MATERIALS AND METHODS

Pasteurella multocida Strains
Pasteurella multocida strain DP1 and FP1 isolated in Kerala, India from a duck and fowl respectively and serotyped as A:1 by Indian Veterinary Research Institute, Izatnagar, India and Pasteurella multocida serotypes A:3, B:2 and a duck isolate LKO, serotype A:1 obtained from Indian Veterinary Research Institute, Izatnagar, India, formed the reference strains for this study.

Oligonucleotide Primers
Two oligonucleotides based on the sequence of P. multocida X-73 OmpH gene, Accession No. U50907 (Luo et al., 1997) were designed using Primer3 software. The primers were custom synthesized by M/s Bangalore Genei India. The sequences of the two primers were as follows:

Amplification of OmpH gene
PCR Conditions
A 50 μL reaction mixture was prepared in 0.2 mL thin walled PCR tube. Five microlitres of template DNA was added to a reaction mixture containing 40 pmoles each of primer OmpH 1 and OmpH 2, 200 μM of each dNTP, 1 X Taq buffer with 1.5 M MgCl₂ and 2 units of Taq DNA polymerase. The amplification reaction was carried out in an automated thermal cycler (Eppendorf Master Cycler, Germany) according to the following program, an initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 15 sec, annealing at 56°C for 1 min, extension at 72°C for 1 min and a final extension at 72°C for 10 min. Bacterial culture lysates was used as template DNA. The product was analysed by 2% agarose gel electrophoresis. Standard molecular size marker low range DNA ruler with fragments 3000, 2500, 2000, 1500, 1000, 600, 300 and 100 bp was used as DNA molecular size marker to ascertain the size of the amplified PCR product.

Restriction Enzyme Analysis of OmpH-PCR Product
The amplified PCR products were subjected to restriction enzyme digestion using restriction enzymes Dra I and Hinf I. The digestion was carried out by a standard method (Sambrook et al., 1989). Briefly restriction enzyme digestion was carried out at 37°C for two h, followed by inactivation of the enzyme at 80°C for 20 min. Electrophoresis of the resultant digest was conducted on 8% acrylamide gels. The gel was viewed on a transilluminator and photographed.

RESULTS

The primer pairs OmpH 1 and OmpH 2, designed to amplify the OmpH gene of P. multocida successfully amplified the OmpH gene of all three serotypes viz., A:1, A:3 and B:2. The amplified product had a size of approximately 1000 bp (Fig. 1).

Specificity of the Primers
Primer pairs OmpH 1 and OmpH 2 did not amplify the DNA prepared from unrelated bacterial species such as Streptococcus zooepidemicus, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Leptospira serogroup canicola serovar canicola.

Restriction Enzyme Analysis
Restriction analysis of the amplified products of serotypes A:1, A:3 and B:2 were carried out using the same restriction endonucleases Dra I and Hinf I. Enzyme Hinf I generated patterns similar in A:3 and B:2 but distinct from A:1, while Dra I profiles were distinct from each other for the three serotypes (Fig. 2).

Fig. 1:	Amplification of OmpH gene of P. multocida Lane 1 Low ragne DNA marker, Lane 2-3 LKO and FPI, Lane 4 DPI, Lane 5 A:3, Lane 6 B:2, Lane 7 Negative control

Fig. 2:	Restriction enzyme profiles of OmpH-PCR products (scrotypes A:1, A:3 and B:2) with Hinf I and Dra I, Lane 1 pBR 322 DNA/Alu digest marker, Lane 2 Undigested OmpH-PCR product, Lane 3-5 A:1, A;3 and B:2 (Hinf I digest), Lane 6-8 A:1, A;3 and B:2 (Dra I digest)

DISCUSSION

The custom designed primers could successfully amplify the OmpH gene of three serotypes viz., A:1, A:3 and B:2. Similar results were reported by Luo et al. (1999) who had reported successful amplification of OmpH genes of the serotypes 1, 3 and 4 to 16 of P. multocida and the product had an approximate size of 1 kilo-base pair.

REA of amplified products of OmpH-PCR with Dra I generated distinct profiles for the three serotypes A:1, A:3 and B:2 while Enzyme Hinf I generated patterns similar in A:3 and B:2 but distinct from A:1. Thus, REA of amplified products of OmpH-PCR with Dra I offers a novel technique for differentiation of various serotypes of P. multocida. If unique patterns for all serotypes could be determined in a similar manner then we can have a serotyping technique that is simple, rapid and straight forward and can be performed in any laboratory that has the capacity to perform PCR. The development of a DNA-based technique for differentiation of serotypes could provide an alternative to conventional serotyping systems and has a potential to overcome the problems associated with the current serotyping techniques, which rely on inconsistent expression of phenotypic traits.

However, further studies have to be carried out with all the different serotypes to know whether profiles unique to each serotype are obtained, before the technique can be put for routine use. To the best of our knowledge this is the first report of the use of PCR-RFLP for differentiation of P. multocida serotypes.

ACKNOWLEDGMENTS

The authors are grateful to the Indian Council of Agricultural Research, New Delhi, India for providing financial support under the All India Network Programme on Haemorrhagic Septicaemia and the Dean, College of Veterinary and Animal Sciences, Mannuthy, Kerala, India for providing facilities to conduct this study. The first author also expresses his sincere thanks to the Dean. Rajiv Gandhi college of Veterinary and Animal Sciences, Pondicherry, for his keen interest and valuable suggestion during the course of this study.