Detection Of Brucella Research Articles

Amperometric Biosensor for Brucella Testing through Molecular Orientation Technology in Combination with Signal Amplification Technology

AbstractBrucellosis is a global zoonotic disease causing abortion and infertility in animals. Based on an oriented immobilization technique of using a biomolecule in combination with a signal amplification technology, a novel biosensor for Brucella detection was designed. Graphene oxide/polypyrrole nanohybrids (GO/Ppy) modified a screen‐printed gold electrode through co‐electrodeposition for the oriented immobilization of a primary antibody (Ab1). Ab1 was successfully immobilized by using cyclic voltammetry (CV). A new second antibody nanoprobe GO/Fe3O4/MB/Ab2 (graphene oxide/nano‐iron oxide/methylene blue/Ab2) was accomplished by using physical and chemical methods to amplify the electrical signal. The successful fabrication of the biosensor was characterized by using SEM, AFM, Raman, and FTIR spectroscopy. The prepared biosensor was also tested in the Brucella solution by using differential pulse voltammetry (DPV). The results showed that the corresponding current was positively correlated with the logarithm of Brucella concentration, with a low detection limit of 2.2×102 CFU/mL. Moreover, the results of the biosensor‘s reproducibility, repeatability, specificity and standard recovery were satisfactory. New methodologies in fabricating the biosensor may be applied to detect other harmful microorganisms.

Evaluating the efficiency of TaqMan real-time PCR and serological methods in the detection of Brucella spp. in clinical specimens collected from suspected patients in Ardabil, Iran

BackgroundThis study aims to evaluate the efficiency of the TaqMan real-time PCR and serological methods in detecting Brucella spp. in clinical specimens that have been collected from suspected patients in Ardabil, Iran. MethodsIn this cross-sectional study, a total of 113 consecutive patients suspected of brucellosis who were referred to the three hospitals in Ardabil province were selected. In the first step, the diagnosis of brucellosis was performed by serological methods including the Rose Bengal slide agglutination test, Wright test, 2-ME test, and BrucellaCapt test. In the next step, TaqMan real-time PCR with primer and probe targeting the bcsp31 gene was used for the detection of Brucella spp. Specificity, sensitivity, and positive and negative predictive values of the TaqMan real-time PCR assay were calculated. ResultsAmong 113 suspected patients with different clinical manifestations, the Rose Bengal slide agglutination test, Wright test, and 2-ME test were positive in 60 cases; however, the BrucellaCapt test titer was 1:160 for one patient. Six patients had high initial serum antibody titers; 2-ME titers of ≥1:640; STA titers of ≥1:1280; BrucellaCapt titers of ≥ 1:2560. Among positive cases, no correlation was observed among gender, age, and life (residence) in urban or rural areas. The TaqMan real-time PCR was positive in 35% of all 60 positive cases. The comparison of the results of the BrucellaCapt and TaqMan real-time PCR methods revealed that 19 out of 54 (35.2%) and 2 out of 6 (33.4%) BrucellaCapt positive cases with titers of >1:320 and ≤ 1:320 were positive, respectively. The sensitivities and specificities of the TaqMan real-time PCR assay were 49.1% and 100% respectively. ConclusionThe sensitivity of the TaqMan real-time PCR assay was low in the diagnosis of brucellosis, while the BrucellaCapt test turned out to be a very valuable, sensitive, and specific test for the diagnosis of brucellosis in suspected patients and, thus, can provide reliable results in medical laboratories.

Development and validation of immunoassay for whole cell detection of Brucella abortus and Brucella melitensis

