Molecular diagnosis of Haemorrhagic Septicaemia-A Review

Pasteurella multocida is associated with hemorrhagic septicaemia in cattle and buffaloes, pneumonic pasteurellosis in sheep and goats, fowl cholera in poultry, atrophic rhinitis in pigs and snuffles in rabbits. Haemorrhagic septicaemia is caused by Pasteurella multocida type B:2, B:2,5 and B:5 in Asian countries and type E:2 in African countries. Pasteurella multocida have five types of capsular serotype i.e. type A, B, D, E and F. Diagnosis of the disease is mainly based on the clinical sign and symptom, post mortem findings. Confirmatory diagnosis is done by isolation and identification of causative agent. A variety of laboratory diagnostic techniques have been developed over the years for pasteurellosis and used routinely in the laboratory. Among these techniques molecular techniques of diagnosis is most important. This technique not only gives diagnosis but it also provides information regarding capsular type of Pasteurella multocida. Techniques which are used for molecular diagnosis of haemorrhagic septicaemia are PCR based diagnosis, Restriction endonuclease analysis (REA), Ribotyping, Colony hybridization assay, Filled alternation gel electrophoresis (FAGE), Detection of Pasteurella multocida by Real Time PCR. Among these techniques real time PCR is most sensitive and specific.


Introduction
Two typing systems for serotyping of Pasteurella multocida isolates are adopted.One for Pasteurellosis is a major disease of cattle and capsular typing by Carter's IHA system and other buffalo occurring as catastrophic epizootics in many somatic typing by the method of Namioka and Murata Asian and African countries resulting into high or Heddleston.Diagnosis of the disease is mainly mortality and morbidity (AHIS, 1997;Mustafa et al., based on the clinical sign and symptom, post mortem 1978;Singh et al., 1996).The disease has been also findings.Confirmatory diagnosis is done by the recorded in poultry, rabbit, pig and wild mammals isolation and identification of causative agent.A (Carigan et al., 1991;Rosen, 1981).Pasteurella variety of laboratory diagnostic techniques have been multocida is associated with hemorrhagic septicaemia developed over the years for pasteurellosis and used in cattle and buffaloes, pneumonic pasteurellosis in routinely in the laboratory.The organism is identified sheep and goats, fowl cholera in poultry, atrophic directly through examination of blood smear from rhinitis in pigs and snuffles in rabbits (De Alwis, affected animal and can be isolated in suitable culture 1996).An annual economic loss in India due to medium in the laboratory.et al., 2005). in cattle, sheep, and pig.Capsular type D of Pasteurella multocida produces atrophic rhinitis in pig 1. PCR based diagnosis and typing: Numerous and snuffles in rabbits.
studies for diagnosis and characterization of Pasteurella multocida have been carried out with alternative to the conventional capsular serotyping variable results.The phenotypic characterization system and used for capsular types determination.The systems by means of morphology, biochemical typing, serogroup specific primers used in this assay were serotyping etc. are very much laborious and time designed following identification, sequence determiconsuming.Even after capsular and somatic antigen nation and analysis of the capsular biosynthetic loci of determination, still few isolates react similarly in both each capsular group.The multiplex capsular PCR the antigens.The PCR based techniques have assay is highly specific and its result correlated well provided the alternative methods of characterization with conventional serotyping results with the to overcoming the limitations of phenotyping.exception of those for some serogroup F strains a. Pasteurella multocida specific PCR assay: The (Townsend et al., 1998(Townsend et al., , 2001)).species specific PCR assay can be applied for The capsular typing of all the isolates were detection of Pasteurella multocida by using template determined by multiplex PCR using capsular types A, B, D, E and F specific primers as mentioned below: as either genomic DNA or bacterial colony or by using 1.CAP A-F 5'-3' TGCCAAAATCGCAGTCAG the direct field samples such as nasal swab, morbid 2.CAP A-R 5'-3' TTGCCATCATTGTCAGTG materials like spleen, one marrow, and heart blood.repeats) are present in 500-1000 copies accounting for specific PCR is 100 % specific for type B serotypes of upto 1% of the genome (Stern et al., 1984) and are Pasteurella multocida isolates.Serotype B cultures present in a wide range of bacteria (Olive and Bean, with the any combination of somatic antigen are 1999).As the REP elements are distributed widely identified by the amplification of a 620 bp fragment across the genome, it produces a multiple banding with the KT SP61: 5'-ATC CGC TAA CAC ACT pattern.ERIC-PCR has been successfully used to CTC-3' and KTT72: 5'-AGG CTC GTT TGG ATT differentiate strains of Pasteurella multocida.The ATG AAG-3' primers (Townsend et al., 1998(Townsend et al., , 2001)).
visual analyses of banding pattern were in range of c. Pasteurella multocida type A specific PCR: 100-900 bp.The band patterns provide DNA Primers for typing of serogroup A strains which fingerprints which allows distinction between species causing number of infection in livestock and poultry and between strains within species.with several somatic types have been reported to be f.Detection of toxigenic Pasteurella multocida: useful in specific identification of isolates.The The Pasteurella multocida capsular type D strain has primers RGPMA5: 5'-AAT GT TTG CGA TAG YCC been identified as causative agent of atrophic rhinitis GTT AGA-3' and RGPMA6: 5'-ATT TGG CGC CAT in pigs and snuffles in rabbits.The toxA gene of ATC ACA GTC-3' gives PCR amplicon size of 564 bp Pasteurella multocida encodes the dermanecrotic which confirms the presence of Pasteurella multocida toxin responsible for atrophic rhinitis.The toxA gene serotype A.
based PCR can be used for direct analysis of toxigenic

d. Multiplex PCR for Pasteurella multocida
Pasteurella multocida without additional hybridicapsular typing: A multiplex PCR assay is a rapid zation.The assay appears to be the most sensitive and expensive electrophoresis equipment, which is effective method for large scale analysis of nasal and generally not available in normal diagnostic tonsillar swabs (Kamp et al., 1996).
3.CAP B-F 5'-3' CATTTATCCAAGCTCCACC Earlier the PCR needed additional hybridization step 4.CAP B-R 5'-3' GCCCGAGAGTTTCAATCC 5.CAP D-F 5'-3' TTACAAAAGAAAGACTAGGAGCCC for increasing the specificity but later with improved 6.CAP D-R 5'-3' CATCTACCCACTCAACCATATCAG PCR technique it became possible to detect as 7.CAP E-F 5'-3' TCCGCAGAAAATTATTGACTC minimum as 10 organisms per reaction.The 8.CAP E-R 5'-3'GCTTGCTTGATTTTGTC Pasteurella multocida can identify all subspecies of 9.CAP F-F 5'-3' AATCGGAGAACGCAGAAATCA 10.CAP F-R 5'-3' TTCCGCCGTCAATTACTCTG Pasteurella multocida.The sensitivity and specificity Sizes of the multiplex PCR amplicons are as follows: of this PCR offer the most compelling argument for Amplicon size Capsular type the use of PCR technology in laboratory to 460bp fragment of DNA.This technique e. REP-PCR and ERIC-PCR: Recently Repetitice has reduced the time for diagnosis of the disease and Extragenic Palindromic (REP) and Enterobacterial also it is specific than traditional one.Repetitive Insertion Consensus (ERIC) PCR have b.HS causing type B specific PCR assay: The PCR been developed for the characterization of Pasteurella amplification can also detect the serotype B specific multocida isolates.REP elements (33 to 40 base pair Pasteurella multocida directly HS causing type B