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Veterinary Microbiology 157 (2012) 471–475

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lation of Actinomyces hyovaginalis from sheep and comparison withlates obtained from pigs

offrey Foster a,*, Peter Wragg b, Mark S. Koylass c, Adrian M. Whatmore c, Lesley Hoyles d

Consulting Veterinary Services, Drummondhill, Stratherrick Road, Inverness IV2 4JZ, UK

imal Health and Veterinary Laboratories Agency – Thirsk Regional Laboratory, Thirsk, North Yorkshire YO7 1PZ, UK

partment of Bacteriology, Animal Health and Veterinary Laboratories Agency – Weybridge, New Haw, Addlestone, Surrey KT15 3NB, UK

d Microbial Sciences Unit, Department of Food and Nutritional Sciences, University of Reading, Whiteknights Campus, Reading RG6 6UR, UK

ntroduction

In 1991, during a study on Actinomyces-like bacterialated from purulent lesions in pigs, two phenotypically

ogenous groups of uncertain taxonomic positionre described (Hommez et al., 1991). The two groupsre labelled groups II and III and differed from onether in respect of colonial appearance, hippuraterolysis, nitrate reduction, b-glucuronidase produc-

and cellobiose fermentation. Actinomyces hyovagi-

is was subsequently described for the isolatesonging to group II, which comprised 11 strains that

all been collected from samples of purulent vaginalcharge or aborted foetuses (Collins et al., 1993). Groupontained 29 strains recovered from samples collected

various non-vaginal sites that included kidney, lung,es, uterus, joint, liver and urinary bladder. Group IIIins, however, were later reported to constitute a

different biotype of A. hyovaginalis on the basis of analysisof tRNA intergenic length polymorphisms, 16S rRNA genesequencing and DNA–DNA hybridization (Storms et al.,2002). In 2003, Aalbeck and colleagues found A. hyova-

ginalis to be the predominant isolate following culture ofdisseminated necrotic lesions in slaughter pigs con-demned during post mortem meat inspection at anabattoir. In the same study A. hyovaginalis was alsorecovered from two of ten pigs with bronchial ectasias.The phenotypic characters of these isolates correspondedto those described by Storms et al. (2002) with theexception of a-galactosidase (Aalbeck et al., 2003). Afurther isolate, from a lung lesion in a four-week-oldpiglet, confirmed an association with lung disease(Reichel and Wragg, 2007).

To date A. hyovaginalis has been predominantlyreported from pigs. We wish to report for the first timethe recovery of A. hyovaginalis recovered from infectedsites in sheep and a moufflon following routine submis-sions to diagnostic veterinary laboratories in the UK andtheir comparison with porcine isolates recovered in thesame laboratories during the same period.

T I C L E I N F O

le history:

ived 5 August 2011

ived in revised form 28 December 2011

pted 2 January 2012

ords:

nomyces hyovaginalis

p

cess

A B S T R A C T

Actinomyces hyovaginalis, an organism initially described from pigs, was recovered from

nine sheep and a moufflon. Further strains of A. hyovaginalis were recovered from five

samples from pigs over the same period. 16S rRNA sequencing and extensive phenotyping

demonstrated high similarity between the ovine and porcine isolates; however differences

with respect to erythritol, adonitol and L-arabitol fermentation were detected. Ovine

isolates were made from various sample sites including abscesses and highlight the

importance of the accurate identification of the various coryneform isolates which affect

sheep. A. hyovaginalis can be added to the growing list of coryneforms which can cause

disease in sheep including Corynebacterium pseudotuberculosis, Trueperella pyogenes and

Arcanobacterium pluranimalium.

� 2012 Elsevier B.V. All rights reserved.

Corresponding author. Tel.: +44 1463 243030; fax: +44 1463 711103.

E-mail address: Geoffrey.Foster@sac.co.uk (G. Foster).

Contents lists available at SciVerse ScienceDirect

Veterinary Microbiology

jo u rn al ho m epag e: ww w.els evier .c o m/lo cat e/vetmic

8-1135/$ – see front matter � 2012 Elsevier B.V. All rights reserved.

10.1016/j.vetmic.2012.01.003

G. Foster et al. / Veterinary Microbiology 157 (2012) 471–475472

2. Materials and methods

2.1. Samples

Sheep and pig carcasses were submitted to diagnosticveterinary laboratories in Scotland and England fornecropsy to determine cause of death and for diseasesurveillance purposes. Further samples were submitted aspus samples mostly collected from suspect caseouslymphadenitis (CLA) cases.