Brucella is alpha-2 Proteobacteria mainly responsible for multi-factorial bacterial zoonotic disease brucellosis with low concentration (10–100 CFU) required to establish the infection. In this study, we developed sandwich ELISA with detection range of 102 to 108 cells mL−1 and limit of detection at 103 cells mL−1 by employing polyclonal rabbit IgG (capture antibody, 10 µg mL−1) and mice IgG (detection antibody, 50 µg mL−1) antibody for its detection. Surface Plasmon Resonance evaluated the interaction of detection antibody with whole cell spiked serum samples at LOD of 102 cells mL−1 along with non co-operative interaction of protein albumin. Further, kinetic evaluation study using detection antibody against cell envelope antigen was performed whereby, Equilibrium Dissociation Constant (KD) and Maximum Binding Capacity (Bmax) were found to be 16.48 pM and 81.67 m° for Brucella abortus S99 and 0.42 pM and 54.50 m° for Brucella melitensis 16 M, respectively. During interference study, sandwich ELISA assay cross-reacted with either of the polyclonal antibody of above Brucella species. Upon validation, no cross-reactivity observed with bacteria-closely related to Brucella. In conclusion, developed semi-quantitative sandwich immunoassay is sensitively rapid in whole cell detection of Brucella and will be useful in development of detection assays from environmental and clinical matrices.

Evaluation of Reactivity of Monoclonal Antibodies Against Omp25 of Brucella spp.

Brucellosis is a serious zoonosis occurring mainly in developing countries, and its diagnosis is largely dependent on serologic detection and bacterial culture. In this study, we developed the murine monoclonal antibodies (mAbs) against a conserved and major outer membrane protein 25 (Omp25) of Brucella species (B. spp.) for use in clinical diagnosis. The mAbs to Omp25 were produced by hybridoma technique, which were utilized for developing various immunoassays for detection of Brucellae, including Western blot (WB), enzyme-linked immunosorbent assay (ELISA), immunochemical staining (ICS), immunofluorescence staining (IFS), and flow cytometry assay (FCM). A number of five mAbs (2B10, 4A12, 4F10, 6C12, and 8F3) specific to Omp25 were selected, including 2 IgG1, 2 IgG2a, and 1 IgG2b. Among them, mAbs 6C12, 8F3, and 4A12 reacted highly with B. melitensis (M5-90), B. abortus (S19, 104M, and 2308), and B. suis strain (S2). No cross-reactivity with Yersinia enterocolitica O:9, Salmonella spp., and Escherichia coli was found. By mapping Omp25 epitopes, mAb 6C12 was found as reacting with a semi-conformational epitope, and mAbs 4A12 and 8F3 as recognizing a different linear epitope, respectively. The paired mAbs were tested for detecting Brucella species, suggesting that 8F3 was suitable for solid phase capture and 6C12 or 4A12 was suitable for conjugation with HRP for detection of Brucella Omp25 in ELISA. The FCM was established by mAb 6C12 for detecting intracellular Brucellae-infected peripheral blood mononuclear cells (PBMCs) from brucellosis patients. In conclusion, mAbs against Omp25 are precious reagents for detection of Brucellae in clinical samples with various immunoassays. mAb 6C12-based FCM could be potentially used for the monitoring of therapeutic efficacy for brucellosis in clinical practice.

Molecular survey of BCSP31 and IS711 using PCR assays in detection of Brucella spp. in raw milk

Brucella spp. as animal pathogens causes human brucellosis, the most momentous zoonotic diseases all over the world that result in economic pressure, human morbidity, and poverty. Brucellosis is commonly transmitted by infected dairy products. The prevalence of Brucella spp. and high consumption of unpasteurized dairy products in urban areas of Iran imposes further studies to clarify the prevalence of Brucella species. This molecular survey was purposefully designed to determine the prevalence of Brucella in raw milk collected from milk sellers using PCR in Zanjan province, Iran. In this molecular cross-sectional study, 73 raw milk samples with a volume of 40 ml gathered (6 samples from sheep and 67 samples from bovine) from raw milk vendors in Zanjan province. DNA of bacteria was extracted from the raw milk samples using (GeneALL, South Korea) kit according to the manufacturer protocol. Specific primers (B4 and B5) were used to the amplification of BCSP31 gene. Five primers (Ba-sp, Bm-sp, Bo-sp, Bs-sp, and IS711-sp) were used to detect Brucella spp. (Brucella abortus, Brucella melitens, Brucella ovis, and Brucella suis) among the positive samples for bcsp31 gene. From the 73 raw milk samples collected, 38 cases (52%) were positive for Brucella genus, 3 cases (7.8%) belonged to Brucella abortus and 2 cases (5.2%) belonged to Brucella melitensis. There were no positive cases for both Brucella ovis and Brucella suis. Results of this study proved that the prevalence of Brucella in raw milk is still a threat to public health, which should have a deeper look at the control strategies and methods of the prevention of brucellosis in the livestock.

