Abstract

Fifteen tissue samples of lymph nodes and spleens from Brucella sero-positive camels were subjected to Four polymerase chain reaction (PCR) tests, which were, abortus melitensis ovis suis (AMOS) PCR, Bruce-ladder multiplex PCR, Multilocus Variable Number of Tandem Repeats (MLVA) and the Real-time PCR (RT-PCR). Extraction of Brucella DNA from tissue samples was done by heat, which was found to be satisfactory for conduction of the tests. Brucella species could be distinguished according to the banding pattern and the amplification fragment length polymorphisms which is a diagnostic tool of specific strains. In this study, we evaluated the performances of newly designed real-time PCR assays using TaqMan probes and targeting the insertion sequence IS711, for the detection of Brucella at genus level. Real-time PCR assays are easy-to-use, produce results faster than conventional PCR systems while reducing DNA contamination risks. The IS711-based real-time PCR assay is specific and highly sensitive and appears as an efficient and reproducible method for the rapid and safe detection of the genus Brucella. MLVA assay has the same advantages of multiplex PCR beside it can differentiate Brucella isolates on biovar level (genotyping). Bruce-ladder PCR assay is recommended for testing the seed cultures commonly used in the production of living Brucella vaccines (Rev-1, S19 and RB51) and in evaluating them in quality control laboratories and also in identification and differentiation of Brucella isolates. PCR techniques are recommended to be used for identification of Brucella instead of isolation, which is dangerous and complex.

1. Introduction

Isolation of Brucella organism from animals was first reported from contagious abortion in cattle and other animal species and the isolate named Bacillus abortus, which was followed by other names, Corynebacterium abortus, Bacterium abortus and Alcaligenes abortus ¹. Then later the name Brucella was suggested to the genus ².

The first isolation of Br. abortus from local cattle in Sudan was from a cow which aborted at Juba dairy farm ³. There after the disease was detected in many parts of the country following isolation of the causative agent or antibody detection.

PCR is fast, can be performed on any body tissue, and can yield positive results as soon as 10 days after inoculation. It was first developed for brucellosis in 1990, using a 635-bp fragment of B. abortus strain 19, ⁴. Subsequently, two major gene sequences have been used as targets: the 16S rRNA gene sequence, ⁵, which represents total genus-specific homology and has been satisfactory in clinical settings ⁶ and the BCSP31 gene, which encodes an immunogenic protein of the external membrane of B. abortus ⁷ and has been extensively studied in clinical practice ⁸.

Real-time PCR offers results in 30 minutes, so it will be the diagnostic tool of the future, ⁹. B. suis identified specific 17 bp repeat on chromosome II (BS1330_II0657 locus). This repeat is common for B. suis bv 1 to 4 and was used to develop a TaqMan probe assay. The average PCR efficiency was determined as 95%. This novel qPCR assay could become a rapid, inexpensive and reliable screening method for large sample pools of B. suis 1 to 4 ¹⁰.

AMOS PCR is one of the first PCR assays to differentiate among Brucella species. AMOS PCR is used for differentiation of Brucella species (abortus, melitensis, ovis, suis). This assay comprises cocktail of five oligonucleotide primers which can identify selected biovars of the four species of Brucella using the polymorphism arising from species-specific localization of the genetic element IS711 in Brucella DNA. ¹¹.

The major advantage of Bruce-ladder PCR is that it can identify and differentiate in a single step most Brucella species as well as the vaccine strains, i.e. B. abortus S19, B. abortus RB51 and B. melitensis Rev.1. In contrast to other PCRs, Bruce-ladder is able to detect also DNA from B. neotomae, B pinnipedialis and B ceti. In addition, B abortus biovars 3, 5, 6, 7, 9 and B. suis biovars 2, 3, 4, 5 can be identified by this new multiplex PCR.

The MLVA assays take advantage of array-length variations in tandem repeats. Owing to the availability of whole genome sequences, tandem repeats can be readily identified, and tested for polymorphism. Most tandem repeats have been evaluated, essentially by three groups (Bricker, Vergnaud, Whatmore and colleagues), and tens of polymorphic loci have been identified. Different selections of such loci, used in MLVA assays, have been proposed to suit different purposes.

The first MLVA assay, named ‘HOOF-Prints’ (hypervariable octameric oligonucleotide fingerprints), was developed by ¹².

The Brucella genome contains a family of tandem repeats sharing the repeat unit‘AGGGCAGT’. Eight highly variable such loci, present in most Brucella species, were selected for use in the HOOF-Print assay. Variations of the repeat numbers at each locus can easily be investigated by amplifying the corresponding regions and subsequent gel electrophoresis or, preferably, capillary electrophoresis, given the short repeat unit size (using A. P. I sequencer). This selection of tandem repeats has a very high discriminatory power and can be useful for local outbreak investigations. However, it cannot provide a species assignment of homogeneity at these loci. The main objective was evaluation of different PCR techniques for diagnosis of Brucella spp.

