USING POLYMERASE CHAIN REACTION TECHNIQUE (PCR) FOR DETECTION BRUCELLA MELITENSIS IN ABORTED EWES' MILK IN NINEVEH, IRAQ

USING POLYMERASE CHAIN REACTION TECHNIQUE (PCR) FOR DETECTION BRUCELLA MELITENSIS IN ABORTED EWES' MILK IN NINEVEH, IRAQ

RAAD A. AL-SANJARY^*; HADEEL A. MOHAMMED^** and MOHAMMAD O. DAHL^**

^* Department of Veterinary Public Health, University of Mosul, Mosul, Iraq

^**Department of Internal and Preventive Medicine, College of Veterinary Medicine, University of Mosul, Mosul, Iraq

Email: ralsanjary61@yahoo.com

INTRODUCTION

Brucellosis is a contagious bacterial infection of livestock, with important effects on both public health and animal health and production. It is continues to be of great health concern and economic importance worldwide (Anonymous, 2001; Ibrahim et al., 2012). Infection in animals frequently results in abortion and diminished milk production (Culter et al., 2005). Brucella organisms localize in the supra-mammary lymph nodes and mammary glands in 80% of infected animals, which continue to excrete these pathogens in their milk throughout their lives and have a significant role in the epidemiology of the diseases (Abd El Razik   et al., 2007). Therefore, man is infected by animal's brucellosis through direct or indirect by ingestion of animal products as after drinking raw milk or eating unpasteurized cheese (Celebi et al., 2007).

The disease presents a great variety of clinical manifestations, making it difficult to diagnose clinically. Therefore, the diagnosis must be confirmed directly by isolation, or indirectly by the detection of immune response against its antigens (Anonymous, 2001). Bacteriological methods depends on the viability and numbers of Brucella in the sample. Thus, culture methods are not always successful as they are time-consuming and the handling of microorganism is hazardous (Refai, 2003; Kazemi et al., 2008) and serological methods are not conclusive; because not all infected animals produce significant levels of antibodies, and because cross-reaction with other bacteria can give false negative results (Abd El Razik et al., 2007; Ilhan    et al., 2008). However, these methods are not wholly satisfactory, PCR assay has been used as an accurate and sensitive assay for detection of Brucella spp in a wide variety of clinical samples; such as, blood, milk, semen, and aborted fetuses even if samples contain low number of brucella (Anonymous, 2001; Dehkordi et al., 2012).

Bucellosis in Mosul, Iraq is a widespread, and many researches have been performed using various diagnostic methods in different samples and different animal species (Al-Farwachi et al., 2009; Al-Obaidi et al., 2009; Rhaymah et al., 2010 and Al-Farwachi et al., 2010). The diagnosis of brucellosis in sheep using serum and bacterial culture by PCR has been done in Mosul (Mohammad, 2006), while Brucella spp antibodies in raw milk has been detected using ELISA in sheep and goat (Al-Obaidi et al., 2009). The purpose of this study was to investigate the viability of the species specific PCR assay as a diagnostic tool for the detection of B. melitensis DNA in the milk of sheep after abortion using specific primersand comparing its results with the Brucella antibodies in serum using Rose-Bengal test in the same animal in Nineveh Province, Iraq.

MATERIALS and METHODS

Fifty aborted ewes were involved in this study. Blood and milk samples were taken from each ewe. Blood samples used to detect Brucella antibodies in serum using Rose-Bengal plate test (RBPT), whereas polymerase chain reaction technique (PCR) were applied on the milk samples to detect Brucella melitensis using one pairs of specific primers for the strain of Br. melitensis.

Preparation of milk samples for PCR:

Milk samples were prepared to improve extraction and purification of bacterial DNA from milk according to Romero and Lopez-Goni (1999): 10 mL of milk centrifuged at 3000 g/min for 15 min. The supernatant was removed and discarded. The pellet was resuspended in 500 μl sterile distil water, mixed with 100 μl buffer (1 mM EDTA, 10 mM Tris-HCl, 50 mM NaCl, 10% sodium dodecyl sulfate and 5 ml of  proteinase K), Vortex and incubated in water bath at 65° C for 5 min, then  centrifuged for 5 min at 3500 g/min.   

DNA extraction:

Br. melitensis DNA was extracted using commercial DNA extraction kit (Sacace Biotechnologies Srl, Italy). According to manufacture instructions, 1.5 ml polypropylene tubes were used, 300 μl of lysis solution and 100 μl of the samples added to each tube, Vortex and incubated at 65° C for 5 min. All tubes centrifuged at 14000g for 5 min. The supernatant carefully transferred by a micropipette into new clean and sterile tubes, then 20 μl of Sorbent added to each tube and incubated at room temperature for 3 min, then centrifuged at 5000g for 30 sec. The supernatant carefully removed and discarded. Thereafter, 300 μl washing Solution 1 added to each tube, vortex vigorously and centrifuged at 8000g for 30 sec., then the supernatant removed and discarded. Then 500 μl washing solution 2 added, vortex vigorously and centrifuged at 8000g for 30 sec, then the supernatant removed. The pellet was resuspended in 50 μl of DNA-eluent, incubated at 65° C for 5 min and vortex periodically. The tubes centrifuged at maximum speed 12000g for 1 min to recover the DNA, which existed in the supernatant and became ready for amplification.

