Molecular detection of Brucella spp . from broth culture of clinical samples in Nigeria : Its role in vaccine quality control

PCR was employed to detect Brucella spp. from broth cultures of clinical samples using a group specific primer based on IS6501 sequence. The sensitivity and specificity of this assay was confirmed by Southern hybridization analysis using a digoxigenin-labeled DNA probe while reproducibility of the analysis was confirmed by repetition of the test. Also, ERI1 and ERI2 primers were used to differentiate Brucella abortus strain 19 from other strains and the relevance in quality control of Brucella vaccine production highlighted.


INTRODUCTION
Brucellosis is a zoonotic disease caused by gramnegative bacteria of genus Brucella.Based on host specificity, six species have been recognized; Brucella abortus (cattle), B. canis (dog), B. melitensis (Goat), B. neotomae (desert wood rats), B. ovis (sheep) and B. suis (pig, reindeer and hare (Meyer and Morgan, 1973;Morgan and Corbel, 1976).Several reports of Brucella species isolated from marine mammals, predominantly, seals and cetaceans have been made (Bricker et al., 2000).Brucella genus is highly homogenous with all members showing greater than 95% homology in DNA-DNA pairing studies, thus classifying Brucella as a monospecific genus (Verger et al., 1985).
In Nigeria, information on the epidemiology of brucellosis suggests that the disease is endemic with varying prevalence rates (Halle and Ajogi, 1997).The herding of cattle, sheep and goat provide conditions, which greatly increase the ease with which infections are transmitted from one animal group to another (Ocholi et al., 1993).Thus, there is a need for an accurate diagnosis for effective control program of brucellosis in Nigeria.
Microbiological isolation and identification are reliable methods of diagnosis for Brucella but are cumbersome and present great risk of infection for laboratory workers (Lopez-Merino et al., 1991).The work of Diaz-Aparicio et al. (1994), Gondswaad et al. (1976), have shown that serological methods of diagnosis are not always sensitive or specific because of cross reactions with related pathogens that often occur.
PCR is known to be of advantage in detecting DNA in pathogenic organisms that have been rendered biologically safe thus reducing the risk of infection of laboratory workers.Leal-Klevezas et al. (1995) have demonstrated the superiority of PCR technique over classical methods of diagnosis such as culture and serology, in its ability to detect small amount of Brucella.
Several molecular methodologies have been employed in the diagnosis of brucellosis: Bricker and Halling (1994), employed PCR assay in the differentiation of B. abortus bv.1, 2 and 4, B. melitensis and B. suis bv. 1 using five oligouncleotide primers, which can identify selected biovars.Also, single step PCR was used for detection of Brucella Spp from blood and milk of infected animals using primers based on the gene encoding for an external membrane protein (Omp-2) (Leal-Klevezas et al., 1995).
This study demonstrates the ability to detect Brucella DNA first from broth culture of clinical samples using PCR analysis and confirmed by Southern Hybridization.

Source of Genomic DNA
The Brucella cultures were made available by the Bacteriology Research Department of the National Veterinary Research Institute, Vom, Nigeria.Broth cultures of the organism were made from clinical samples grown on serum dextrose agar.

Controls
The positive control was standard brucella DNA obtained from Applied Biotechnology (onderstepoort) Laboratory, South Africa while the negative control was a broth culture of a suspected case of brucellosis proven negative microbiologically and by PCR.

DNA preparation
DNA was extracted from broth cultures of Brucella using a modified method of extraction of Brucella DNA according to Romero et al. (1995).500 ml of the broth culture was aliquoted into a micro centrifuge tube; 500 µl of buffer 1 (20 mM NaCl, 20 mM EDTA, 20 mMTris-HCl pH = 7.5, 0.5% Triton x -100) was added and left on ice for 30 min.The mixture was then centrifuged at 12000 rpm for 15 min at room temperature and the supernatant was discarded.The pellets were washed by adding 500 µl of 1x saline sodium citrate and vortexed and then centrifuged at 12000 rpm for 15 min and supernatant discarded.500 µl of buffer 2 (10 mM NaCl, 50 mM EDTA, 50 mM Tris-HCl ph = 7.5, 1% sodium dodecyl sulphate) was added and vortexed, then 20 µl of proteinase k (20 mg/µl) was added and incubated at 50°C overnight to digest the cells.Digestion of cells was enhanced by boiling at 100°C for 5 min in a water bath (Precision: Scientific Inc.) 500 µl of biophenol (Phenol, chloroform and isoamyl alcohol (25:24:1)) then vortexed and centrifuged at 12000 rpm for 10 min.
The aqueous layer was transferred into a clean tube and 1/3 of the volume 3.5 M ammonium acetate and 2 volumes of absolute ethanol was added, mixed and centrifuged at 12000 rpm for 10 min to precipitate the DNA.The supernatant was discarded and the pellets (DNA) washed by adding 200 µl of 70% ethanol then centrifuged at 12000 rpm for 10 min.The supernatant was again discarded and DNA pellets dried in a heating block (BIBBY: Sterilin) at 56°C, and then dissolved in 30 µl of nuclease free water (Promega ®) and stored at -20°C.
These were designed based on the IS6501 sequence of Brucella by Ouahrani-Bethach et al. (1996) and obtained courtesy of the Applied Biotechnology (Onderstepoort) Laboratory, South Africa.

