performed on autoclaved biomass by using previously described methods (Staneck & Roberts, 1974; Grund & Kroppenstedt, 1989; Saddler et al., 1991). Physiological tests were evaluated by using the media of Gordon et al. (1974). Allantoin hydrolysis was evaluated in the basal medium suggested by Gordon et al. (1974) for aesculin hydrolysis. Phosphatase activity was evaluated by using the method of Kurup & Schmitt (1973). Temperature range for growth was determined on slants of ATCC medium no. 172 agar (Cote et al., 1984). Genomic DNA was isolated, purified and sequenced following previously described procedures (Labeda & Kroppenstedt, 2000). A phylogenetic tree was constructed within the ARB software environment for sequence data, developed by Wolfgang Ludwig and Oliver Strunk, Lehrstuhl für Mikrobiologie, University of Munich, Germany (), using evolutionary distances by the method of Kimura (1980) and linkages by the neighbour-joining method of Saitou & Nei (1987). Genomic DNA was isolated and DNA relatedness between Amycolatopsis mediterranei NRRL B-3240^T and the equine isolates was determined spectrophotometrically in duplicate as described previously (Labeda, 1998). All three strains were found to have meso-diaminopimelic acid as the diamino acid in hydrolysates and arabinose and galactose as the primary sugars in whole-cell hydrolysates. The predominant phospholipid was phosphatidylethanolamine with small amounts of phosphatidylmethylethanolamine, while the primary menaquinones were MK-9(H₂) and MK-9(H₄). These chemotaxonomic properties are very characteristic of members of the genus Amycolatopsis. Fatty acid methyl ester profiles of all three strains (Table 1) consisted of straight- and branched-chain fatty acids, also typical of Amycolatopsis. Moreover, these micro-organisms produce branching vegetative mycelium that fragments into ovoid rod-shaped arthrospores, which is typically observed for members of this genus. Morphological and chemotaxonomic properties of the strains are in good agreement with those described by Lechevalier et al. (1986), when the genus Amycolatopsis was first proposed.

Table 1. Fatty acid content of equine Amycolatopsis species Species: 1, A. kentuckyensis NRRL B-24129T; 2, A. lexingtonensis NRRL B-24131T; 3, A. pretoriensis NRRL B-24133T. Values are percentages of total fatty acids present; minor components are not shown.

The 16S rRNA gene sequences determined in this study for equine isolates NRRL B-24129^T, NRRL B-24131^T and NRRL B-24133^T and A. mediterranei NRRL B-3240^T have been deposited in GenBank under accession numbers AY183357, AY183358, AY183356 and AY184424, respectively. Phylogenetic analysis indicates that all of the equine isolates are closely related to A. mediterranei NRRL B-3240^T with a bootstrap value of 95 % by neighbour-joining analysis, as can be seen clearly in Fig. 1. Tree topographies from the maximum-parsimony and maximum-likelihood methods were very similar. The topographies strongly suggest that the strains represent individual species, with 16S rRNA gene sequence similarities between them ranging from 99·2 to 99·8 % and similarity to A. mediterranei NRRL B-3240^T ranging from 98·8 to 99·4 %. Determinations of DNA relatedness among these strains and with A. mediterranei NRRL B-3240^T, as shown in Table 2, confirm that these strains represent distinct novel species, based on whole-genomic DNA relatedness of significantly less than 70 % among all three equine strains and with A. mediterranei NRRL B-3240^T.

(39K): Fig. 1. Phylogenetic tree of the genus Amycolatopsis, calculated from 16S rDNA sequences by using Kimura's evolutionary distance method (Kimura, 1980) and the neighbour-joining method of Saitou & Nei (1987). Bootstrap values, expressed as percentages of 500 replications, are given at branch-points. Bar, 0·01 nucleotide substitutions per site.

Table 2. DNA relatedness (%) between A. mediterranei NRRL B-3240T and equine Amycolatopsis species Strains: 1, A. mediterranei NRRL B-3240T; 2, A. kentuckyensis NRRL B-24129T; 3, A. lexingtonensis NRRL B-24131T; 4, A. pretoriensis NRRL B-24133T.

Gross morphological characteristics and differential physiological properties of these strains (as shown in Table 3) are consistent with molecular systematic observations and demonstrate clearly that the strains are different from each other, as well as from other described species in the genus Amycolatopsis. It is proposed that the following novel species should be created with their respective type strains: Amycolatopsis kentuckyensis NRRL B-24129^T, Amycolatopsis lexingtonensis NRRL B-24131^T and Amycolatopsis pretoriensis NRRL B-24133^T. Formal descriptions of each of these species follow. Additional actinomycete strains isolated from equine placentas in Kentucky during the 1999 and 2002 breeding seasons are currently under study to determine if additional strains that are representative of these new taxa can be found, as well as additional novel species.

Table 3. Differential properties of A. kentuckyensis, A. lexingtonensis and A. pretoriensis compared with previously described species of the genus Amycolatopsis Taxa: 1, A. kentuckyensis NRRL B-14129T; 2, A. lexingtonensis NRRL B-24131T; 3, A. pretoriensis NRRL B-24133T; 4, A. mediterranei NRRL B-3240T; 5, Amycolatopsis alba NRRL 18532T; 6, Amycolatopsis albidoflavus IMSNU 22139T; 7, Amycolatopsis azurea NRRL 11412T; 8, Amycolatopsis coloradensis NRRL 3218T; 9, Amycolatopsis eurytherma DSM 44348T; 10, Amycolatopsis fastidiosa NRRL B-16697T; 11, Amycolatopsis japonica DSM 44213T; 12, Amycolatopsis methanolica NBRC 15065T; 13, Amycolatopsis orientalis subsp. orientalis NRRL 2450T; 14, Amycolatopsis rubida JCM 10871T; 15, Amycolatopsis sacchari DSM 44468T; 16, Amycolatopsis sulphurea DSM 46092T; 17, Amycolatopsis thermoflava NBRC 14333T. +, Positive; -, negative; W, weak reaction.