2.2. Isolation and identification of bacterial isolates

All tissues selected for culture were plated on Columbiaagar (Oxoid, Basingstoke, UK) supplemented with 5% sheepblood incubated at 37 8C in a capnophilic atmosphere andMacConkey agar (Oxoid) incubated aerobically at the sametemperature. In addition samples from pigs were alsocultured on chocolate agar (Oxoid). In some casesanaerobic culture was made on pre-reduced fastidiousanaerobe agar (Oxoid). Isolates were initially checked forGram morphology, production of catalase and growth wascompared in aerobic, capnophilic and anaerobic atmo-spheres. Actinomyces-like colonies which were Grampositive irregular rods, catalase negative, capnophilicand non-haemolytic were further tested with the APICoryne, ID 32Strep, API 50CH and API ZYM kits (BioMer-ieux, Basingstoke, UK) according to the manufacturer’sinstructions.

2.3. Sequencing

Strains P31/96/2, S45/96/1, P60000/99/1, S577/98/3and S60396/99/3 were sent to the University of Readingand strain P60074/02/02 was sent to the Animal Healthand Veterinary Laboratories Agency for determination oftheir 16S rRNA gene sequences as described by Hoyleset al. (2004). The closest known relatives of the isolateswere determined by performing searches against an in-house database of Actinomyces and related sequences. Amultiple sequence alignment was created using CLUS-TALW within Geneious 4.6.1 (http://www.geneious.com),and corrected manually to omit gaps from the 50 and 30

ends from further analyses. A distance matrix (Kimura 2-correction parameter) was calculated using DNADIST, and

the phylogenetic tree constructed using the neighbour-joining (NEIGHBOR) method (Felsenstein, 1989). Stabilityof groupings was estimated by bootstrap analysis (500replications) using SEQBOOT, DNADIST, NEIGHBOR andCONSENSE (Felsenstein, 1989). The phenogram wasvisualized using DRAWGRAM (Felsenstein, 1989). Thesequences determined in this study have been depositedin GenBank: P60000/99/1, FR874094; S577/98/3,FR874093; S60396/99/3, FR874095; S45/96/1,AJ234065; P31/96/2, AJ234064; P60074/02/02,HE577000.

3. Results

Since 1996, eight strains of non-haemolytic Actino-

myces-like bacteria sharing similar colonial and pheno-typic properties have been recovered from sheepsubmitted from farms at various locations in the Highlandsand Islands of Scotland. Further similar strains wereisolated from the consolidated lung of a 20-month-oldmule sheep in Yorkshire, England and from an abscess in amoufflon which was housed in Scotland. The organismswere typically recovered in profuse growth mostly in pureculture but along with other organisms on four occasions.Details of the source for each isolate along with any otherorganisms recovered are given in Table 1.

Isolates were slow-growing and enhanced by theaddition of 5–10% CO2, producing pinpoint non-haemoly-tic colonies at 48 h. Over the same period A. hyovaginalis

isolates were recovered from five pig samples at differentlocations around the UK (Table 2). Isolates from pigstypically produced larger colonies of around 1 mm after48 h. All the sheep, moufflon and pig isolates grewanaerobically and were catalase negative. API profilesobtained with the API Coryne, ZYM and ID 32Strep kits forthe sheep isolates were typical of A. hyovaginalis and couldnot be distinguished from those obtained from pig isolatesover the same period (Table 3). API 50CH results arepresented in Table 4 and revealed differences between pigstrains and the sheep and moufflon strains with respect toerythritol, adonitol and L-arabitol fermentation. 16S rRNAgene sequencing of selected strains (P31/96/2, S45/96/1,P60000/99/1, S577/98/3, S60396/99/3 and P60074/02/02)confirmed their identity as A. hyovaginalis. All sequences

Table 1

Sources of sheep Actinomyces hyovaginalis isolates and other organisms recovered from the same site.

Ref. no. Site Other isolates

S45/96/1 Brain abscess None

S577/98/3 Chest-pericardial abscesses Streptococcus ovis, Trueperella pyogenes, Fusobacterium necrophorum subsp.

necrophorum, Bacteroides macacae

S60396/99/3 Liver, lung Liver – Streptococcus dysgalactiae, coliforms

Lung – Trueperella pyogenes

S601281/00/1 Abscess None

S603814/04/2 Pus ex-face Arcanobacterium pluranimalium. Anaerobic culture not attempted

S605243/06/1 Submandibular pus None. Anaerobic culture not attempted

S606198/07/2 Mandibular swelling None. Anaerobic culture not attempted

15/S47/01/09 Lung None. Anaerobic culture not attempted

S608653/10/2 Lung Mannheimia haemolytica, Fusobacterium necrophorum subsp. necrophorum,

Prevotella melaninogenica, Bacteroides sp.

