Detection Of Brucella Research Articles

Label-free electrochemical immunosensor for rapid and highly sensitive detection of Brucella abortus in serum

Accurate and efficient detection of Brucella is crucial to control the disease and mitigate its impact on animal health and human safety. In this study, a simple and rapid label-free electrochemical immunosensor for the detection of Brucella abortus was fabricated based on the principle of current decrease after specific capture of the bacteria. A certain proportion of multi-walled carbon nanotubes (MWCNTs), dopamine hydrochloride (DA), and gold nanoparticles (AuNPs) were modified on the surface of a gold disc electrode. Then, Brucella abortus antibody and bovine serum albumin (BSA) were immobilized in sequence to complete the assembly of the sensor. The electrodes from various assembly steps were characterized by a cyclic voltammetry (CV) curve. The immunosensor exhibited high sensitivity with a wide linear range between 10 CFU/mL and 108 CFU/mL. Additionally, the sensor displayed good repeatability, stability, and interference resistance. The recovery rates for low, medium, and high concentrations of bacteria were all within the range of 90 % to 100 %, indicating a high level of accuracy. The method established in this study can provide reference and assistance for early diagnosis of Brucella abortus in clinical practice.

Rapid Colorimetric Quantita2tive Portable Platform for Detection of Brucella melitensis Based on a Fluorescence Resonance Energy Transfer Assay and Nanomagnetic Particles.

Brucellosis is a bacterial zoonotic disease that requires major attention for both health and financial facilities in many parts of the world including the Mediterranean and the Middle East. The existing gold standard diagnosis relies on the culturing technique, which is costly and time-consuming with a duration of up to 45 days. The Brucella protease biosensor represents a new detection approach that will lead to low-cost point-of-care devices for sensitive Brucella detection. In addition, the described diagnostic device is portable and simple to operate by a nurse or non-skilled clinician making it appropriate for the low-resource setting. In this study, we rely on the total extracellular protease proteolytic activity on specific peptide sequences identified using the FRET assay by high-throughput screening from the library of peptide (96 short peptides such as dipeptides and tripeptides) substrates for Brucella melitensis (B. melitensis). The B. melitensis-specific protease substrate was utilized in the development of the paper-based colorimetric assay. Two specific and highly active dipeptide substrates were identified (FITC-Ahx-K-r-K-Ahx-DABCYL and FITC-Ahx-R-r-K-Ahx-DABCYL). The peptide-magnetic bead nanoprobe sensors developed based on these substrates were able to detect the Brucella with LOD as low as 1.5 × 102 and 1.5 × 103 CFU/mL using K-r dipeptide and R-r dipeptide substrates, respectively, as the recognition element. The samples were tested using this sensor in few minutes. Cross-reactivity studies confirmed that the other proteases extracted from closely related pathogens have no significant effect on the sensor. To the best of our knowledge, this assay is the first assay that can be used with low-cost, rapid, direct, and visual detection of B. melitensis.

Development of a nanobiosensor for rapid detection of Brucella based on graphene quantum nanoparticles

Brucellosis is a highly contagious disease that affects public health and wildlife species worldwide. The efficient determination is required to reduce the transmission of infection in human and animals. In this research, a new nano-biosensor was developed for diagnosis of Brucella abortus (B. abortus) bacteria based on graphene quantum nanoparticles (GQNPs), magnetic nanoparticles (MNPs) and polyclonal antibody. First, GQNPs and MNPs were synthesized and conjugated with B. abortus polyclonal antibody (named here IgG1 and IgG2, respectively). The morphology and size of the synthesized nanoparticles were characterized using SEM, DLS and FTIR. Then, the GQNP-IgG1 and MNP-IgG2 conjugates were added to B. abortus to form a sandwich structure. After isolating the sandwiches by a magnet, the detection method was approved successfully using a fluorescence spectrophotometer as a quick and precise procedure for detection of B. abortus.

