A Multiplex PCR for the Detection of Brucella spp. and Salmonella abortus ovis from aborted ovine fetus
A. Sharifzadeh1 , A. Doosti1, Kh . Khaksar3
1- Department of Microbiology, Faculty of Veterinary Medicine,
Islamic Azad University, Shahrekord, Iran.
2- Shahrekord Veterinary Clinic Net work .
Brucella spp. and Salmonella abortus ovis are important causes of ovine abortion. Both bacteria can be serologically diagnosed, but many factors may cause false positive and negative results. Direct methods based on bacteriological isolation are usually applied, but they are difficult, time consuming and dangerous. Polymerase chain reaction (PCR) has been successfully described for the detection of Brucella spp. and Salmonella abortus ovis.
The detection of these agents in aborted ovine fetuses by multiplex PCR is described. The mPCR was applied to 54 fetal stomach contents. Of the tested samples 14 were totally negative, 10 resulted positive for Brucella spp., 24 came back positive for Salmonella abortus ovis and 6 resulted positive for both. Simplicity and the possibility of detection of both bacteria in a single tube reaction support the use of the mPCR in the routine diagnosis.
Key-words: Multiplex PCR, Brucella spp, Salmonella abortus ovis, ovine fetus.
Brucella spp. and Salmonella abortus ovis are widely distributed around the world. Reproductive problems such as abortions and premature births may be the only clinical signals in pregnant ewes. Both diseases can be diagnosed by detection of serum specific antibodies but these methods are presumptive because many factors may cause false positive and negative results.
Direct methods based on the demonstration of these bacteria in the host are the most precise diagnostic procedures. Bacteriological isolation is usually employed, but it is difficult, time consuming and dangerous for the operators (4,5,7,11).
After the development of the polymerase chain reaction (PCR), some papers described its use for the diagnosis of Brucella spp. (3,10) and Salmonella abortus ovis (1,6).
Multiplex PCR (mPCR) is a PCR derived procedure where multiple target DNA sequences can be detected in a single reaction (9).
This paper describes a mPCR using novel primers for the detection of both Brucella spp. and S. a. ovis DNA in aborted ovine fetuses.
2. Materials and methods
2.1 Reference bacterial strains
B. abortus strain 119-3 and S. a. ovis were kindly supplied by Dr. Tadjbakhsh Hassan, Laboratory of Bacterial Diseases of the Faculty of Veterinary Medicine of the University of Tehran, Iran.
2.2 Ovine clinical samples
Clinical samples from 54 aborted ovine fetus were sent under refrigeration to the Biological Research Centre of Islamic Azad University of Shahrekord for bacteriological examination. All samples consisted of abomasal contents. Whole abomasal contents were stored at -20 until DNA extraction.
2.3.1 Extraction protocol
Genomic DNA was directly isolated from Abomasal contents by cinnagen DNA TM kit (IRAN)
2.3.2 DNA amplification
PCR assays for the detection of Brucella spp. (PCR/Bruce) and S. a. ovis (PCR/SAO) was performed. For the expected size of amplicons 243 bp for Brucella spp. and 172 bp for S. a. ovis the mPCR assay employed the novel primers of PCR assays: Bruce CTA TTA TCC GAT TGG TGG TCT G- and
Bruce GGT AAA GCG TCG CCA GAA GG - for Brucella spp., and SAO GCC GAA GAT GAG TGT GTC CAG TT- + SAO CCG TGT TCT TAC CCA CCG TAT 3 for S.a. ovis.
The mPCR assay was carried out in microtubes under following conditions: initial denaturation at 97º C for 4 minutes by 30 cycles of denaturation at 94º C for 1 minute, annealing at 57º C for 40 seconds, extension at 72º C for 40 seconds and final extension at 72º C for 3 minutes.
2.3.3 Visualization of PCR products
The PCR products were visualized after electrophoresis in 2% agarose gels and stained with ethidium bromide (12).
A molecular weight marker with 100 bp increments (100bp ladder fermentas ) was used as size standards .
Of the 54 samples tested, 14 were totally negative, 10 resulted positive for Brucella spp., 24 resulted positive for Salmonella abortus ovis and 6 resulted positive for both agents.
Figure 1 shows the positive results for Brucella spp. and S. a. ovis in ovine abortion samples, compared with positive and negative controls.
Figure 1) Multiplex PCR for detection of Brucella spp. and
Salmonella abortus ovis. Lane 1 to 3, different samples from
aborted ovine fetus and lane 4 to 5 are positive and
negative controls respectivily.
Brucella spp. and S. abortus ovis are largerly present in Iran and both agents induce abortion in infected animals.
The diseases spread in a herd mostly through contact with infected discharges from an aborting ewe and its fetus.
Bacterial isolation is a tedious process influenced by a number of factors, including highly fastidious growth requirements, low number of viable organisms in the sample, delay in transportation (leading to putrefaction) and treatment with chemotherapeutics (5).
Additionally, a prolonged incubation period may lead to isolation’s failure.
The mPCR technique has been increasingly used as a supplementary method in Brucella and Salmonella a. ovis diagnosis by simultaneously amplifying more than one locus in the same reaction. mPCR is a rapid and convenient screening assay, with both clinical and research applications.
Simultaneous detection of two major pathogenic bacteria in fetal stomach contents has been demonstrated in the present study by analyzing a single sample using mPCR.
The results show that developed mPCR assay was able to successfully detect Brucella spp. and Salmonella abortus ovis.
The following two main reasons could be listed to recommend the use of the mPCR for routine diagnosis of Brucella spp. and S. abortus ovis in ovine abortions: a) the simplicity and speed of the procedure, b) the possibitity of detecting both agents in a single tube reaction.
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First Published June 2007