2. Materials and Methods

Fifteen tissue samples of lymph nodes and spleens from Brucella seropositive camels were used for the tests. Three Brucella reference strains were used as controls these were: Brucella abortus biovar 1 strain 19 was obtained, as dried seed, from the United States Department of Agriculture, Ames, Iowa, USA. Brucella abortus biovar 3 strain Tulya, B. melitensis biovar 1 strain 16M were obtained from the FAO/WHO Collaborating Centre for Reference and Research on Brucellosis, New Haw, Addestone, UK.

DNA was purified from 15 tissue samples, using Invitrogen Pure link genomic DNA kit and the protocol recommended by the manufacturer. As a starting material, small pieces of tissue (max. 20 mg) were used. Seventy two hours Brucella cultures of reference strains were harvested with appropriate amounts of phenol saline and washed in PBS. Three cycles of freezing at -20°C and thawing at 60°C were carried out. The DNA was pelleted at 12,000 g at 4°C for 15 minutes, dissolved in 50 μl Tris-EDTA and stored at -20°C until used.

The samples and controls were subjected to the 4 PCR techniques, which were AMOS, Bruce-ladder, Real-Time and MLVA.

AMOS PCR was used for differentiation of Brucella species (abortus, melitensis, ovis, suis). Five oligonucleotide primers were used.

Cycling conditions were, cycle 1, 94°C- 4 min., cycle 2, 94°C- 1min., 68°C- 1min., 72°C- 1 min. Cycle 2 repeated 40 times. Cycle 3, 72°C- 3 min.

Visualisation was done on 1.5% agarose-gel stained with ethidium-bromide. Species determination was based on differences in sizes of amplified DNA fragment. Brucella abortus isolates produced 498 bp, Brucella melitensis 731 bp, Brucella ovis 976 bp and Brucella suis 285 bp band.

This assay was used with a multiplex eight pairs of primers in the same reaction tube ¹³.

Two samples were tested using this method in the presence of a negative control (without DNA) and a positive control (B. suis DNA).

Cycle condition as follow: initial denaturation at 95°C for 7 min., template denaturation at 95°C for 35 sec., annealing at 64°C for 45 sec., extension at 72°C for 3 min. and final extension at 72°C for 6 min.

The PCR products (7 μl) were analyzed by electrophoresis (120 V for 1 hour) in a 1.5% agarose gel in TBE buffer (89 mM Tris/HCl, 89 mM boric acid, 2.0 mM ethylene diamino tetra-acetic acid [EDTA], pH 8.0).

1 kb plus DNA ladder was used as a molecular size marker. Bands were visualized in illuminator with UV light after staining with ethidium bromide. Interpretation of the results depended on the band bp size produced.

MLVA-16 ¹⁴ was used for molecular typing of Brucella isolates. For this purpose, DNA from samples was purified and concentration and purity of obtained DNA were determined (as described previously).

Sixteen different loci were amplified by PCR using Taq Master mix PCR kit from Qiagen. Total volume of reaction was 25 µL, with final concentration of primers 0.5 µl and DNA input of approximately 20 ng. All forward primers were labeled with FAM fluorescent dye at 5’ position, which enabled fragment size determination using capillary electrophoresis.

Cycling condition as follows: initial denaturation at 94°C for 5 min, then 30 cycles of denaturation at 94°C for 30 sec., annealing at 60°C for 30 sec., extension at 72°C for 1 min. and final extension at 72°C for 5 min.

Capillary electrophoresis was performed in ABI 310 Genetic Analyzer, using POP 4 polimer and GS LIZ1200 Size Standard (Applied Biosystems). PCR products were diluted 1/100 and 0.5 µl mixed with 0.5 µl LIZ1200 Size Standard and 11 µl of HiDi Formamide (Applied Biosystems), denaturated at 95°C for 3 minutes and immediately transferred on an ice rack for 2 minutes. The mixture was loaded into the machine and appropriate running module was selected. Obtained raw data was analyzed with Gene Mapper software (Applied Biosystems) and fragment size for each locus was determined. Based on the results for fragment sizes, number of repeats for each locus was inferred, using table from MLVA web based database (https://minisatellites.u-sud.fr/MLVAnet/index.php?&largeur=1280).

Real-time PCR (RT-PCR) provides means of detecting and quantifying DNA targets by monitoring PCR product accumulation during cycling as indicated by increased fluorescence.

The performances of a designed real-time PCR assay using TaqMan probes and targeting the insertion sequence IS711, for the detection of Brucella at genus level was evaluated.

The real-time PCR assay was compared to previously describe conventional PCR assays targeting the same gene.