Detection of Brucella in Dermacentor Ticks of Wild Boar with Brucellosis

Brucellosis is a sanitary and economically relevant disease affecting humans, livestock, and wildlife. Ticks have been suggested as vectors, long-term carriers, and amplifiers of Brucella. In this study, ticks from wildlife ungulate hosts living in hunting reserves of a central region of Spain were collected during a 6-year period, pooled, and screened for Brucella spp. by PCR. Aiming to correlate Brucella spp. DNA presence in ticks with Brucella spp. infections in wildlife ungulate hosts, liver samples from deceased wildlife ungulates coming from the hunting reserves showing a positive result for Brucella in ticks were tested using a commercial ELISA. In total, 229 tick pools from wild boar (Sus scrofa, n = 176; 76.8%, 95% CI 70.9%–81.8%), red deer (Cervus elaphus, n = 40; 17.4%, 95% CI 13.1%–22.9%), mouflon (Ovis orientalis musimon, n = 7; 3.06%, 95% CI 1.49%–6.17%), and fallow deer (Dama dama, n = 6; 2.62%, 95% CI 1.21%–5.60%) were analyzed. PCR results showed that 3.93% (95% CI 2.08%–7.30%) tick pools (9/229) from 16.6% hunting reserves (7/41) screened yielded a positive PCR result for Brucella. All positive ticks were Dermacentor (Dermacentor marginatus or Dermacentor reticulatus) collected from wild boar. Ticks collected from wild boars were positive to Brucella in a relative percentage of 5.10% (95% CI = 1.61–11.4) in 2018 and of 7.59% (95% CI = 2.79–15.6) in 2021 (6-year prevalence of 5.17%, 9/176). ELISA showed positive results in three wild boars coming from two out of seven hunting reserves (28.5%) with a positive PCR for Brucella in ticks. To conclude, Brucella spp. DNA can be detected in Dermacentor ticks parasitizing wild boars living in hunting reserves harboring Brucella spp.-seropositive wild boars. This study provides evidence that the contribution of arthropod vectors should be considered in the epidemiology of brucellosis in wildlife.

DNA Sensor for the Detection of Brucella spp. Based on Magnetic Nanoparticle Markers.

Due to the limitations of conventional Brucella detection methods, including safety concerns, long incubation times, and limited specificity, the development of a rapid, selective, and accurate technique for the early detection of Brucella in livestock animals is crucial to prevent the spread of the associated disease. In the present study, we introduce a magnetic nanoparticle marker-based biosensor using frequency mixing magnetic detection for point-of-care testing and quantification of Brucella DNA. Superparamagnetic nanoparticles were used as magnetically measured markers to selectively detect the target DNA hybridized with its complementary capture probes immobilized on a porous polyethylene filter. Experimental conditions like density and length of the probes, hybridization time and temperature, and magnetic binding specificity, sensitivity, and detection limit were investigated and optimized. Our sensor demonstrated a relatively fast detection time of approximately 10 min, with a detection limit of 55 copies (0.09 fM) when tested using DNA amplified from Brucella genetic material. In addition, the detection specificity was examined using gDNA from Brucella and other zoonotic bacteria that may coexist in the same niche, confirming the method's selectivity for Brucella DNA. Our proposed biosensor has the potential to be used for the early detection of Brucella bacteria in the field and can contribute to disease control measures.

A recombinase polymerase amplification-SYBR Green I assay for the rapid and visual detection of Brucella.

Brucellosis is a zoonosis caused by Brucella, which poses a great threat to human health and animal husbandry. Pathogen surveillance is an important measure to prevent brucellosis, but the traditional method is time-consuming and not suitable for field applications. In this study, a recombinase polymerase amplification-SYBR Green I (RPAS) assay was developed for the rapid and visualized detection of Brucella in the field by targeting BCSP31 gene, a conserved marker. The method was highly specific without any cross-reactivity with other common bacteria and its detection limit was 2.14 × 104CFU/mL or g of Brucella at 40°C for 20min. It obviates the need for costly instrumentation and exhibits robustness towards background interference in serum, meat, and milk samples. In summary, the RPAS assay is a rapid, visually intuitive, and user-friendly detection that is highly suitable for use in resource-limited settings. Its simplicity and ease of use enable swift on-site detection of Brucella, thereby facilitating timely implementation of preventive measures.

