BAELE, A

Chiers, Koen

Decostere, Annemie

Haesebrouck, Freddy

Hermans, Katleen

Moyaert, Hilde

TAVEMIER, P

English

Ghent University Academic Bibliography

www.elsevier.com/locate/vetmicVeterinary Microbiology 124 (2007) 184–186Short communicationAn unusual Actinobacillus equuli strain isolated from a rabbitwith Tyzzer’s diseaseH. Moyaert *, A. Decostere, M. Baele, K. Hermans, P. Tavernier,K. Chiers, F. HaesebrouckDepartment of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine,Ghent University, Salisburylaan 133, B-9820 Merelbeke, BelgiumReceived 8 January 2007; received in revised form 30 March 2007; accepted 4 April 2007AbstractActinobacillus equuli was isolated in pure culture from the liver and lungs of an adult rabbit with Tyzzer’s disease(Clostridium piliforme). Based on the haemolytic features on blood agar plates, a positive reaction in the CAMP-test, hydrolysisof esculin, the inability to ferment L-arabinose, tDNA-PCR and sequencing of the 16S rRNA gene, the isolate was classified as A.equuli subsp. haemolyticus biovar 1. However, the aqxA gene, characteristic for haemolytic A. equuli strains, was not detectedby PCR.# 2007 Elsevier B.V. All rights reserved.Keywords: Actinobacillus equuli; RabbitActinobacillus equuli, a member of the familyPasteurellaceae, is a small, pleomorphic, non-motileGram-negative rod. It is the etiological agent of a rare,but frequently fatal septicaemia in neonatal foals alsoknown as ‘‘sleepy foal disease’’ which is spreadworldwide (Rycroft and Garside, 2000; Berthoudet al., 2002). Besides this acute form of disease, A.equuli can be associated with other types of infectionsin both foals and adult horses, such as abortion,enteritis, peritonitis, pleuropneumonia, pericarditis,periorchitis and arthritis (Webb et al., 1976; Dill et al.,1982; Al-Mashat and Taylor, 1986; Belknap et al.,* Corresponding author. Tel.: +32 9 2647434; fax: +32 9 2647494.E-mail address: Hilde.Moyaert@UGent.be (H. Moyaert).0378-1135/$ – see front matter # 2007 Elsevier B.V. All rights reserveddoi:10.1016/j.vetmic.2007.04.0131988; Peremans et al., 1991; Collins et al., 1994;Patterson-Kane et al., 2001). A. equuli has beensubdivided in two different subspecies, designated A.equuli subsp. haemolyticus (formerly described asBisgaard Taxon 11), which is haemolytic andenhances the haemolytic effect of staphylococcal b-haemolysin (CAMP-positive) and A. equuli subsp.equuli, which is non-haemolytic and CAMP-negative(Christensen et al., 2002). The haemolytic strains havemainly been described in horses, but are notspecifically associated with ‘‘sleepy foal disease’’,which is typically linked with the non-haemolyticstrains (Kuhnert et al., 2003). A. equuli subsp. equuliwas not only isolated from horses, but has also beenassociated with abortion and endocarditis in pigs.H. Moyaert et al. / Veterinary Microbiology 124 (2007) 184–186 185(Jones and Simmons, 1971; Werdin et al., 1976).Moreover, the organism was isolated from thenasopharynx, conjunctiva and middle ear of guineapigs, rats and mice (Lentsch and Wagner, 1980). A.equuli is additionally considered as a potentialopportunistic pathogen of humans, especially inimmunocompromised people or people who areoccupationally exposed to infected horses or pigs(Ashhurst-Smith et al., 1998; Vaz et al., 2001).In this report, we describe the isolation of anunusual A. equuli strain from the liver and lungs of anadult rabbit with Tyzzer’s disease.An adult, male pet-rabbit (Oryctolagus cuniculus)weighing 4.1 kg, died after a 4-day history of anorexiaand depression. The animal had not been treated withan antimicrobial agent.At necropsy, a fibrinous pericarditis as well asnumerous 1-mm-diameter necrotic foci scatteredwithin the liver parenchyma were observed. Histolo-gical examination of haematoxylin-eosin stainedsections of formaldehyde (10%) fixed, paraffinembedded liver tissue samples revealed a focalextensive necrotizing hepatitis. In Warthin-Starry-stained sections of the liver, numerous large, elongatedbacilli were observed within the cytoplasm of viablehepatocytes at the margins of necrotic foci. The latterfinding is diagnostic for Tyzzer’s disease, a rapidlyprogressing, acute hepatitis caused by Clostridiumpiliforme (Ganaway et al., 1971; Kelly, 2000).Liver and lung samples of the animal werebacteriologically examined. Cultures of these sampleson Columbia agar with 5% sheep blood (Oxoid,Basingstoke, UK) yielded profuse and pure growth ofhaemolytic bacteria which produced greyish-whitecolonies. Gram staining revealed small, pleomorphicGram-negative rods. The organism was oxidase,catalase, urease and CAMP-positive, but negativefor L-arabinose fermentation. In a bile containingmedium, hydrolysis of esculin was observed. Based onthese phenotypic characteristics, the isolate wasidentified as A. equuli subsp. haemolyticus biovar 1(Christensen et al., 2002).Using tDNA-PCR, the organism showed anidentical pattern to A. equuli and A. suis (Baeleet al., 2000). Sequencing of the 16S rRNA gene (Baeleet al., 2001) yielded a fragment of 1214 bp (accessionnumber: EF141826) which showed 99% similarity toA. suis and A. equuli subsp. haemolyticus and 98%similarity to A. equuli subsp. equuli. Subsequently,aqxA, apxICA and apxIICA PCR-assays were carriedout (Frey et al., 1995; Berthoud et al., 2002). In thesePCR-assays, A. equuli subsp. haemolyticus CCUG19799T, an A. suis field isolate and A. pleuropneu-moniae serotypes 1–12 reference strains were used ascontrols. None of these PCR-assays yielded anamplicon with DNA of the rabbit isolate, while theexpected amplicons were obtained with DNA of thecontrol strains (Frey et al., 1995; Kuhnert et al., 2003).Within the genus Actinobacillus, haemolysis isobserved for A. pleuropneumoniae, A. equuli subsp.haemolyticus and A. suis. In contrast with A.pleuropneumoniae and most of the A. equuli strains,all A. suis strains have the ability to ferment L-arabinose. However, these closely related speciescannot unequivocally be separated based on pheno-typic characteristics alone. Additionally, 16S rRNAgene sequence comparison and tDNA-PCR are notable to separate A. equuli subsp. equuli, A. equulisubsp. haemolyticus and A. suis at the (sub)specieslevel. Therefore, PCR typing for RTX (repeat in toxin)genes is recommended (Kuhnert et al., 2003;Christensen and Bisgaard, 2004). Indeed, A. equulisubsp. equuli does not contain apxICA, apxIICA noraqxA genes. In contrast, A. equuli subsp. haemolyticusstrains contain the aqxA gene and neither apxICA norapxIICA genes, while A. suis isolates contain apxICAand apxIICA but no aqxA genes (Kuhnert et al., 2003).A. pleuropneumoniae serotypes 1, 5a, 5b, 9, 10, 11 and14 contain the apxICA gene and apxIICA is present inall serotypes except in serotypes 10 and 14 (Frey et al.,1995; Schaller et al., 2001).Phenotypically, the rabbit isolate was identified asA.equuli subsp. haemolyticus biovar 1. Contradictorily,since we could not detect amplicons of the aqxA gene,our isolate would be identified as A. equuli subsp.equuli. The haemolytic activity of our isolate and itsability to enhance the haemolytic effect of staphylo-coccal b-haemolysin indicate the presence of an RTX-toxin (Devenish et al., 1992). The negative result in theaqxA-specific PCR might be caused by a DNA-mutation in one or both primer binding sites, resulting infailure of primer annealing and amplification. Alter-natively, one might speculate on the presence of adifferent RTX-toxin in this rabbit-isolate.Our finding that this A. equuli strain was isolated inpure culture from both the lungs and the liver mightH. Moyaert et al. / Veterinary Microbiology 124 (2007) 184–186186suggest that it contributed to the disease, probably as asecondary invader. Recently, a comparable case hasbeen reported by Meyerholz and Haynes (2005): a5-year-old pet rabbit died after a 3-day history ofanorexia and depression. This rabbit suffered from anActinobacillus capsulatus septicaemia, secondary tothe gastric stasis syndrome (‘‘wool block’’). Veterinar-ians and diagnosticians need to be aware of Actinoba-cillus species as a potentially under-diagnosed bacterialpathogen of lagomorphs, especially since Actinoba-cillus spp. may be misidentified as Pasteurella spp.,which are quite common in rabbits (Lentsch andWagner, 1980; DeLong and Manning, 1994).AcknowledgmentsWe are grateful to Serge Verbanck and Arlette Vande Kerckhove for their technical assistance.ReferencesAl-Mashat, R.R., Taylor, D.J., 1986. Bacteria in enteric lesions ofhorses. Vet. Rec. 118, 453–458.Ashhurst-Smith, C., Norton, R., Thoreau, W., Peel, M.M., 1998.Actinobacillus equuli septicaemia: an unusual zoonotic infec-tion. J. Clin. Microbiol. 36, 2789–2790.Baele, M., Baele, P., Vaneechoutte, M., Storms, V., Butaye, P.,Devriese, L.A., Verschraegen, G., Gillis, M., Haesebrouck, F.,2000. Application of tRNA intergenic spacer PCR for identifica-tion of Enterococcus species. J. Clin. Microbiol. 