S603837/04/1 Moufflon abscess None

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G. Foster et al. / Veterinary Microbiology 157 (2012) 471–475 473

re more similar to A. hyovaginalis UZCOR144 biotype IInBank accession no. AJ293302; 98.6–99.4% sequenceilarity) than A. hyovaginalis NCFB 2983T (GenBankession no. X69616; 98.2–98.8% sequence similarity). Inition, the strains shared a high degree of similarity withh other (Fig. 1).

iscussion

Our findings report for the first time A. hyovaginalis inep and a moufflon. The isolates were recovered fromgnostic material submitted to UK veterinary labora-es dating back to 1996. In seven of the ten cases, A.

vaginalis was cultured from abscesses (Table 1),ically in profuse growth and often in pure culture,icating their significance for their ovine hosts. Thesene strains of A. hyovaginalis produced smaller coloniesCSBA than isolates recovered from similar materialmitted from pigs in the UK over the same time period.ther consistent differences between ovine and porcineins could not be detected using API 20Strep, 32Strep

Coryne kits; however, carbohydrate tests with the APIH system did detect differences between these hosts,h the sheep and moufflon isolates alone producingative erythritol, adonitol and L-arabitol reactions (TableErythritol has been reported as a variable characteristicporcine A. hyovaginalis elsewhere (Aalbeck et al., 2003),

adonitol and L-arabitol reactions were consistentlyorted as positive for strains from pigs in other studies

le 2

rces of pig Actinomyces hyovaginalis isolates and other organisms recovered from the same site.

f. no. Site Other isolates

1/96/2 Foetal stomach, lung, liver, placenta Streptococcus hyovaginalis

0000/99/1 Joint abscess Streptococcus dysgalactiae subsp. equisimilis, Fusobacterium

necrophorum subsp. fundiliforme

00074/02/2 Umbilicus Streptococcus suis

/P243/8/03 Uterine fluid from sow with pyometra None

/P196/10/06 Lung abscess Mycoplasma hyopneumoniae. Anaerobic culture not attempted

le 3

Coryne, 32Strep and ZYM profiles for Actinomyces hyovaginalis isolates

sheep and pigs.

f. no. Coryne 32Strep ZYM

5/96/1 0570763 66006000000 2402070(2506070)a

77/98/3 0570763 66116001100 2402070(2406170)

0396/99/3 0570761 66006001100 2402010(2402070)

01281/00/1 0570763 66116001100 2402070(2406070)

03814/04/2 0570763 66016001100 2402050(2506070)

05243/06/1 0570763 66116001100 2002070(2406070)

06198/07/2 0570763 66116001100 2402050(2406350)

/S47/01/09 0570763 66116001310 2006050(2506170)

08653/10/2 0570763 66016001100 2402050(2406070)

03837/04/1 0570763 66106001300 2402170(2402370)

1/96/2 0570761 66106001220 2402270(6402370)

0000/99/1 0570761 66106003300 2402370(2406370)

00074/02/2 0570761 66116003300 2402170(2506370)

/P243/8/03 0570761 66106001310 2402170(2402370)

/P196/06 0570761 66106001310 2402370(2406370)

Table 4

API 50CH results for Actinomyces hyovaginalis isolates from sheep and

pigs.

Well Carbohydrate Sheep

(n = 9)

Moufflon

(n = 1)

Pig

(n = 5)

1 Glycerol 2/9 + +

2 Erythritol � � +

3 D-Arabinose + + +

4 L-Arabinose + + +

5 Ribose + + +

6 D-Xylose + + +

7 L-Xylose � � �8 Adonitol � � +

9 b-Methyl-D-xyloside � � �10 Galactose 2/9 + +

11 Glucose + + +

12 Fructose + + +

13 Mannose + + +

14 Sorbose � � �15 Rhamnose 4/9 � �16 Dulcitol � � �17 Inositol � � �18 Mannitol � � �19 Sorbitol � � �20 a-Methyl-D-mannoside � � �21 a-Methyl-D-glucoside � � 1/5

22 N-Acetylglucosamine 2/9 � +

23 Amygdalin 2/9 � +

24 Arbutin 1/9 + �25 Aesculin + + +

26 Salicin + + +

27 Cellobiose + + +

28 Maltose + + +

29 Lactose + + +

30 Melibiose � � �31 Sucrose + + +

32 Trehalose 1/9 � �33 Inulin � � �34 Melizitose � � �35 Raffinose 1/9 � �36 Starch + + 1/5

37 Glycogen + + 2/5

38 Xylitol � � �39 Gentiobose 1/9 + 3/5

40 Turanose + + +

41 Lyxose 8/9 + 4/5

42 Tagatose � � �43 D-Fucose � � �44 L-Fucose 7/9 + +

45 D-Arabitol � � �46 L-Arabitol � � +

47 Gluconate � � �48 2-Ketogluconate � � �

API ZYM scores are first recorded for strong reactions only, whilst

bers in parenthesis include both strong and weak reactions.49 5-Ketogluconate + + +

G. Foster et al. / Veterinary Microbiology 157 (2012) 471–475474

(Aalbeck et al., 2003; Hommez et al., 1991), indicating thatnegative results for these substrates may be specific toovine isolates.

