Journal of Research in Biology
Identification of Animal Pasteurellosis by PCR Assay
Keywords:
Culture lysate, genomic DNA, Pasteurella multocida, PCR .
ABSTRACT:
Diagnosis of pasteurellosis has become difficult, as there are five different capsular types and 16 somatic types. Molecular techniques like PCR are adapted nowadays for rapid and accurate diagnosis in early stage of the disease and also it provides useful information for epidemiological studies. The present study was conducted to study the efficiency of polymerase chain reaction (PCR) in the identification of P. multocida isolates and evaluation of different PCR methods viz., (i) PCR using genomic DNA (ii) PCR using culture lysate and (iii) PCR by colony touch method. In the present study P. multocida specific PCR was performed by using KMT1SP6 and KMT1T7 oligos. These oligos amplified the genomic DNA from P. multocida isolates only. All the three methods produced PCR amplified product at 460 bp and colony touch method was found to be the best method.
895-899 | JRB | 2013 | Vol 3 | No 3
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Authors:
Venkatesan PS, Deecaraman M and Vijayalakshmi M.
Institution:
Department of IBT,
Dr. M.G.R. Educational & Research Institute, Department of IBT, Maduravoyal,
Chennai - 600095.
Corresponding author:
Venkatesan PS.
Email:
venkyvet74@gmail.com
Web Address:
http://jresearchbiology.com/documents/RA0332.pdf.
Dates:
Received: 04 Feb 2013 Accepted: 05 Mar 2013 Published: 30 Apr 2013
Article Citation:
Venkatesan PS, Deecaraman M and Vijayalakshmi.
Identification of animal Pasteurellosis by PCR assay.
Journal of Research in Biology (2013) 3(3): 895-899
Journal of Research in Biology
An International Scientific Research Journal
Original Research
INTRODUCTION
The various forms of pasteurellosis caused by Pasteurella multocida are the major health problem for livestock population worldwide. Diagnosis of pasteurellosis has become difficult, as there are five different capsular types and 16 somatic types. Molecular techniques like PCR are adapted nowadays for rapid and accurate diagnosis in early stage of the disease and also it provides useful information for epidemiological studies. Pasteurellosis has high impact on economic status of Indian farmers. The overall incidence rate of haemorrhagic septicaemia (HS) was reported as 6.4 per lakh population during 1974-86, resulting in losses exceeding ten million rupees annually (Dutta et al., 1990; singh et al., 1996).
Isolation and identification of P. multocida from specimens like fresh tissues or heart blood followed by the performance of various biochemical and serological methods have been used to study P. multocida. These include catalase, indole, oxidase and sugar fermentation tests. Due to time consuming procedure and limitations of these methods, molecular techniques like polymerase chain reaction (PCR) were adapted nowadays. PCR has advantages over the conventional techniques in rapidity, sensitivity and specificity to identify the P. multocida. The present study was conducted to assess the efficiency of PCR in the identification of P. multocida from poultry and ruminants and to evaluate the different methods in PCR assay viz. PCR using genomic DNA, PCR using culture lysate and PCR by colony touch method.
MATERIALS AND METHODS
Isolation and Identification of P. multocida
Fifty two samples were collected from various geographical areas of Tamil Nadu, India. Specimens such as heart blood swab, liver, spleen and long bones collected from various animals, were streaked directly onto 5% sheep blood agar and Pasteurella multocida selective agar as reported earlier (Moore et al., 1974) and incubated at 37°C with 5-10 % CO2 for 24-48 h. Plates were examined for colonies, the suspected colonies were subjected to grams staining, and biochemical test as per standard techniques. Standard vaccine strain of P. multocida P52 (B:2) was taken as reference strain. Pathogenicity test in mice were carried out for all the fifteen isolates and PCR was performed for all the isolates.
Isolation and Purification of Genomic DNA
A 900 µl cell suspension of each sample were resuspended in 100 µl of 10x Tris-EDTA (TE) buffer (pH 8.3) with 10 mg of lysozyme and were incubated at 37°C for 1.5 h. Bacterial cultures were treated with 10 µl of proteinase K (10 mg/ml) and incubated at 50°C for 1 h. The nucleic acid was extracted with phenol-chloroform-isoamyl alcohol followed by ethanol precipitation as per the method of Sambrook et al., (1989) and Sachithanandam et al., (2011).
PCR Using Culture Lysate
One Milliliter of 18 h broth culture or take few freshly grown pure colonies from blood agar plate and suspend in 500 µl sterile distilled water and centrifuge at 4000 g for 1 minute and collect the pellet. The pellet was washed with sterile distilled water, resuspended in 100 µl sterile distilled water and boiled for 10 min. The samples were centrifuged to sediment cell debris and 10 µl of the supernatant was used in the PCR reaction.
