Veterinary Microbiology 144 (2010) 484–486

Short communication

Use of citrate adonitol agar as a selective medium for the isolation ofEscherichia fergusonii from a captive reindeer herd

Geoffrey Foster a,*, Judith Evans b, Morten Tryland c, Simon Hollamby d, Isabel MacArthur a,Emma Gordon b, Jane Harley e, Katja Voigt a,1

a SAC Consulting Veterinary Services, Drummondhill, Stratherrick Road, Inverness IV2 4JZ, UKb SAC Epidemiology Research Unit, Animal Health Group, SAC Research, West Mains Road, Edinburgh EH9 3JG, UKc Norwegian School of Veterinary Science, Section of Arctic Veterinary Medicine, Stakkevollveien 23, N-9010 Tromsø, Norwayd R(D)SVS School of Veterinary Studies, Hospital for Small Animals, University of Edinburgh, Easter Bush Veterinary Centre, Roslin, Midlothian EH25 9RG, UKe Strathspey Veterinary Centre, Forest Road, Grantown-on-Spey PH26 3JJ, UK

A R T I C L E I N F O

Article history:

Received 5 June 2009

Received in revised form 8 January 2010

Accepted 14 January 2010

A B S T R A C T

Escherichia fergusonii is an emerging potentially zoonotic organism which has been

recovered from a broad range of human and animal sources. Efforts to recover E. fergusonii

from mixed flora hitherto however have been constrained by the lack of a suitable

selective medium for its isolation. This paper reports for the first time the recovery of E.

fergusonii from reindeer carcases in a wildlife park and the use of citrate adonitol agar to

selectively screen for the presence of this organism in faecal samples from further animals

in the park, and reindeer in their natural habitat in Norway.

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Escherichia fergusonii was first described for strains ofEnterobacteriaceae previously known as Enteric Group 10(Farmer et al., 1985). The organism can be distinguishedfrom other members of the genus by several phenotypictests, which include positive adonitol, amygdalin andcellobiose fermentation and negative sorbitol reaction(Farmer et al., 1985; Huys et al., 2003). The original speciesdescription for E. fergusonii was based on isolates that hadbeen recovered from a wide range of human sourcesincluding blood, urine, faeces and an abdominal wound aswell as from unspecified sources in three pigs, a cow, ahorse and a turkey (Farmer et al., 1985). In the years since,E. fergusonii has occasionally been reported from furtherhuman cases including septicaemic patients (Freney et al.,1987; Funke et al., 1993) and a potential to cause diarrhoeawas demonstrated by Chaudhury et al. (1999). More

* Corresponding author. Tel.: +44 463 243030; fax: +44 463 711103.

E-mail address: Geoffrey.Foster@sac.co.uk (G. Foster).1 Current address: Clinic for Ruminants, Ludwig-Maximilians Uni-

versity Munich, Sonnenstr. 16, 85764 Oberschleissheim, Germany.

0378-1135/$ – see front matter � 2010 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetmic.2010.01.014

recently 24 cases of bacteraemia due to E. fergusonii werereported from England, Wales and Northern Ireland (Anon,2009). In animals, Bain and Green (1999) reported theisolation of profuse growths of E. fergusonii from sevenbovine and ovine cases clinically suggestive of salmonel-losis. Further reports have included pure growths of E.

fergusonii from the caecae of ostriches with fibrino-necrotic typhlitis, fibrinous peritonitis and multifocalserosal haemorrhages with isolation also from blood, liverand spleen (Herraez et al., 2005) and the isolation of theorganism within a mixed growth from a goat with weightloss and diarrhoea (Hariharan et al., 2007). More recentlysurveillance of enteric samples from cattle, sheep and pigswas carried out in the North of England for E. fergusonii

(Wragg et al., 2009). In this study the organism wasisolated from pigs, cattle and sheep with or without entericdisease.

Hitherto reports of the isolation of E. fergusonii haverelied on its isolation on non-selective or differential mediasuch as MacConkey agar or sorbitol MacConkey agar(Wragg et al., 2009). Such agars are sufficient in most casesfor the recovery of E. fergusonii from infected sites, which

G. Foster et al. / Veterinary Microbiology 144 (2010) 484–486 485

would normally be considered sterile in healthy animals,however they lack sensitivity when culturing samples witha heavy bacterial flora such as enteric material. The lack ofan effective selective medium for E. fergusonii is a majorimpediment to methods for its isolation from sampleslikely to harbour a high background flora such as thosecollected from enteric sites. The use of an effectiveselective medium could significantly increase the sensi-tivity of methods for the isolation of E. fergusonii therebyproviding greater opportunity for exploring the epide-miology of this emerging organism.