Serological and molecular survey of brucellosis and chlamydiosis in dromedary camels from Tunisia

The present sero-epidemiological survey was designed and conducted to scrutinize the current status of camel-related brucellosis and chlamydiosis in Tunisia. Whole blood and serum samples were collected from 470 dromedaries (Camelus dromedarius) from eight different Tunisian governorates. Serum samples were subjected to indirect enzyme-linked immunosorbent assay (iELISA). The detection of Brucella and Chlamydia DNA was performed using conventional PCR targeting the bcsp-31 and 16S rRNA gene, respectively. Overall, 10/470(2.12%) and 27/470 (5.75%) camels were revealed seropositive to Brucella and Chlamydia, respectively. Multivariate logistic regression analysis showed different risk factors associated with these infections. Meaningful high rates of seropositivity of brucellosis (9.5%; p=0.000; OR=64.193) and chlamydiosis (22.6%; p=0.000; OR=42.860) were noted among camels showing previous abortions in particular for aged females. Besides, Chlamydia seropositivity is significantly important during winter (12.5%; p= 0.009; OR= 27.533), and in camels raised in small farms (11.4%, p= 0.000, OR=86.052). Molecular analysis revealed no positivity from all analyzed blood samples. These findings indicate the involvement of camels in the epidemiology of these abortive infectious diseases. This raises awareness and serious public health concern for infectious camel diseases in order to develop further diagnostic improvements and effective control strategies.

Reliable and highly specific techniques for the detection of Brucella spp. antibodies in camel milk

Reliable and highly specific techniques for the detection of Brucella spp. antibodies in camel milk

Development of antigen-capture ELISA using monoclonal antibodies for the detection of brucellae in milk

In this study, a Brucella antigen-capture ELISA (Ag-cELISA) prototype was developed. To study the validity of the developed Ag-cELISA, milk samples collected from Brucella-positive goats (n=120) and cattle (n= 64), as well as from unknown Brucella-status cattle (n=105) and sheep (n=65) herds were tested by Ag-cELISA, I-ELISA, and culture method. All Brucella-positive samples were confirmed using PCR. It was found that the developed Ag-cELISA could detect 50-100 bacteria per well (equivalent to 103 to 2×103 cells per mL) as the lowest limit of detection (LOD) and was therefore considered moderately sensitive to detect brucellae in milk. In an infected goat herd, out of 120 milk samples, 41, 32, and 17 were positive by Ag-cELISA, I-ELISA, and culture, respectively. Ag-cELISA detected 15 positive cases out of 17 culture-positive milk samples. Two culture-positive milk samples were not detected in Ag-cELISA. The relative sensitivity and specificity between Ag-cELISA and I-ELISA were 78% and 100%, respectively. In an infected cow herd, out of 64 milk samples, 32, 23, and 11 were found positive by Ag-cELISA, I-ELISA, and culture, respectively. Ten out of 11 culturally positive milk samples were found positive by Ag-cELISA. The relative sensitivity and specificity between the Ag-cELISA and I-ELISA were 71.9% and 100%, respectively. From randomly collected 105 cow and 110 sheep milk samples from herds of unknown Brucella-infection status, three (2.85%) and five (4.5%) samples were found positive using Ag-cELISA, respectively. These results showed that Ag-cELISA could be used to detect brucellae in milk more practically and safely than bacterial culture. On the other hand, this information re-affirms that milk can be an important source of brucellosis and creates a public health risk in humans; therefore, increased public awareness is of utmost importance.

Immunoassay-based evaluation of rOmp28 protein as a candidate for the identification of Brucella species.