38, 4201–4207.Baele, M., Chiers, K., Devriese, L.A., Smith, H.E., Wisselink, H.J.,Vaneechoutte, M., Haesebrouck, F., 2001. The gram-positivetonsillar and nasal flora of piglets before and after weaning. J.Appl. Microbiol. 91, 997–1003.Belknap, J., Arden, W., Yamini, B., 1988. Septic periorchitis in ahorse. J. Am. Vet. Med. Assoc. 192, 363–364.Berthoud, H., Frey, J., Kuhnert, P., 2002. Characterization of Aqxand its operon: the haemolytic RTX determinant of Actinoba-cillus equuli. Vet. Microbiol. 87, 159–174.Christensen, H., Bisgaard, M., 2004. Revised definition of Actino-bacillus sensu stricto isolated from animals. A review withspecial emphasis on diagnosis. Vet. Microbiol. 99, 13–30.Christensen, H., Bisgaard, M., Olsen, J.E., 2002. Reclassification ofequine isolates previously reported as Actinobacillus equuli,variants of A. equuli, Actinobacillus suis or Bisgaard taxon11 and proposal of A. equuli subsp. equuli subsp. nov. and A.equuli subsp. haemolyticus subsp. nov. Int. J. Syst. Evol. Micro-biol. 52, 1569–1576.Collins, M.B., Hodgson, D.R., Hutchins, D.R., 1994. Pleural effu-sion associated with acute and chronic pleuropneumonia andpleuritis secondary to thoracic wounds in horses: 43 cases(1982–1992). J. Am. Vet. Med. Assoc. 205, 1753–1758.DeLong, D., Manning, P.J., 1994. Bacterial diseases. In: Manning,P.J., Ringler, D.H., Newcomer, C.E. (Eds.), The Biologyof Laboratory Rabbit. Academic Press, San Diego, pp.129–170.Devenish, J., Brown, J.E., Rosendal, S., 1992. Association of theRTX proteins of Actinobacillus pleuropneumoniae with haemo-lytic, CAMP, and neutrophil-cytotoxic activities. Inf. Immun.60, 2139–2142.Dill, S.G., Simoncini, D.C., Bolton, G.R., Rendano, V.T., Crissman,J.W., King, J.M., Tennant, B.C., 1982. Fibrinous pericarditis inthe horse. J. Am. Vet. Med. Assoc. 180, 266–271.Frey, J., Beck, M., van den Bosch, J.F., Segers, R.P.A.M., Nicolet, J.,1995. Development of an efficient PCR method for toxin typingofActinobacillus pleuropneumoniae strains. Mol. Cell. Probes 9,277–282.Ganaway, J.R., Allen, A.M., Moore, T.D., 1971. Tyzzer’s disease ofrabbits: isolation and propagation of Bacillus piliformis (Tyzzer)in embryonated eggs. Infect. Immun. 3, 429–437.Jones, J.E., Simmons, J.R., 1971. Endocarditis in the pig caused byActinobacillus equuli: a field and an experimental case. Br. Vet.J. 127, 25–29.Kelly, A., 2000. Tyzzer’s disease in a 11-day-old foal. Can. Vet. J.41, 491–492.Kuhnert, P., Berthoud, H., Christensen, H., Bisgaard, M., Fey, J.,2003. Phylogenetic relationship of equine Actinobacillus speciesand distribution of RTX toxin genes among clusters. Vet. Res.34, 353–359.Lentsch, R.H., Wagner, J.E., 1980. Isolation of Actinobacilluslignieresii and Actinobacillus equuli from laboratory rodents.J. Clin. Microbiol. 12, 351–354.Meyerholz, D.K., Haynes, J.S., 2005. Actinobacillus capsulatussepticemia in a domestic rabbit (Oryctolagus cuniculus). J.Vet. Diagn. Invest. 17, 83–85.Patterson-Kane, J.C., Donahue, J.M., Harrison, L.R., 2001. Septi-caemia and peritonitis due to Actinobacillus equuli infection inan adult horse. Vet. Pathol. 38, 230–232.Peremans, K., Verschooten, F., De Moor, A., Desmet, P., 1991.Monoarticular infectious arthritis in the horse: 34 cases. J. Eq.Vet. Sci. 11, 27–32.Rycroft, A.N., Garside, L.H., 2000. Actinobacillus species and theirrole in animal disease. Vet. J. 159, 18–36.Schaller, A., Djordjevic, S.P., Eamens, G.J., Forbes, W.A., Kuhn, R.,Kuhnert, P., Gottschalk, M., Nicolet, J., Frey, J., 2001. Identi-fication and detection of Actinobacillus pleuropneumoniae byPCR based on the gene apxIVA. Vet. Microbiol. 79, 47–62.Vaz, R., Kennedy, K., Peel, M., 2001. Septicemia in a horse trainercaused by Actinobacillus equuli. Clin. Microbiol. Newsl. 23,151–153.Webb, R.F., Cockram, F.A., Pryde, L., 1976. The isolation ofActinobacillus equuli from equine abortion. Aust. Vet. J. 52,100–101.Werdin, R.E., Hurtgen, J.P., Bates, F.Y., Borgwardt, F.C., 1976.Porcine abortion caused by Actinobacillus equuli. J. Am. Vet.Med. Assoc. 169, 704–706.

An unusual Actinobacillus equuli strain isolated from a rabbit with Tyzzer's disease

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An unusual Actinobacillus equuli strain isolated from a rabbit with Tyzzer's disease

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