Adonitol fermentation is a feature which is typical of E.

fergusonii but which is rare amongst Escherichia coli andother strains of Enterobacteriaceae (Huys et al., 2003; Holtet al., 1994). Adonitol fermentation is the basis for citrateadonitol agar, a medium which was developed for theselective isolation of another adonitol fermenting memberof the Enterobacteriaceae, enterotoxigenic E. coli (ETEC)K99 (Pohl et al., 1984). Adonitol fermenting colonies ofETEC K99 appear yellow on this medium, while most otherfaecal bacteria either do not grow or appear as bluecolonies. Citrate adonitol agar therefore represents amedium with significant potential for the selectiveisolation of E. fergusonii from faeces or other specimenswhich would typically contain a high background flora. Wehad used citrate adonitol agar for many years for thesuccessful recovery of ETEC K99 from bovine faecalsamples. In order to investigate its suitability for recoveryof E. fergusonii, we inoculated citrate adonitol agar plateswith 22 field isolates collected from reindeer, cattle, sheep,pig and poultry which had originally been recovered onMacConkey agar. All E. fergusonii strains tested producedyellow colonies.

We wish to report the recovery of E. fergusonii fromreindeer for the first time and the use of citrate adonitolagar to selectively screen for faecal carriage of thisorganism in this animal species.

In September 2007, a male yearling tundra reindeercarcase (Rangifer tarandus tarandus) was submitted to SACVeterinary Services Inverness for post-mortem examina-tion. This animal originated from a free-range wildlife parkand had died the day prior to submission following aprolonged period of weight loss and diarrhoea. A post-mortem investigation was carried out with follow-uproutine bacteriological culture of liver and ileum, para-sitological, histopathological and trace element andvitamin E analysis. E. fergusonii was recovered on routinecultures in scant mixed growth from the liver and in aprofuse mixed growth from the ileum. The animal wasdiagnosed with selenium and copper deficiency andoesophageal necrobacillosis. No other significant patho-gens were isolated. (Full details of this and other reindeerdeaths will be provided in a separate paper.)

The significance of the E. fergusonii infection in this casewas unclear and the question arose as to whether theorganism was present in other members of the tundrareindeer group, which included a number of animalsexhibiting signs of ill thrift and intermittent diarrhoea, orindeed in other in-contact species in the wildlife park suchas forest reindeer (Rangifer tarandus fennicus), red deer(Cervus elaphus), bukhara deer (Cervus elaphus bactrianus),

elk (Cervus canadensis), yak (Bos grunniens), European bison(Bison bonasus), mouflon (Ovis musimon), Przewalski’shorse (Equus przewalskii) and kiang (Equus kiang).

In order to further investigate the occurrence of E.

fergusonii in animals in the wildlife park, we collected andcultured faecal samples from tundra reindeer and otherresident species. In total, 35 samples were collected duringthe months September–December 2007. They weredelivered to the laboratory on the day of collection andcultured on MacConkey agar (Oxoid, Basingstoke, UK) andcitrate adonitol agar prepared in-house according to therecipe of Pohl et al. (1984) using Simmons citrate agar(Becton-Dickinson, Oxford, UK) supplemented with 2%adonitol. Plates were incubated aerobically at 37 8C andexamined for yellow colonies after 24 and 48 h. Suspectcolonies were selected for oxidase and indole testingfollowed by confirmation with API 20E according to themanufacturer’s database (BioMerieux, Basingstoke, UK).

E. fergusonii was recovered from 9 of the 35 faecessamples screened as follows: 8/14 tundra reindeer, 1/3 yakand 0/18 other species sampled. The citrate adonitol agarsuccessfully suppressed the growth of non-target organ-isms while E. fergusonii presented as yellow colonies whichcould be easily selected for further identification.