DNA Amplification:

For amplifying Br. melitensis DNA, the primers were used as an essential PCR elements which give bands (731 bp), and the primers sequences were 5'-AAA, TCG, CGT, CCT, TGC, TGG, TCT, GA-3' and the reverses were 5'-TGC, CGA, TCA, CTT, AAG, GGC, CTT, CAT-3' (Mohammad, 2006). The amplification reaction mixture (final volume: 25 µl) includes: 2.5 µl PCR buffer [10 mM Tris-HCl (pH 8), 50 mM KCl , 1.5 mM MgCl₂], 2.5 µl deoxynucleoside triphosphate (200 µM) (Germany, Eppendrof), 2.8 µl foreword  primer, 2.6 µl reverse primer, 1 U of Taq DNA polymerase (Germany, Eppendrof), and 100 ng genomicDNA. The volume was completed to 25 µl with deionized distil water.

The amplification was carried out by the thermalcycler (Germany, Eppendrof) according to the commercial kit (Sacace Biotechnologies Srl, Italy). The following program was started when the temperature reaches 95° C: one cycle at 93° C for 5 min, 40 cycles of 90° C for 60 sec, 60 °C for 60 sec, 72° C for 60 sec. the final incubation was one cycle at 72° C for 10 min.

Analysis of results was based on the presence or absence of specific bands of amplified DNA in agarose gel 2% (U.S.A., Ammersham Bioscience) stained with ethidum bromide. The length of specific amplified DNA fragments for Br. melitensis (731 bp) was visualized using Gel documenting system.

Statistical Analysis

The data was analyzed statistically using SPSS 16.0 (SPSS, 2007). The Goodness-of-fit between RBPT and PCR was measured by McNemar's Chi-square test (Ott and Longnecker, 2004), whereas the agreement between the two was identified using Kappa Statistic (Viera and Garrett, 2005).

RESULTS

The total number of animals that gave positive reaction by RBPT was 35 out of 50 serum samples (70%), while by PCR was 34 out of 50 milk samples (68%). Statistically, this result indicates no significant difference between RBPT and PCR (P<0.01).

The agreement between RBPT and PCR was measured by comparing the results of the two tests in each animal of this study. The observed agreement was 0.66 (66%), while Kappa was 0.206 indicating slight agreement between the two tests (Table 1).

Table 1: Comparison between the number and percentage of the RBPT and PCR results in 50 sheep.

DNA amplification of Br. melitensis using Species-specific Primers electro-phorized on (1.2 %) agarose gel indicates positive sample that showed single band of 731 bp (Fig. 1).

Fig. 1: Result of DNA amplification of Br. melitensis using Species-specific Primers electrophorized on (1.2 %) agarose gel. A single band of 731 bp in lanes (1, 2, 3, 5, 6, 7, 9, 11) indicates positive sample, while lanes (4, 8, 10) representing negative samples. Lane (M) indicates the DNA Ladder 100bp as DNA marker.              

DISCUSSION

Brucellosis is a major zoonotic disease endemic in many parts of the world. It is characterized by chronic infection in animals leading to abortion and infertility. The disease is caused by Brucella which is gram negative, non spore forming, facultative intracellular organism (Cardoso      et al., 2006).

In the present study, a PCR has been compared with one of  serological  methods for detection of brucellosis, RBPT is the most sensitive and rapid screening test used in detection of Brucella infection in animals, but it may produce false-positive and false-negative results (Ilhan    et al., 2008). PCR has been used as an accurate and sensitive assay for detection of Brucella spp in a wide variety of clinical samples even if samples contain low number of brucella (Anonymous, 2001; Dehkordi et al., 2012).

In this research, there were differences between results of RBPT and PCR (Table 1). Studying milk samples using PCR to same animals their serums gave positive reactions by RBPT indicates that 74.3% positive with both tests (Table 1), which confirm that the infection of that animals caused by Br. melitensis, since we used one pairs of specific primers for the strain of Br. melitensis. Furthermore, there was 25.7% of milk samples presented negative reaction by PCR, while their samples gave positive reaction by RBPT (Table 1), which may assign to antibodies of other bacteria, not Br. Melitensis, or may produced due to B. abortus S19 vaccination or exposure to gram-negative bacteria, including include Vibrio cholerae O1, E. coli O:157, Salmonella group N (O:30) and Yersinia enterocolitica O:9, which has lipopolysaccharide (LPS) O-chains similar to those of brucellae (Charisis, 1998, OIE, 2004, Munoz et al., 2005).

Furthermore, Out of 15 animals their serum negative by EBPT there were 8 (53.3%) milk samples positive by PCR (Table 1) which may indicates early incubation of disease that results in false negative RBPT (Radostits      et al., 2007 ). Furthermore, the false negative of RBPT in this research me referred to testing of animals immediately after abortion, since many of owners test their animals immediately after abortion. On the other hand, there were 7 milk samples (46.7%) present negative reaction by PCR out of 35 animals their serum also negative by EBPT (Table 1).

According to results of present research, the percentage of Br. melitensis infection in aborted ewes in Nineveh is 68%, which support by Anonymous (2001) who indicates that Br. melitensis is the main etiological agent of brucellosis in small ruminants. Furthermore, presence of positive results on milk sample by PCR means that the animals shed the bacteria in their milk, since PCR detect the organism itself (Ilhan et al., 2008) and the PCR was found highly sensitive  and specific for identification of Brucella from milk even if  as low as 40 cells/ml of milk (Romero et al., 1995).

The study concluded that using an accurate tests, such as species-specific PCR technique, is an important for reflecting an accurate incidence; since PCR revealed that Br. melitensis existed in aborted ewe's milk in a high percentage in Nineveh, Iraq, which has a public health importance.

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