The TaqMan Assay portfolio comprehensive set of products was used. Gene-specific probe, primer sets and master mix were used.

The 7500 Fast Real-Time PCR System with specialized optical system enables easy and accurate calibration, Easy-to-use, spontaneous software.

The TaqMan probe–based chemistry, the gold standard in allelic discrimination and quantitative gene expression offering high sensitivity, specificity, and reproducibility.

PCR reaction for detection of Brucella genome was targeting the IS711 ¹⁵. PCR reaction was set using BioRad iScript TM One Step RT PCR using the following protocol:

Prepared reaction mix was cycled and fluorescence was measured in Bio Rad IQ5 real time machine under following cycling conditions: Cycle 1, 95°C for10 min, Cycle 2, 95°C for 15 sec. and 60°C for 1 min. Cycle 2 was repeated 40 times. Acquisition of fluorescent data was at the end of the annealing step (60°C for 1 min).

3. Results

1- AMOS PCR

Brucella AMOS PCR test was evaluated to determine its accuracy in differentiating Brucella spp. obtained from field samples and the vaccine strain 19. All five samples were negative by AMOS PCR (Figure 1).

Out of the five, two tissue samples tested by Bruce-ladder which measured five fragments, of 1,682, 794, 587, 450,and 152 bp in size in electrophoresis gel, that indicated they were B. abortus species, (Figure 2).

3- MLVA_16 PCR

The tissues tested with MLVA gave different repeat with the different 16 loci. These raw data inferred to the gene MAB, (Data Base). (Table 1 and Figure 3) Result obtained by MLVA (Data Base) confirmed that isolates were clustering to the species abortus which had been isolated from neighboring countries from different animal species.

4- RT PCR qPCR (protocol for detection of IS711), Brucella isolates from camels

Real-time PCR diagram showed detection and quantification of DNA targets by monitoring PCR product accumulation, measured by increased fluorescence during cycling (Table 2 and Figure 4 and Figure 5).

4. Discussion

In this study four molecular biological tests were conducted and evaluated for detection of Brucella in seropositive camels.

AMOS PCR is the most widely used assay. The assay makes use of one common primer attached in the IS711element and a species-specific primer that binds to unique sequence flanking that insertion site resulting in species discrimination due to different amplicon sizes. The assay was subsequently modified to include the vaccine strains S19 and RB51 ¹¹ a significant improvement given the critical need to distinguish field and vaccine isolates in eradication programs ¹⁶ and some later modifications to primers were reported to improve performance ^{16, 17}.

The new multiplex PCR bruce-ladder, is an assay that can differentiate all six classical species, Brucella isolates from marine mammals and the vaccine strains B. abortus RB5, S19 and B. melitensis Rev 1. Generated amplicons of different sizes resulting in a unique profile for Brucella abortus biovar 6 following agarose gel electrophoresis.

In this study, we evaluated the performances of newly designed real-time PCR assays using TaqMan probes and targeting the insertion sequence IS711, for the detection of Brucella at genus level. Real-time PCR assays are easy-to-use, produce results faster than conventional PCR systems while reducing DNA contamination risks.

The IS711-based real-time PCR assay is specific and highly sensitive and appears as an efficient and reproducible method for the rapid and safe detection of the genus Brucella. These results agreed with ¹⁸ who stated that IS711 real-time PCR assay is a specific and sensitive tool for detection of Brucella spp. infections. For this reason they propose the employment of IS711 real-time PCR as a complementary tool in brucellosis screening programs and for confirmation of diagnosis in doubtful cases.

MLVA assay has the same advantages of multiplex PCR beside it can differentiate Brucella isolates on biovar level (genotyping)). In the near future, it’s necessary to speculate that international data bases containing MLVA data of thousands of strains will be produced and MLVA should become a routine assay for any new isolate and it is a useful tool for epidemiological tracing of brucellosis. Also, this findings confirmed that B. abortus and in particular biovar 6, is an etiological agent most frequently isolated from camels in Eastern and central parts of Sudan. As a final point, Bruce-ladder PCR assay is recommended for testing the seed cultures commonly used in the production of living Brucella vaccines (Rev-1, S19 and RB51 vaccines) and in evaluating them in quality control laboratories and also in identification and differentiation of Brucella isolates.

These results were agreed with the results of ¹⁹, who detected B. abortus and B. melitensis from lymph nodes of camels. Also ²⁰ found Brucella in Sudanese camel sera.

5. Conclusion

It can be concluded that B. abortus biovar 6 is an etiological agent isolated from camels in eastern and central parts of Sudan. PCRs used for confirmation of camel field isolates were found to be in 100% agreement with the conventional bio typing methods.

Acknowledgments

The authors thanks APHA, Weybridge. UK for sponsoring this study and appreciate the allowance to do this work in their laboratory.

Competing Interests

The authors declare that they have no conflict of interests.

References