From September to December 2007, two further tundrareindeer carcases from the tundra reindeer group werenecropsied. Animal 1 was euthanased after an accidentbut had hitherto appeared clinically healthy. Sterilecultures were obtained with lung, liver and spleen andE. fergusonii was recovered from small intestine, largeintestine and faeces on citrate adonitol agar, but was notdetected on MacConkey agar. There was also a moderateto high trichostrongyle burden, and evidence of copperand selenium deficiency. Animal 2 had died after aprolonged period of weight loss, ill thrift and intermittentdiarrhoea. The animal presented with various liverabscesses, a lung abscess and secondary peritonitis onpost-mortem examination. A profuse growth of Fusobac-

terium necrophorum was obtained from the abscessmaterial and a necrotic lesion under the tongue. E.

fergusonii was isolated within a mixed growth from liverand small intestine, large intestine and faeces samples onMacConkey agar and citrate adonitol agar from thisanimal, while aerobic cultures were negative from lungtissue. No other significant bacterial pathogens wereisolated. There was a high endoparasite burden andevidence of copper and selenium deficiency. A thirdanimal, which had recently been imported from theNetherlands, died after a short period of blood-staineddiarrhoea in April 2008. At gross post-mortem examina-tion the animal presented with severe congestion andhaemorrhage of the abomasum, small and large intestine,and a full thickness abomasal ulcer. Lung and liver fromthis animal produced small growths of E. coli andBibersteinia trehalosi and E. fergusonii was detected fromsmall and large intestine and abomasums on citrateadonitol agar only, but not MacConkey agar. A profusegrowth of Clostridium sordellii was isolated from theabomasum. No other significant bacterial pathogens wererecovered, and viral and parasitic diseases were also ruledout. The copper and selenium status of this animal was

G. Foster et al. / Veterinary Microbiology 144 (2010) 484–486486

adequate. A stress-related gastro-enteropathy due to therecent movement was postulated in this animal.

Histopathology was carried out on a full range of tissuesincluding small and large intestine from all four animals.No organisms resembling E. fergusonii were located athistopathological examination of gastrointestinal or livertissue from the initial case or animals 1 and 3. In animal 2,gram stain of the gastrointestinal lesions showed mixedbacteria, mainly gram negative filamentous bacilli resem-bling Fusobacterium, which was considered the primarypathogen involved, but also small gram negative bacilliwhich would be consistent with the morphology of E.

fergusonii or other Enterobacteriaceae.The reindeer were not mixed withany of the other species

in the wildlife park, although they had been moved aboutvarious different enclosures that had seen different speciespreviously, including bison and Prezwalski horses, mouflonand Soay sheep grazing on those areas at some stage.

In order to investigate whether E. fergusonii might be amember of the normal flora of tundra reindeer, faecalsamples were collected directly from the anus of 40 livesemi-domesticated reindeer in their natural habitat inTroms County, Norway, in February 2009 as part of a studyinto infectious keratoconjunctivitis (Tryland et al., 2009).Four to five weeks prior to sampling, the animals weregathered from their natural winter pasture, on whichlichen and other plants under the snow cover constitutethe main food item, and corralled for supplementaryfeeding with pelleted food for reindeer (Norske Felleskjøp,Oslo, Norway). The animals sampled comprised four malesand 36 females and ranged in age from 10 months to 4years old (mean 1.9 years). Samples were seeded on citrateadonitol agar and processed as above. E. fergusonii was notrecovered from any of the samples collected from semi-domesticated reindeer in Norway.

This paper reports for the first time the recovery of E.

fergusonii from reindeer. The importance and role of E.

fergusonii as a potential secondary or primary pathogen inreindeer and other species as well as the organism’sprevalence within the intestinal flora of clinically healthyanimals requires further investigation. The lack of aselective medium for the isolation of E. fergusonii fromsample materials with a high background flora such asfaeces has represented a major hurdle to its detection.Isolation has previously relied on MacConkey agar,although Wragg et al. (2009) used sorbitol MacConkeyagar in combination with this medium. In this study,citrate adonitol agar significantly reduced the growth ofbackground flora and enhanced recovery of E. fergusonii

from faeces samples collected from animals at a Scottishwildlife park.

The ability of citrate–adonitol agar to allow therecovery of small numbers of E. fergusonii from faecalsamples in this study demonstrates its significant potential

as a first step towards characterising the epidemiology ofthis potential emerging zoonotic organism. We intend touse citrate–adonitol agar in further studies to investigatethe prevalence and potential significance of E. fergusonii infarm animal species.

Acknowledgements

We thank the Royal Zoological Society of Scotland andthe staff of the Highland Wildlife Park for their support ofthis study. We gratefully acknowledge the cooperation ofreindeer herders in Troms County, Norway. Histopatholo-gical examination was carried out by our colleagues FionaHowie (SAC consulting Edinburgh) and Mark Dagleish(Moredun Research institute, Edinburgh). We are alsograteful to Terje D. Josefsen, National Veterinary Institute,Norway for valuable discussion on reindeer diseases andnutrition. This work was part-funded by the ScottishGovernment as part of its Public Good Veterinary andAdvisory Service.

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