Introduction. Brucellosis is an important bacterial zoonosis, re-emerging as a serious public health concern in developing countries. Two major species, Brucella melitensis and Brucella abortus, cause recurrent facile infection in human. Therefore, rapid and accurate diagnosis for early disease control and prevention is needed in areas with low disease burden.Hypothesis. This study evaluated the sandwich enzyme-linked immunosorbent assay (ELISA) (S-ELISA) immunoassay for potential use of whole-cell (WC) and recombinant outer-membrane protein (rOmp28)-derived IgG polyclonals in sensitive detection of Brucella.Aim. Immunoassay-based WC detection of Brucella species in important sub-clinical matrices at lower limits of detection.Methodology. We purified recombinant rOmp28 with Ni-NTA gel affinity chromatography and produced IgG polyclonal antibodies (pAbs) using BALB/c mice and New Zealand white female rabbits against different antigens (Ags) of Brucella. Checkerboard sandwich ELISA and P/N ratio (optical density of 'P' positive test sample to 'N' negative control) were used for evaluation and optimization of the study. The pAbs were characterized using Western blot analysis and different matrices were spiked with WC Ag of Brucella.Results. Double-antibody S-ELISA was developed using WC Ag-derived rabbit IgG (capture antibody at 10 µg ml-1) and rOmp28-derived mice IgG (detection antibody at 100 µg ml-1) with a detection range of 102 to 108 cells ml-1 and a limit of detection at 102 cells ml-1. A P/N ratio of 1.1 was obtained with WC pAbs as compared to 0.6 and 0.9 ratios with rOmp28-derived pAbs for detecting B. melitensis 16M and B. abortus S99, respectively. An increased P/N ratio of 4.4 was obtained with WC Ag-derived rabbit IgG as compared to 4.2>4.1>2.4 ratios obtained with rabbit IgGs derived against cell envelope (CE), rOmp28 and sonicated antigen (SA) of Brucella with high affinity for rOmp28 Ag analysed on immunoblots. The rOmp28-derived mice IgG revealed two Brucella species at P/N ratios of 11.8 and 6.3, respectively. Upon validation, S-ELISA detected Brucella WCs in human whole blood and sera samples with no cross-reactivity to other related bacteria.Conclusion. The developed S-ELISA is specific and sensitive in early detection of Brucella from different matrices of clinical and non-clinical disease presentation.

Development of a novel single-tube SYBR Green real-time PCR assay for simultaneous detection of Brucella spp. and Listeria monocytogenes by melt curve analysis

Brucella spp. and Listeria monocytogenes are recognised as important food safety and public health concerns. We provide a unique single-tube real-time platform for simultaneous yet differential detection of Brucella spp. and L. monocytogenes in milk using melt curve analysis. Melting curve analysis revealed two different peaks at melting temperatures of 79.91 ± 0.56 °C and 87.51 ± 0.26 °C for L. monocytogenes and Brucella spp., respectively. The technique detected only L. monocytogenes and Brucella spp., with no different melt curves for other bacterial pathogens. The limit of detection (LoD) in spiked raw milk demonstrated that the duplex test detects the target pathogens up to 10 cfu mL−1 milk. The findings show that the newly proposed method is easy-to-use, sensitive, specific, and a viable diagnostic tool for testing a wide range of foods that are concomitantly contaminated with L. monocytogenes and Brucella spp.

Next-generation sequencing in the diagnosis of neurobrucellosis: a case series of eight consecutive patients

BackgroundNeurobrucellosis (NB) presents a challenge for rapid and specific diagnosis. Next-generation sequencing (NGS) of cerebrospinal fluid (CSF) has showed power in detection of causative pathogens, even some infrequent and unexpected pathogens. In this study, we presented 8 cases of NB diagnosed by the NGS of CSF.MethodsBetween August 1, 2018 and September 30, 2020, NGS was used to detect causative pathogens in clinically suspected central nervous system (CNS) infections. Data on demographics, clinical features, and laboratory tests, imaging results and NGS results were collected and reviewed.ResultsAmong the presented 8 patients, Brucella was rapidly detected using NGS of CSF within 1–4 days, despite those eight patients had variable medical history, disease course, clinical manifestations, laboratory tests and imaging findings. NGS showed the sequence reads corresponded to Brucella species were 8 to 448, with genomic coverage of 0.02 to 0.87%. The relative abundance was 0.13% to 82.40% and sequencing depth was 1.06 to 1.24. Consequently, patients were administered with 3 to 6 months of doxycycline, ceftriaxone and rifampicin, double or triple combination, supplemented with symptomatic therapy and were fully recovered except for case 1.ConclusionNGS of CSF provides a powerful tool in detection of Brucella in a prompt and specific manner, and can be considered for first-line diagnostic use in practice.

