Abdul Rahman, Sheikh Omar

Zakaria, Zunita

Universiti Putra Malaysia Institutional Repository

Agricultural SciencesBacteriological Studies of Dichelobacter Nodosus Isolated from Footrot Infected Sheep*Sheikh Omar Abdul Rahman; Zunita ZakariaFaculty of Veterinary MedicineUniversiti Putra Malaysia43400 UPM, Serdang, SelangorMalaysiaE-mailofCorrespondingAuthor:omar@vet.upm.edu.myKey words: Dichelobacter nodosus, footrot, sheep.IntroductionFootrot is a contagious disease of ru-minants particularly sheep and goatsalthough cattle and deer may also beaffected (Beveridge et al., 1941). Thedisease has been known for the last twocenturies in many parts of the world,however, it has only been detected inMalaysia in the last five years. Thefirst case of footrot occurred in InstitutHaiwan Kluang (IHK) in 1994 andbecoming an increasingly importantdisease in Malaysia. Vaccination withcommercial vaccine did reduce theprevalence but the strength and dura-tion of the immunity achieved is lim-ited (Egerton et al. and Meritt, 1973).Extensive studies are being carried outon footrot disease to obtain a compre-hensive knowledge about the etiologi-cal agent and to understand the situa-tion in the country.Main objectives of the project were tocharacterise Dichelobacter nodosusfound in Malaysia and to explore thepossibility of developing a serogroup.specific vaccine of high potency. Thisinvolves the production of fimbriae byrecombinant DNA in a host that is lessfastidious growth requirement and isable to grow to a much higher densitythan Dichelobacter nodosus. Apartfrom that, to study epidemiology of thedisease in Malaysia as well, the patho-genesis of the bacteria in causing thedisease.Materials and MethodsIsolations of Dichelobacter nodosuswere made from several sheep farms inMalaysia. The isolates obtained wereidentified and confirmed as D. nodosusby polymerase chain reaction us~ngspesies specific primers. These Is?lateswere subjected to laboratory tests IIIorder to characterise and differentiatethem. Two conventional tests i.e. elas-tase and gelatin gel tests (Link andMorris, 1996) were used to assess thevirulence of the isolates. Moleculartechniques, which include pulsed fieldgel electrophoresis analysis, were alsodone to further differentiate the iso-lates. The isolates were also tested ontheir susceptibility to different antimi-crobial agents. In developing specificrecombinant vaccines, detail studieswere done on the fimbrial gene of theorganism. The fimbrial gene of theisolates was detected by PCR tech-nique. The D. nodosus fimbrial genewere then cloned and expressed inanother host i.e. Pseudomonas aerugi-nosa. Antigenicity of the recombinantvaccine was determined by WesternBlot.Epidemiology studies were carried outat three farms for one year (twelvevisits each farm). Footrot lesions werescored and sampled for D. nodosusisolations and characterisation. Patho-genicity studies were done according togross lesion scores. Skin biopsies weretaken for pathological studies by lightand electron microscope.Results and DiscussionTwelve Dichelobacter nodosus wereisolated from 30 sheep with footrotlesions. Diagnosis was done success-fully by Gram-stain method whilepolymerase chain reaction (PCR)method confirmed the isolates as Di-chelobacter nodosus by producing asingle product of approximately 780basepairs. All isolates, although ob-tained from distant locations, were ofserogroup B. Among the antibioticstested, penicillin G proved to be themost effective antibiotic with MICx,%of 0.023 Ilglml. Generally, the isolatesexhibited variation in the laboratorycharacteristics although they had beenisolated from the similar lesion score.Some of the isolates, which appeared tohave the capability of causing virulentfootrot in-vitro, failed to show clinicalsigns of virulent form of footrot . Thiswas probably due to the frequent topi-cal regimen adhered to and results ofthe vaccination programme by the farmmanagement. All isolates were foundnot to contain plasmid by standardplasmid extracting method. This indi-cates that the genes coding for viru-lence of the isolates were not palsmid-mediated. Molecular typing of theisolates was successfully carried