PCR Using Colony Touch Method
A single pure colony grown on agar plates was used to perform PCR. A pipette tip was lightly touched onto a colony and then suspend in PCR amplification mixture.
PCR Technique
The species-specific primers KMT1SP6 and KMT1T7 designed by Townsend et al., (1998) were used in this study to amplify the gene sequences in P. multocida.
Primers 1 KMT1SP6 5’-GCT GTA AAC GAA CTC GCC AC- 3’
Primers 2 KMT1T7 5’- ATC CGC TAT TTA CCC AGT GG-3’
PCR mixture was prepared using PCR kit obtained from FINNZYME, Finland. The 50 µl of reaction mixture was prepared with 10 µl template DNA, 10ng of each primers, 200 µM concentration of each dNTPs, 10x PCR buffer and 1 unit Taq DNA polymerase. PCR amplification was carried out in an automated thermal cycler (Perkin Elmer Gene AMP PCR system 2400) with the following thermal programme. Initial denaturising at 95°C for 4 min followed by 30 cycles of denaturising at 95°C for 1 min., annealing at 55°C for 1 min., extension at 72°C for 1 min. and final extension at 72°C for 9 min, were carried out. After amplification, PCR products were checked in 1.5% agarose gel electrophoresis along with the standard molecular weight marker (Lambda DNA Hind III digest and ϕ X 174 DNA Hae III digest; FINNZYME, Finland).
The biochemical tests were carried out as per the standard procedure followed in Arun kumar et al., (2012)
RESULTS
Out of total collection of 52 suspected samples, procured from cattle sheep, goat and poultry, 15 samples were confirmed as P. multocida based on biochemical tests (Table 1) and PCR. All the P. multocida isolates were pathogenic to mice and dies within 24 h. PCR was performed for all the 15 isolates by 3 methods viz., colony touch method, culture lysate and with genomic DNA. P52 strain of P. multocida, obtained from the Institute of veterinary preventive medicine (IVPM) Ranipet, Tamil Nadu, taken as a positive control and the following bacteria Escherichia coli, Clostridium chauvoei, Salmonella enteritidis, Salmonella typhimurium, Bacillus anthracis,
Staphylococcus aureus and Klebsiella spp. used as negative controls. The expected amplification size of 460 bp was obtained in all the 15 isolates. PCR amplification was noticed at approximately 460 bp by all the three methods and in all the 15 isolates as like that of positive control (figure 1). No amplification product was observed in negative controls (figure 1). Molecular weight of PCR product was estimated based on the standard molecular weight marker.
DISCUSSION
The 15 isolates of P.multocida collected from different places and sources of origin produced approximately 460 bp amplified product as that of reference strain P52, but no amplified product was noticed among the negative controls. It is concluded that the primers were highly specific to P. multocida isolated from various sources. The above result agrees with the previous reports of earlier workers (Townsend et al., 1998; Hunt et al., 2000; Miflin and Blackall, 2000; OIE manual, 2000; Dutta et al., 2001). In this study the amplified product of approximately 460 bp was observed using three different methods viz. colony touch method, culture lysate method and purified genomic DNA method (figure 1). The intensity of the amplified PCR product varies (figure 1), due to the variation in DNA concentrations. Townsend et al., (1998) reported that PCR using colony touch method produced amplification
product approximately at 460 bp and the intensity of the amplified product varied due to inconsistency of the DNA concentration. Dabo et al., (2000) reported that the boiled cell extract method has the advantages of simplicity and rapidity in the identification of P. multocida isolates. Since the PCR amplified product of 460 bp was noticed in all samples of poultry and ruminants, using oligos KMT1SP6 and KMT1T7, the oligos are considered as specific to P. multocida affecting all species of poultry and ruminants. Considering the cost and time involved in the preparation and purification of genomic DNA, the colony touch method has advantages of simplicity and rapidity for epidemiological surveys involving large number of P. multocida isolates. PCR using colony touch method would be an adaptable easy to perform method in regional laboratories for rapid diagnosis of HS and FC from field cases without the need to obtain pure culture and extensive biochemical and serological tests.
ACKNOWLEDGEMENT
The authors thank the Head, department of microbiology, Madras Veterinary College, Chennai, for providing the facilities to carry out this work.
