Abdul Rahman, Sheikh Omar

Mutalib, Abdul Rahim

Radu, Son

Zakaria, Zunita

Universiti Putra Malaysia Institutional Repository

UPM Research Report 1998: Volume 2ISOLATION AND CHARACTERISATION OFDICHELOBACTER NODOSUS FROM FOOTROTINFECTED SHEEP IN MALAYSIASheikh Omar Abdul Rahman, Zunita Zakaria,Abdul Rahim Mutalib and Son RaduFaculty of Veterinary MedicineUniversiti Putra Malaysia, 43400 UPM, Serdang, Selangor,MalaysiaKeywords: footrot, Dichelobacter nodosus, sheep,Malaysia.IntroductionDichelobacter nodosus is the causative agent of severefootrot, a contagious debilitating disease which affects rumi-nants particularly sheep and goats. The disease is character-ised by separation of the hom of the hoof from the underly-ing soft tissues resulting in lameness, loss of body conditionand reduced wool production (Rood et al. 1996). The firstcase of footrot in Malaysia was detected in early 1994 at In-stitut Haiwan Kluang farm and the disease is now known tobe present in several other farms. Information on the etio-logical agent of the disease is important to understand thesituation in Malaysia enabling of suitable measures to controland if possible to eradicate footrot. This study involved iso-lation, identification and characterisation of D. nodosus iso-lates.Materials and MethodsD. nodosus isolates were obtained from sheep suffering fromfootrot from three farms in Johor, Kedah and Terengganu.Lesion materials were inoculated onto 4% hoof agar (HA)plates (Egerton and Roberts, 1971) and incubated anaerobi-cally at 37°C for 3-4 days. Suspected colonies were purifiedon 4% HA. Serogrouping was done by slide agglutinationtest as described by Claxton et al. (1983) and the serotypewas determined by the microtitre plate agglutination test.Confirmation of field isolates was done by PCR using spe-cific primers. The elastase and gelatin gel test were appliedto isolates in order to determine the virulence. Isolates in-oculated on elastin agar were observed for their elastase ac-tivity on 4, 7, 11, 17,21 and 24 days post-incubation (p.i.).Isolates were recorded positive on a particular day p.i. if azone of elastin clearance was observed around the inoculum.Proteases were recorded as either thermostable or thermola-bile depending on whether a zone of gelatin clearance wasobserved after 16 minutes of prior incubation of a brothculture at 68°C. The pulsed field electrophoresis (PFGE)technique (Chu et al. 1986) was used for genomic typing ofD. nodosus isolates.Results and DiscussionD. nodosus was isolated from 12 of 30 infected sheep withfootrot lesion score 2 from three separate farms. They weredesignated Mal 1 to Mal 12. Diagnosis was done by Gram-staining while laboratory confirmation was accomplished byPCR technique. All 12 isolates reacted positively in the PCRmethod by producing a single product of 783 base pair. Allisolates, although obtained from different locations, werefound to be of serogroup B (10 isolates were B2 serotype, 2isolates were Bl serotype). The occurrence of a single sero-116group in Malaysia out of nine serogroups currently recog-nised for D. nodosus, is rather unique. In other countries suchas Australia, New Zealand and Britain, at least three sero-groups were present in their flocks (Claxton et al. 1983). Re-s~lt~ of serotyping later showed that the isolates were quitedlst~nct from the fo~r B group prototype strains. Althoughthe Isolates were designated to a certain serotype within theserogroup, they exhibited low agglutinating titres for theprototype antisera. These results indicated that the isolatespos~essed only a f~w shared antigens with the prototypestrains, thus possessmg less agglutinating epitopes with pro-totypes antisera. The assessment of the virulence of eachisolate was done by two standard conventional methods,'namely the elastase and gelatin-gel tests. Both tests showeda high level of agreement and repeatability. Therefore eitherof the tests is suitable for use in determining the virulence ofisolates. Generally the isolates exhibited a variation in thelaboratory characteristics although they had been isolatedfrom lesions with a similar score. Mal 1, Mal 8 and Mal 12isolates which appeared to have the ability to cause virulentfootrot w!t~ their positive elastase and thermostable proteasech~actens.tlcs, c~used only a mild form of the disease pro-ducmg lesions With score 2. This manifestation is probablydue to the constant topical treatment regimen adhered to bythe farm management and the result of the vaccination pro-gramme causing the bacteria not to fully express its viru-lence. PFGE analysis was used to compare chromosomalDNA restriction patterns in order to differentiate betweenisolates. It was f?und that D. nodosus isolates could readilybe analysed by this technique. Significant patterns were gen-erated by three GC-rich enzymes (ApaI, SfiI and SmaI). Aconsiderable genetic diversity was found among the D. nodo-sus isolates (eight genome types form 12 isolates). It is ofinterest that although opportunities for cross infection be-twee.n flocks were high, no particular genome type was pre-dorrunant among the flocks studied.ConclusionsD. nodosus isolates in Malaysia which belong to a single se-rogroup. (serogroup B), are variable in their capability tocause different degrees of footrot. The isolates also demon-strated multiple genotypes even though they were obtainedfrom the same flock. It is highly probable that the isolateswere from diverse so~rces: The source of this heterogeneitycould not be defined III this study, although it is most likelythe. res~lt o~ b?th multiple environmental exposure and ge-netic drift within the population.ReferencesChu, G., Vollrath, D., Davis, R.W. 1986. Separation of large DNAmolecules by contour .clamped homogenous elactric field. Sci.234, 1582-1585.Claxton, P.D., Ribeiro, L.A. and Egerton, 1.R. 1983. Classificationof Bacteroides nodosus by agglutination tests, Aust. Vet. J.60(11).331-334.Egerton, 1.R. and Roberts, D.S. 1971. Vaccination against ovinefootrot. J. Compo Path. 81, 179-185.R~, 1.1., Howarth, P.A., ~aring, V.• Billington, SJ .• Yong, W.K.,LIU,D., Palmer, M.A., Pitman, DR, Links. 1., Stewart, DJ. andVaughan, J.I. 199~. Comparison of gene probe and conventionalmethods for the differentiation of ovine footrot isolates of D no-dosus. Vet. Micro. 52: 127-141. .Supported by 1RPA Grant 01-02-04-0211

Isolation and characterisation of Dichelobacter nodosus from footrot infected sheep in Malaysia

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Isolation and characterisation of Dichelobacter nodosus from footrot infected sheep in Malaysia

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