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

¹ Microbial Genomics and Bioprocessing Research Unit, National Center for Agricultural Utilization Research, USDA Agricultural Research Service, 1815 North University Street, Peoria, IL 61604, USA
² Livestock Disease Diagnostic Center, Department of Veterinary Science, University of Kentucky, Lexington, KY 40511, USA
³ Golden Vetlab, PO 1537, Alberton, South Africa

Correspondence
D. P. Labeda
labedadp{at}mail.ncaur.usda.gov

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Actinomycete strains isolated from lesions on equine placentas from two horses in Kentucky and one in South Africa were subjected to a polyphasic taxonomic study. Chemotaxonomic and morphological characteristics indicated that the isolates are members of the genus Amycolatopsis. On the basis of phylogenetic analysis of 16S rDNA sequences, the isolates are related most closely to Amycolatopsis mediterranei. Physiological characteristics of these strains indicated that they do not belong to A. mediterranei and DNA relatedness determinations confirmed that these strains represent three novel species of the genus Amycolatopsis, for which the names Amycolatopsis kentuckyensis (type strain, NRRL B-24129^T=LDDC 9447-99^T=DSM 44652^T), Amycolatopsis lexingtonensis (type strain, NRRL B-24131^T=LDDC 12275-99^T=DSM 44653^T) and Amycolatopsis pretoriensis (type strain, NRRL B-24133^T=ARC OV1 0181^T=DSM 44654^T) are proposed.

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of Amycolatopsis kentuckyensis NRRL B-24129^T, Amycolatopsis lexingtonensis NRRL B-24131^T, Amycolatopsis pretoriensis NRRL B-24133^T and Amycolatopsis mediterranei NRRL B-3240^T are AY183357, AY183358, AY183356 and AY184424, respectively.

	INTRODUCTION

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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.

	METHODS

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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 (http://www.arb-home.de), 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).

	RESULTS AND DISCUSSION

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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.

View larger version (39K): [in this window] [in a new window]   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.

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 -D-glucoside, raffinose, rhamnose, salicin, D-sorbitol, sucrose and xylose. Acid is not produced from meso-erythritol, mannitol, melezitose or methyl -xyloside. Grows in the presence of up to 5 % (w/v) NaCl. Temperature range for growth is 15–42 °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 -D-glucoside, raffinose, rhamnose, salicin, sucrose and xylose. Acid is produced weakly from dulcitol, erythritol and mannitol. Acid is not produced from melezitose, methyl -xyloside, sorbitol or trehalose. Grows in the presence of 5 % (w/v) NaCl. Temperature range for growth is 15–42 °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 -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 -xyloside. Grows in the presence of 5 % (w/v) NaCl. Temperature range for growth is 15–37 °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.

	ACKNOWLEDGEMENTS

 
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.

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Cote, R., Dagget, P.-M., Gantt, M. J., Hay, R., Jong, S.-C. & Pienta, P. (1984). ATCC Media Handbook. Rockville, MD: American Type Culture Collection.

Donahue, J. M. & Williams, N. M. (2000). Emergent causes of placentitis and abortion. Vet Clin North Am Equine Pract 16, 443–456.[Medline]

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Giles, R. C., Donahue, J. M., Hong, C. B. & 7 other authors (1993). Causes of abortion, stillbirth, and perinatal death in horses: 3,527 cases (1986–1991). J Am Vet Med Assoc 203, 1170–1175.[Medline]

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Grund, E. & Kroppenstedt, R. M. (1989). Transfer of five Nocardiopsis species to the genus Saccharothrix Labeda et al. 1984. Syst Appl Microbiol 12, 267–274.

Hong, C. B., Donahue, J. M., Giles, R. C., Jr & 7 other authors (1993). Etiology and pathology of equine placentitis. J Vet Diagn Invest 5, 56–63.[Abstract/Free Full Text]

Kim, B., Sahin, N., Tan, G. Y. A., Zakrzewska-Czerwinska, J. & Goodfellow, M. (2002). Amycolatopsis eurytherma sp. nov., a thermophilic actinomycete isolated from soil. Int J Syst Evol Microbiol 52, 889–894.[Abstract]

Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120.[CrossRef][Medline]

Kurup, P. V. & Schmitt, J. A. (1973). Numerical taxonomy of Nocardia. Can J Microbiol 19, 1035–1048.[Medline]

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Labeda, D. P. & Kroppenstedt, R. M. (2000). Phylogenetic analysis of Saccharothrix and related taxa: proposal for Actinosynnemataceae fam. nov. Int J Syst Evol Microbiol 50, 331–336.[Abstract]

Lechevalier, M. P., Prauser, H., Labeda, D. P. & Ruan, J.-S. (1986). Two new genera of nocardioform actinomycetes: Amycolata gen. nov. and Amycolatopsis gen. nov. Int J Syst Bacteriol 36, 29–37.[Abstract/Free Full Text]

Saddler, G. S., Tavecchia, P., Lociuro, S., Zanol, M., Colombo, E. & Selva, E. (1991). Analysis of madurose and other actinomycete whole cell sugars by gas chromatography. J Microbiol Methods 14, 185–191.

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