Southern hybridization
Southern hybridization analysis was used as a confirmatory technique for the PCR.Briefly, nylon membrane (Sigma ®), 3 mm filter paper (Whatman : England) and paper towels (sigma®) were cut to size (4 x 6 inches) and the edges and that of the gel were cut at the top-left corner for easy, identification.The gel was incubated in 0.25 M HCl for 15 min and then washed briefly with sterile distilled water and incubated with 20xSSC for a few minutes.Downward capillary transfer was then done overnight according to Koetsier et al. (1993).
The gel was later stained with ethidium bromide (5 µl) (Promega ®) and visualized under UV-light to confirm complete transfer of DNA to the nylon membrane.The nylon membrane was washed in 2-x SSC, air-dried and stored at 4°C between two 3 mm filter papers.
A digoxigenin labeled probe was prepared using a re-amplified amplicon of Brucella strain S19 DNA purified using Agarose Gel DNA Extraction Kit (Roche ®).The purified DNA was washed by adding 2 volumes of absolute ethanol and kept at -20°C overnight then centrifuged at 14000 rpm for 15 min and supernatant discarded.This was repeated using 70% ethanol and then air-dried and 30 µl of nuclease free water was added.
Labelling and testing of the Probe (DNA) was done using High Prime DN Labelling and Detection Starter Kit 1 (Roche®) following the manufacturer's instruction.
Prehybridization and hybridization reaction was carried out on the above mentioned nylon membrane followed by stringency washes and then immunological detection, all according to manufacturer's instruction on the DIG High Prime DNA Labelling and Detection Starter Kit 1.

RESULTS AND DISCUSSION
Detection to the species level was not effected as the primers (1SP 1 and 1SP 2 ) are group specific and therefore will only amplify DNA sequences common to the Brucella genus.Going by the result of this study as shown in (Figure 1), the Brucella DNA (group specific) was amplified while non-Brucella DNA was not amplified using the above mentioned primers which are specifically designed for amplification of DNA of Brucella genus.It is note worthy that Brucella was once considered to be related to the genera Bordetella and Alcaligenes (Johnson and Sneath, 1973).Ouahrani et al. (1996), demonstrated that the IS6501 (ISP 1 and ISP 2 ) primers have around 10 times sensitivity compared to conventional method of PCR employed by Fekete et al. (1990).Thus, the effect of contamination by foreign DNA is insignificant as they will not be amplified especially at 650 bp fragment, ruling out the problem created by cross reactions related pathogen with Brucella using serological methods.
As a confirmatory test Southern Hybridization analysis indicated bands formed as a result of hybridization of Brucella samples (DNA) to the probe (a digoxigeninlabelled known positive Brucella DNA).Moreover, the negative control showed no band, as there was no hybridization to the probe (Figure 2).Lanes 1, 2 and 6 revealed a negative result as there were no observable bands formed as a result of hybriddization indicating that the PCR technique used is specific and will only detect the DNA of Brucella.The result of the Southern hybridization analysis is significant as cross hybridization by contaminant which sometimes occur in Southern hybridization analysis thereby making interpretation of result difficult (Van kuppeveld et al., 1994) was overcome by the use of purified PCR product (Brucella DNA) for probe making to enhance sensitivity and specificity.Figure 2b shows the agarose gel electrophoresis of PCR product employed in the Southern hybridization test which serves as a confirmatory evidence of PCR reaction specificity.
In addition, we were able to differentiate by exclusion, B. abortus strain 19 from other strains of B. abortus using the primers ERI 1 and ERI 2 which amplifies a 178 bp fragment which represent an intact eri sequence in all B. abortus strain except strain 19 (Bricker and Halling, 1995).Samples were considered as abortus strain 19 if a 500 bp fragment was amplified with the IS7119 and AB primers and not by ERI 1 and ERI 2 set of primers (Figure 3).This may be of importance in the identification and/or confirmatory analysis of quality control program of Brucella vaccine production where B. abortus strain 19 is used.
Brucella vaccine production in Nigeria involves the use of B. abortus strain 19.Thus, the exclusion of other B. abortus strain which could act as contaminant during vaccine production process and the confirmation of the   presence of the vaccine strain are of utmost importance in quality control procedure and quality assurance of Brucella vaccine produced.Furthermore, the control of brucellosis is among other factors, dependent on the effective vaccination of live-stock with quality assured vaccine.
All amplifications were repeated two times with same result indicating the reproducibility of this assay.Although, the need for PCR detection of Brucella DNA to the specie level and direct detection from clinical samples cannot be over emphasized this result indicates the specificity and sensitivity of our PCR analysis of Brucella genus using cultures of Brucella colonies isolated from infected samples.
Considering the current epidemiology of brucellosis in developing countries like Nigeria and the zoonotic importance of this disease, the use of PCR assay for accurate detection is paramount where specificity and sensitivity are not compromised.
Also, a successful fight against brucellosis and the role of Brucella in biological warfare and agro-terrorism as pointed out by Bricker (2002) makes molecular diagnosis a vital tool that must also be accessed by developing countries.Furthermore, a reliable diagnostic and quality control tool such as described in this work should be employed in order to ensure proper control of the disease in animal and human population.

Figure 2 .
Figure 2. a. Agarose gel electrophoresis of PCR product employed in Southern hybridization.The sensitivity of this test was confirmed by Southern hybridization Figure 2b.All PCR analysis was performed under the same condition.b.Southern hybridization of Brucella cultures.Lanes 3, 4 and 5 show bands as a result of positive hybriddization reaction, while lanes 1, 2 and 6 show no bands indicating a negative result.Lane 7 is positive control while lane 8 is the negative control.