Description of Amycolatopsis kentuckyensis sp. nov.
Amycolatopsis kentuckyensis (ken.tuc.ky.en'sis. N.L. fem. adj. kentuckyensis from Kentucky, named after the place of origin, state of Kentucky, USA).

Well-developed, yellow-orange to brownish-orange substrate mycelium is produced on most media. Aerial mycelium that ranges from light orangish-white to greyish orange-white in colour is produced on most media. A faint brownish soluble pigment is produced on some media. Chemotaxonomic characteristics are typical of the genus Amycolatopsis. Casein, aesculin, gelatin, hypoxanthine, tyrosine, urea and hippurate are hydrolysed or decomposed. Adenine, allantoin, starch and xanthine are not hydrolysed or decomposed. Nitrate is not reduced. Phosphatase is produced. Acetate and citrate are assimilated. Benzoate, lactate, malate, mucate, oxalate, propionate, succinate and DL-tartrate are assimilated weakly, if at all. Acid is produced from adonitol, arabinose, cellobiose, dextrin, dulcitol, D-fructose, D-galactose, D-glucose, glycerol, myo-inositol, lactose, maltose, D-mannose, melibiose, methyl α-D-glucoside, raffinose, rhamnose, salicin, D-sorbitol, sucrose and xylose. Acid is not produced from meso-erythritol, mannitol, melezitose or methyl β-xyloside. Grows in the presence of up to 5 % (w/v) NaCl. Temperature range for growth is 1542 °C.

The type strain is NRRL B-24129^T (=LDDC 9447-99^T=DSM 44652^T). Isolated from an equine placenta in Lexington, Kentucky. Implicated in nocardioform placentitis in mares.

Description of Amycolatopsis lexingtonensis sp. nov.
Amycolatopsis lexingtonensis (lex.ing.ton.en'sis. N.L. fem. adj. lexingtonensis from Lexington, named after the place of origin, Lexington, Kentucky, USA).

Abundant dark orange-brown to dark reddish-brown substrate mycelium is produced on most media. Copious aerial mycelium is produced on most media, ranging in colour from light yellow to purplish-tan. A dark red to reddish-brown soluble pigment is produced on most media tested. Chemotaxonomic characteristics are typical of the genus Amycolatopsis. Casein, aesculin, gelatin, hypoxanthine, tyrosine, urea and hippurate are hydrolysed or decomposed. Adenine, allantoin, starch and xanthine are not hydrolysed or decomposed. Nitrate is reduced. Phosphatase is produced. Acetate, citrate, oxalate and propionate are assimilated. Benzoate, lactate, malate, mucate, succinate and DL-tartrate are not assimilated. Acid is produced from adonitol, arabinose, cellobiose, dextrin, D-fructose, D-galactose, D-glucose, glycerol, myo-inositol, maltose, D-mannose, melibiose, methyl α-D-glucoside, raffinose, rhamnose, salicin, sucrose and xylose. Acid is produced weakly from dulcitol, erythritol and mannitol. Acid is not produced from melezitose, methyl β-xyloside, sorbitol or trehalose. Grows in the presence of 5 % (w/v) NaCl. Temperature range for growth is 1542 °C.

The type strain is NRRL B-24131^T (=LDDC 12275-99^T=DSM 44653^T). Isolated from an equine placenta in Kentucky.

Description of Amycolatopsis pretoriensis sp. nov.
Amycolatopsis pretoriensis (pre.tor.i.en'sis. N.L. fem. adj. pretoriensis from Pretoria, named after the place of origin, Pretoria, South Africa).

Well-developed greyish-yellow to orange-brown substrate mycelium is produced on most media. Abundant production of white to orange-white aerial mycelium occurs on most media. Faint soluble pigments are produced on some media, such as yeast extract/malt extract agar. Chemotaxonomic characteristics are typical of the genus Amycolatopsis. Casein, aesculin, gelatin, hypoxanthine and hippurate are hydrolysed or decomposed. Tyrosine and urea are decomposed weakly. Adenine, allantoin, starch and xanthine are not hydrolysed or decomposed. Nitrate is not reduced. Phosphatase is produced. Acetate is assimilated. Benzoate, citrate, lactate, oxalate, propionate and succinate are assimilated weakly. Malate, mucate and tartrate are not assimilated. Acid is produced from arabinose, cellobiose, dextrin, dulcitol, erythritol, D-fructose, D-galactose, D-glucose, glycerol, myo-inositol, lactose, maltose, D-mannose, melibiose, methyl α-D-glucoside, raffinose, rhamnose, salicin, sucrose, trehalose and xylose. Acid is produced weakly from D-sorbitol. Acid is not produced from adonitol, mannitol, melezitose or methyl β-xyloside. Grows in the presence of 5 % (w/v) NaCl. Temperature range for growth is 1537 °C.

The type strain is NRRL B-24133^T (=ARC OV1 0181^T=DSM 44654^T). Isolated from an equine placenta in Pretoria, South Africa.

The able technical assistance of E. N. Hoekstra with physiological characterization, DNA isolation and purification and 16S rDNA sequencing is gratefully acknowledged. Names are necessary to report factually on available data; however, USDA neither guarantees nor warrants the standard of the product and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.