out bypulsed field gel electrophoresis analy-sis. Significant patterns were gener-ated by three GC-rich enzymes dis-criminating the isolating into eightgenome types. Isolates from the sameflock were also shown to possessvariation in their PFGE profiles.The gene coding for fimbriae was suc-cessfully amplified and cloned in a T-vector. An expression vector was cho-sen based on the fimbrial gene orienta-tion and appropriate restriction en-zymes were selected in order to designa recombinant plasmid. The fimbrialgene was recloned into the expressionvector downstream the lac promoter.The plasmid containing the D. nodosusfimbrial gene was then successfullytransformed in the surrogate host,Pseudomonas aeruginosa. Electropho-retic and western blot analysis showedthat Pseudomonas aeruginosa con-taining recombinant plasmid (with D.nodosus fimbrial gene) expresses D.nodosus fimbriae instead of its ownfimbriae. Preliminary tests were doneby injecting these purified recombinantfimbriae in sheep. Results showed thatthere were high levels of antibodyproduction against D. nodosus.ConclusionsDichelobacter nodosus isolates inMalaysia that belong to a single sero-group (serogroup B), are variable intheir capability of causing differentdegrees of footrot. The isolates alsodemonstrate multiple genotypes eventhough they were obtained from thesame flock. The development of spe-cific footrot vaccine looks very127UPM Research Report 1997-2000, Section 2-Extended Abstractspromising as we have already suc-ceeded in expressing D. nodosus fim-briae in a surrogate host, Pseudomonasaeruginosa that is less fastidious andfast growing. This specific vaccine,which has commercial value world-wide, will be able to control and pre-vent the disease.Benefits from the studyResults from this project will havesignificant impacts for the animal in-dustry in particularly sheep industry inMalaysia, and especially the productionof serogroup specific vaccine. Thespecific vaccine will be able to elicithigh level of antibody production insheep thus protecting sheep fromfootrot disease. One master studenthad completed her study under thisproject. At present, there are two Ph.D.candidates pursuing their degrees inthis field of study.Literature cited in the textBeveridge, W.I.B. 1941. Footrot in sheep: Atransmissible disease due to infection withFusiformis nodosus, Commonwealth ofAustralia; Bulletin: 140.Egerton, J.R. and Merritt, G .C. 1973.Serology of footrot: antibodies againstFusiformis nodosus in normal, affected,vaccinated and passively immunisedsheep. Aust. Vet. J. 49: 139-145.Project Publications inRefereed JournalsLinks, I.J. and Morris, S. 1996. Assessmentof gelatin gel and elastase tests fordetection of protease activity ofDichelobacter nodosus isolates fromovine footrot. Vet. Microbiol. 51: 305-318.Sheikh Omar, A.R., Zakaria, Z. and AI-Jashamy, K. Ovine footrot in Malaysia:the diversity of the causative agentDichelobacter nodosus. 200!. (accepted)The Science Conference 200 I , YemeniScientific Research Foundation.Zakaria, Z., Sheikh Omar, A.R., Mutalib,A.R., Mohd. Azrni, ML and Son Radu.200 I. Analysis of fimbrial subunit gene ofDichelobacter nodosus isolated from128footrot infected sheep in Malaysia.(submitted to the Asia Pacific Journal ofBiotechnology).Zakaria, Z., Ahmad, N., Mutalib, A.R., AI-Jashamy, K., and Sheikh Omar, A.R.200!. Evaluation of different dna templatepreparations for detection ofDichelobacter nodosus by polymerasechain reaction. (submitted to Biomedicalletters).Project Publications inConference ProceedingsZakaria, Z., Ahmad, N., Mutalib, A.R., AI-Jashamy, K. and Sheikh Omar, A.R. 2001Detection of Dichelobacter nodosus directfrom footrot lesion materials bypolymerase chain reaction. (accepted)25th Malaysian Microbiology Symposium,Pahang, 9 - 12 September 2001.Graduate ResearchZunita Zakaria. 1998. VeterinaryMicrobiology. [M. Se]. Universiti PutraMalaysia

Bacteriological studies of Dichelobacter nodosus isolated from footrot infected sheep

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Bacteriological studies of Dichelobacter nodosus isolated from footrot infected sheep

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