REFERENCE
Arun Kumar JM, Lakshmi A, Sangeetha Rani V and Sailaja B. 2012. Isolation and characterization of feather degrading bacteria from poultry waste. Journal of Research in Biology. 2(7): 676-682.
Blackall PJ and Miflin JK. 2000. Identification and typing of Pasteuruella multocida; A Review. Avian Pathology 29(4):271-287.
Dabo SM, Confer A and Lu YS. 2000. Single primer polymerase chain reaction fingerprinting for Pasteurella multocida isolates from laboratory rabbits. American Journal of Veterinary Research 61(3):305-309.
Dutta J, Rathore BS, Mullick SG, Singh R and Sharma GC. 1990. Epidemiological studies on occurrence of haemorrhagic septicaemia in India. Indian Veterinary Journal 67(10): 893-899.
Dutta TK, singh VP and Kumar AA. 2001. Rapid and Specific diagnosis of animal pasteurellosis by using PCR assay. Indian Journal of Comparative Microbiology, Immunology and Infectious Diseases 22(1):43-46
Hunt ML, Alder B and Townsend KM. 2000. The molecular biology of Pasteurella multocida. Veterinary Microbiology 72(1-2):3-5.
Manual of Standards for diagnostics tests and vaccines. 2000. Office International Des Epizootics Manual, France. 446-456.
Sachithanandam V, Mohan PM, Dhivya P, Muruganandam N, Baskaran R, Chaaithanya IK and Vijayachari P. 2011. DNA barcoding, phylogenetic relationships and speciation of Genus: Plectropomus in Andaman coast. Journal of research in Biology. 1( 3): 179-183.
Sambrook J, Fritisch EF and Mamiatis T. 1989. Molecular cloning: a laboratory manual, Cold spring harbor press, Plainview, N.Y. 2nd ed. 3.
Singh VP, Kumar AA, srivastava SK and Rathore BS. 1996. Significance of Haemorrhagic Septicaemia in Asia: India. International workshop on diagnosis and control of Haemorrhagic Septicaemia. Bali, Indonasia. May 28-30.
Townsend KM, Frost AJ, Lee CW, Papadimitrion JM and Dawkins HJS. 1998. Development of PCR assays for species and type specific identification of Pasteurella mutlocida isolates. Journal of Clinical Microbiolgy 36(4):1096- 1100.
Venkatesan et al., 2013
896 Journal of Research in Biology (2013) 3(3): 895-899
Venkatesan et al., 2013
Journal of Research in Biology (2013) 3(3): 895-899 897
Tests |
Name of the Isolates |
||||||||||||||
D1P |
D2P |
FP |
GP |
HP |
KP |
LP |
NP |
OP |
AS |
CS |
TS |
YS |
BG |
MC |
|
Hemolysis on Blood agar |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
Growth on MacConkey agar |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
Motility |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
Gelatin Liquefaction |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
Methyl Red Test |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
H2S (Hydrogen sulphide) |
+ |
+ |
- |
- |
+ |
+ |
+ |
- |
+ |
+ |
+ |
+ |
+ |
+ |
- |
Catalase |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
Oxidase |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
Nitrate Reduction |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
Indole |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
Lysine Decarboxylase |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
Ornithine Decarboxylase |
- |
+ |
+ |
+ |
+ |
+ |
- |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
Urease |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
Pyrase |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
Esculin Hydrolysis |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
VT (Voges Proskaeur |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
Phenylalanine |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
β-Galactosidase (ONPG) |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
β-Glucuronidase |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
α-Galactosidase |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
β-Xylosidase |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
N-acetyl β-D-glucosaminedase |
- |
- |
+ |
+ |
+ |
+ |
- |
+ |
- |
- |
+ |
+ |
+ |
+ |
+ |
Table 1. Biochemical Profiles for the Identification of Pasteurella multocida Isolates
+ : Positive, - : Negative,
Venkatesan et al., 2013
898 Journal of Research in Biology (2013) 3(3): 895-899
Figure 1: Pasteurella multocida – specific PCR (PM-PCR) assay
CS TS YS BG MC PC NC M
460bp→
D1P D2P FP GP HP PC NC M
460bp→
KP LP NP OP AS PC NC M
These figures illustrate fragments specifically amplified by PCR in all the P. multocida isolates by means of the primers KMT1SP6 and KMT1T7. Variation in the intensity of the amplified product was observed, due to variation in DNA concentration of each sample.
D1P, D2P, FP, GP, HP, KP, LP, NP, OP, AS, CS, TS, YS, BG, and MC are the names of P. multocida isolates.
Venkatesan et al., 2013
Journal of Research in Biology (2013) 3(3): 895-899 899
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