A rapid direct-differential agglutination assay for Brucella detection using antibodies conjugated with functionalized gold nanoparticles

Brucellosis is the most widespread and serious zoonotic disease worldwide which affects livestock, sylvatic wildlife, marine dwellers, and humans. It is acquired through Alphaproteobacteria which belong to the genus Brucella and is categorized as a potential bio-threat agent. In this study, we developed a rapid and direct differential whole cell (WC) agglutination-based assay for its on-field detection. The recombinant outer membrane (rOmp28) protein-derived specific mice IgG polyclonal antibodies (pAbs) of Brucella were purified using affinity chromatography and conjugated with functionalized gold nanoparticles (AuNPs) for rapid agglutination. A positive blot of 32 kDa protein revealed specific immuno-reactivity of rOmp28-pAbs using immunoblot analysis. For the synthesis of AuNPs, the conventional “Turkevich method” was optimized at a concentration < 1 mM of gold precursor for obtaining 50-nm-sized particles. Also, their physico-chemical characteristics were analyzed using UV-visible spectrophotometry, Fourier transform infra-red spectroscopy (FT-IR), Raman spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential (ζ, ZP), and fluorescence spectroscopy. Furthermore, these AuNPs were functionalized with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to prepare modified carboxylated AuNPs. For bioconjugation with Brucella rOmp28 IgG pAbs, antibody-conjugated functionalized AuNP constructs were prepared and characterized using FT-IR analysis with strong N–H deformations. Subsequently, these bioconjugated AuNPs were used to develop a direct-differential slide agglutination assay with a detection limit of 104 CFU mL−1. The sensitivity of this assay was compared with standard double-antibody sandwich ELISA (S-ELISA) using rOmp28 IgG pAbs with an LOD of 103 CFU mL−1 and a detection range of 102–108 CFU mL−1. No intraspecies cross-reactivity was observed based on evaluation of its specificity with a battery of closely related bacterial species. In conclusion, the increased sensitivity and specificity of the developed agglutination assay obtained using bioconjugated functionalized AuNPs is ≥ 98% for the detection of Brucella. Therefore, it can be used as an alternate rapid method of direct WC detection of bacteria as it is simple, robust, and cost-effective, with minimal time of reaction in the case of early disease diagnosis.

Comparative Surface Interaction Study to Detect Brucella melitensis 16M Using Biosensor Transducer Modifications with 4-MBA, ZnONPs/AuNPs, ZnONPs/AuNPs/4-MBA