Until recently A. hyovaginalis had been exclusivelyassociated with pigs with different phenotypic groups forvaginal and foetal isolates (Collins et al., 1993) and non-vaginal strains (Storms et al., 2002; Aalbeck et al., 2003). In2009, however, A. hyovaginalis was reported from a case oflymphadenitis in a goat (Schumacher et al., 2009).Unfortunately limited phenotypic characteristics wereprovided by the authors of the goat isolate so comparisonscannot be made with the ovine strains of A. hyovaginalis.

CLA caused by Corynebacterium pseudotuberculosis is animportant disease of small ruminants for which somecountries, including the UK, provide accreditationschemes. Our results demonstrate that A. hyovaginalis

can present as the sole pathogen in abscesses of sheep or inmixed growth with other aerobic and anaerobic isolates.Six of nine isolates of A. hyovaginalis from sheep wererecovered from samples collected from abscess material, atleast three of which were submitted for suspected CLA.Taken together with its recovery from a case of caprinelymphadenitis (Schumacher et al., 2009) and an abscess ina moufflon from this study, A. hyovaginalis should beconsidered as yet another coryneform bacterium that is a

potential cause of skin abscesses and other sites in smallruminants. It is important that diagnostic laboratoriesdistinguish coryneform isolates from skin abscesses ofsmall ruminants in particular, which may be due to A.

hyovaginalis, C. pseudotuberculosis, Trueperella pyogenes

and Arcanobacterium pluranimalium, particularly whereCLA accreditation schemes are in place. Indeed on one ofthe farms included in this study (S606198/07/2), swabswere submitted from abscesses of four sheep with A.

hyovaginalis, C. pseudotuberculosis and T. pyogenes eachrecovered from single animals (unpublished results),emphasising the importance of sampling several animalsto ensure detection of C. pseudotuberculosis where CLA issuspected. A lack of haemolysis readily distinguishes A.

hyovaginalis from the strongly b-haemolytic T. pyogenes

and A. pluranimalium as well as the more slowly developinghaemolysis for C. pseudotuberculosis, which is also dis-tinguished further from A. hyovaginalis and T. pyogenes byits positive catalase reaction.

Acknowledgements

This work was funded by the Scottish Government aspart of its Public Good Veterinary and Advisory Service.VLA surveillance activities receive funding as part of the

Fig. 1. Neighbour-joining tree, based on an analysis of �1330 bases of the 16S rRNA gene, showing the relationships of the sheep and pig isolates with their

nearest phylogenetic relatives. Type species of other members of the Actinomycetaceae were used to root the tree. Bootstrap values are shown at the nodes

and are expressed as a percentage of 500 replicates. Bar, sequence similarity.

DefwoDev

Ref

Aalb

Coll

Fels

G. Foster et al. / Veterinary Microbiology 157 (2012) 471–475 475

ra FFG Scanning Surveillance Programme. Sequencingrk at the AHVLA was supported by Research &elopment Internal Investment Fund Project RD0016.

erences

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Hommez, J., Devriese, L.A., Miry, C., Castryck, F., 1991. Characterisation oftwo groups of Actinomyces-like bacteria from purulent lesions in pigs.J. Vet. Med. B 38, 375–380.

Hoyles, L., Collins, M.D., Falsen, E., Nikolaitchouk, N., McCartney, A.L.,2004. Transfer of members of the genus Falcivibrio to the genusMobiluncus, and emended description of the genus Mobiluncus. Syst.Appl. Microbiol. 27, 72–83.

Reichel, R., Wragg, P., 2007. Isolation of Actinomyces hyovaginalis from alung lesion in a pig. Vet. Rec. 160, 203.

Schumacher, V.L., Hinckley, L., Gilbert, K., Risatti, G.R., Londono, A.S.,Smyth, J.A., 2009. Actinomyces hyovaginalis-associated lymphadenitisin a Nubian goat. J. Vet. Diagn. Inv. 380–384.

Storms, V., Hommez, J., Devriese, L.A., Vaneechoutte, M., De Baere, T.,Baele, M., Coopman, R., Verschraegen, G., Gillis, M., Haesebrouck, F.,2002. Identification of a new biotype of Actinomyces hyovaginalis intissues of pigs during diagnostic bacteriological examination. Vet.Microbiol. 84, 93–102.