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 .

Abstract

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.

Introduction

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 mPCR

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 .

 

3. Results

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.

 

4. Discussion

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.

References

1- Beuzon, C.R., Schiaffino, A., Leori, G., Cappuccinelli, P., Rubino, S., Casadesus, J. 1997. Identification of Salmonella abortus ovis by PCR amplification of a serovar–specific IS200 element. Appl. Env. Microbiol., 63(5), pp. 2082-2085

2- Fekete, A.J., Halling, S.M. 1990. Preliminary development of a diagnostic test for Brucella using polymerase chain reaction. J. Appl. Bacteriol., 69, pp . 216–227

 

3- Herman, L., Ridder, H. 1992. Identification of Brucella spp. by using the polymerase chain reaction. Appl. Environ. Microbiol., 58, pp . 2099–2101

 

4- Kirkbride, C. A. 1990. Laboratory diagnosis of livestock abortion. Iowa State University Press, Ames, IA, pp. 260

5- LeyLa, G., Kadri, G., Umran, 0. 2003. Comparison of polymerase chain reaction and bacteriological culture for the diagnosis of sheep brucellosis using aborted fetus samples.

Vet. Microbiol. 93, pp.53-61

6- Masala, G., Porcu, R., Daga, C., Denti, A., Scanu, G., Patta, C., Tola, S. 2007.  Detection of pathogens in ovine and caprine abortion samples from Sardinia, Italy, by PCR. J. Vet. Diag. Invest., 19(1), pp.96-98

7 – Nielsen, K.J., Duncan, J.R. 1990. Animal brucellosis. CRC Press, Boca Raton, FL, pp. 453

8- Ocholi, R.A., Kwaya, J.K.P., Ajogi, I., Bale, J.O.O. 2005. Abortion due to Brucella in sheep in Nigeria. Rev. Sci. Tech. OIE 24(3), pp.973-979

9- Richtzenhain, L.J., Cortez, A., Heinemann, M.B., Soares, R.M., Sakamoto, S.M., Vasconcellos, S.A., Higa, Z.M., Scarcelli, E., Genovez, M.E. 2002. Multiplex PCR for the detection of Brucella spp. and Salmonella abortus ovis. DNA from aborted ovine fetuses. Vet. Microbiol., 87(2), pp.139-147

10– Romero, C., Ggamzo, M., Lopez–Goni, L. 1995. Specific detection of brucella DNA by PCR. J.Clin.Microbiol., 33, pp. 615 – 617

11- Salehi, M., Pishva, E., Salehi, R., Rahmani, M. 2006. Isolation of Brucella abortus using PCR-RFLP analysis. Iranian J. Pub. Health, 35(4), pp.22-27

12- Sambrook, J., Fritsch, E.F., Maniatis, T. 1989. Molecular cloning: a laboratory manual. 2nd edition. Cold Spring Harbor Laboratory Press, New York, p. 2100

 

© Copyright Priory Lodge Education Limited 2007

First Published June 2007

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