Brucellosis is a zoonotic disease endemic in developing countries and caused by gram-negative bacteria of genus Brucella infecting both livestock and humans. Brucella melitensis and Brucella abortus are important species representing largest biotypes world-wide. Therefore, modified detection strategies and advancement in potential analytical tools are required to monitor its rapid prevalence. In this study, we aim to modify gold-transducer of Surface Plasmon Resonance biosensor with combination of metal oxide nanomaterials and chemical probe to detect recombinant outer membrane ‘rOmp28’ protein antigen of Brucella melitensis 16M in concentration-dependent surface interactions. We synthesized Zinc (ZnONPs) and Gold (AuNPs) nanoparticles using standard ‘Hydrothermal and Turkevich Methods’ and their crystalline structure, chemical property and morphology were analysed using UV-Visible Spectrophotometry, FT-IR, Powder-XRD, SEM-EDX and TEM-SAED. For immobilizing specific rOmp28 derived IgG-pAbs on modified Au-transducer, rOmp28 protein was expressed and purified using Ni-NTA gel affinity chromatography for producing pAbs in BALB/c mice. Three modifications of Au-transducer with 4-MBA, ZnONPs/AuNPs and ZnONPs/AuNPs/4-MBA were subjected for immobilization and SPR biosensing was performed with rOmp28 Ag at detection range of 0.1 μg mL-1 to 0.01 fg mL-1. Limit of detection observed with ZnONPs/AuNPs/4-MBA combination was 0.1 fg mL-1 by relative increase in SPR response angle at 0.1 μg mL-1 in the order 83.7° < 98.9° < 179.2° for 4-MBA < ZnONPs/AuNPs < ZnONPs/AuNPs/4-MBA respectively. In conclusion, metal oxide nanomaterials in combination with biosensor are suitable in sensitive and specific interaction of antigen displaying lowest LODs and enhanced biosensor response for on-field real-time Brucella detection in both clinical and non-clinical disease scenario.

A rapid and sensitive triplex-recombinase polymerase amplification for simultaneous differentiation of Brucella abortus, Brucella melitensi s, and Brucella suis in sera and foods.

Brucella is the causative agent of brucellosis and can be transmitted to humans through aerosolized particles or contaminated food. Brucella abortus (B. abortus), Brucella melitensis (B. melitensis), and Brucella suis (B. suis) are the most virulent of the brucellae, but the traditional detection methods to distinguish them are time-consuming and require high instrumentation. To obtain epidemiological information on Brucella during livestock slaughter and food contamination, we developed a rapid and sensitive triplex recombinant polymerase amplification (triplex-RPA) assay that can simultaneously detect and differentiate between B. abortus, B. melitensis, and B. suis. Three pairs of primers (B1O7F/B1O7R, B192F/B192R, and B285F/B285R) were designed and screened for the establishment of the triplex-RPA assay. After optimization, the assay can be completed within 20 min at 39°C with good specificity and no cross-reactivity with five common pathogens. The triplex-RPA assay has a DNA sensitivity of 1-10 pg and a minimum detection limit of 2.14×104-2.14×105 CFU g-1 in B. suis spiked samples. It is a potential tool for the detection of Brucella and can effectively differentiate between B. abortus, B. melitensis, and B. suis S2, making it a useful tool for epidemiological investigations.

Brucella Phagocytosis Mediated by Pathogen-Host Interactions and Their Intracellular Survival.

The Brucella species is the causative agent of brucellosis in humans and animals. So far, brucellosis has caused considerable economic losses and serious public health threats. Furthermore, Brucella is classified as a category B bioterrorism agent. Although the mortality of brucellosis is low, the pathogens are persistent in mammalian hosts and result in chronic infection. Brucella is a facultative intracellular bacterium; hence, it has to invade different professional and non-professional phagocytes through the host phagocytosis mechanism to establish its lifecycle. The phagocytosis of Brucella into the host cells undergoes several phases including Brucella detection, formation of Brucella-containing vacuoles, and Brucella survival via intracellular growth or being killed by host-specific bactericidal activities. Different host surface receptors contribute effectively to recognize Brucella including non-opsonic receptors (toll-like receptors and scavenger receptor A) or opsonic receptors (Fc receptors and complement system receptors). Brucella lacks classical virulence factors such as exotoxin, spores, cytolysins, exoenzymes, virulence plasmid, and capsules. However, once internalized, Brucella expresses various virulence factors to avoid phagolysosome fusion, bypass harsh environments, and establish a replicative niche. This review provides general and updated information regarding Brucella phagocytosis mediated by pathogen-host interactions and their intracellular survival in host cells.

Highly sensitive detection of Brucella in milk by cysteamine functionalized nanogold/4-Mercaptobenzoic acid electrochemical biosensor

Highly sensitive detection of Brucella in milk by cysteamine functionalized nanogold/4-Mercaptobenzoic acid electrochemical biosensor

Brucella spp. distribution, hosting ruminants from Greece, applying various molecular identification techniques

BackgroundBrucellosis still remains an endemic disease for both livestock and human in Greece, influencing the primary sector and national economy in general. Although farm animals and particularly ruminants constitute the natural hosts of the disease, transmission to humans is not uncommon, thus representing a serious occupational disease as well. Under this prism, knowledge concerning Brucella species distribution in ruminants is considered a high priority. There are various molecular methodologies for Brucella detection with however differential discriminant capacity. Hence, the aim of this survey was to achieve nationally Brucella epidemiology baseline genotyping data at species and subtype level, as well as to evaluate the pros and cons of different molecular techniques utilized for detection of Brucella species. Thirty-nine tissue samples from 30 domestic ruminants, which were found positive applying a screening PCR, were tested by four different molecular techniques i.e. sequencing of the 16S rRNA, the BP26 and the OMP31 regions, and the MLVA typing panel 1 assay of minisatellite markers.ResultsOnly one haplotype was revealed from the 16S rRNA sequencing analysis, indicating that molecular identification of Brucella bacteria based on this marker might be feasible solely up to genus level. BP26 sequencing analysis and MLVA were in complete agreement detecting both B. melitensis and B. abortus. An interesting exception was observed in 11 samples, of lower quality extracted DNA, in which not all expected MLVA amplicons were produced and identification was based on the remaining ones as well as on BP26. On the contrary OMP31 failed to provide a clear band in any of the examined samples.ConclusionsThe present study reveals the constant circulation of Brucella bacteria in ruminants throughout Greece. Further, according to our results, BP26 gene represents a very good alternative to MLVA minisatellite assay, particularly in lower quality DNA samples.

Molecular detection of Brucella spp. in clinical samples of seropositive ruminants in Bosnia and Herzegovina

Brucellosis is one of the most common zoonotic diseases worldwide that is endemic to Bosnia and Herzegovina (B&H) and remains an emerging public and animal health concern in this country. Diagnostic testing of brucellosis in ruminants in B&H relies exclusively on serological methods. The present study was conducted to determine the presence of Brucella spp. in clinical samples of seropositive ruminants by Real-time PCR assay. Of the 135 samples tested, 37% were positive by Real-time PCR. The frequency of detection of Brucella-DNA in the samples collected from aborted animals (n = 20/20; 100%) was significantly higher (P < 0.00001) when compared to asymptomatic animals (n = 30/115; 26%). Among asymptomatic animals, 31.1% of cattle were positive for Brucella-DNA, followed by sheep (23.4%) and goats (16.6%). The results of this research underline the limitations of the current control policy and indicate the need for additional diagnostic methods required for more effective brucellosis control program.

Molecular detection of Brucella spp. in ruminant herds in Greece.

Brucellosis is a worldwide distributed infectious disease. Ruminants and other animal species (swine, dogs, equids, etc.), as well as wild mammals, can be affected. The disease can be transmitted to humans through the food chain or by direct contact with infected animals. Because of the relatively high economic burden due to abortions within a herd, significant efforts have been employed and hence the disease in most European countries has been eradicated. Accordingly, Greece applies both control and eradication programs concerning small ruminants (sheep and goats) and bovines depending on the geographical area. Current challenges in the standard antibody-based laboratory methods used for Brucella detection are the failure to differentiate antibodies against the wild strain from the ones against the vaccine strain Rev1 and antibodies against B. melitensis from those against B. abortus. The aim of the study was to reexamine and combine previously published protocols based on PCR analysis and to generate a rapid, not expensive, and easy to perform diagnostic tool able to confirm the doubtful results delivered from serology. For this reason, 264 samples derived from 191 ruminants of the farm and divided in 2 groups (male/female) were examined with a modified DNA extraction and PCR protocol. Molecular examination revealed the presence of Brucella spp. in 39 out of 264 samples (derived from 30 animals). In addition, Brucella spp. was detected in infected tissues such as testicles, inguinal lymph nodes, fetal liver, and fetal stomach content.