Seroepidemiological Study on Some Zoonotic Agents Associated with Fever of Unknown Origin in Human

Fever is not a disease in itself but it is a symptom that indicates a disease in the body. Fever of unknown origin (FUO) was defined as a temperature of 38.3 °C or higher with a minimum duration of 3 weeks without an established diagnosis after an intensive 1-week investigation in the hospital. The current study was carried out for detection of some possible zoonotic causes of FUO. A total of 200 serum samples collected from patients attending Fever hospitals were subjected to serological examination for detection of Brucella antibodies by Rose Bengal plate test (RBPT) as well as detection of antibodies of Toxoplasma gondii, Leishmania infantum and Rickettsia conorii by Enzyme Linked Immunosorbent Assay (ELISA). It was recorded that the overall frequency of detection of Brucella was 9% and males (14.6%) showed higher detection rate compared to that of females (4.5%). In addition, the highest detection rate was observed in age group 45 – 65 years (15.9%) followed by the age group 15 - < 30 years (9.2%) then the age group 30 - < 45 years (5%) with statistical significant association between these frequencies. Also, the overall frequency of detection of T. gondii Abs was 6% and females (6.3%) showed higher detection frequency compared to that of males (5.6%) with statistical significant association. Moreover, the highest detection rate was observed in age group 15 - < 30 years (7.9%) followed by the age group 45 – 65 years (6.8%) then the age group 30 - < 45 years (3.8%) with statistical significant association between these frequencies. Serological investigation of L. infantum clarified that only three samples showed serological reaction with percentage of 1.5 %. In addition, males’ frequency was higher than that of females (2.24 % vs 0.9%) with non-significant association between them. Moreover, the age group impact on the detection rate also showed non-significant association. Finally, serological investigation of Rickettsia conorii also clarified that only three samples showed serological reaction with percentage of 1.5%. In addition, males’ frequency was higher than that of females (2.25 % vs 0.9%) with non-significant association between them.

Functionalization of anti-Brucella antibody based on SNP and MNP nanoparticles for visual and spectrophotometric detection of Brucella

An Immuno-Nano-Biosensor with high sensitivity was designed based on iron and silica nanoparticles to detect B. abortus. Briefly explain, primary polyclonal antibody (IgG1) was conjugated on surface magnetic nanoparticles (MNPs) to form MNP-IgG1. Secondary polyclonal antibody (IgG2) and Horseradish Peroxidase enzyme were conjugated on silica nanoparticles (SNPs) to form HRP-SNP-IgG2. HRP-SNP-IgG2. MNP-IgG1 and HRP-SNP-IgG2 were added to B. abortus. The MNP-IgG1-B.abortus-IgG2-SNP-HRP complex was isolated from the reaction mixture using a magnet. After that, tetramethylbenzidine was added to the complex. The reaction was stopped with HCl and investigated using UV-Vis spectrophotometry. The nanoparticles' structure and size were investigated using SEM and DLS. Immuno-Nano-Biosensor sensitivity and specificity were determined. The SEM and DLS results indicated that the SNPs, MNPs, HRP-SNP-IgG2 and MNP-IgG1 size and structure were 35, 44, 60 and 56nm, respectively. In addition, a good linear correlation was observed at 102-107CFUmL-1 concentrations, which their linear equation and regression were Y=0.3×+0.18 and R2 0.982, respectively. The limitation of detecting B. abortus was 160CFUmL-1. Finally, the results demonstrated that those designed Immuno-Nano-Biosensor could be specifically detected B. abortus and B. melitensis in real samples.

Neurobrucellosis in a common bottlenose dolphin (Tursiops truncatus) stranded in the Canary Islands

BackgroundBrucella spp. isolation is increasingly reported in cetaceans, although associated pathologies, including lesions of the musculoskeletal and nervous systems, are less frequently described. Concerning the nervous system, Brucella sp. infection causing meningitis, meningoencephalitis or meningoencephalomyelitis have been extensively reported in striped dolphins (Stenella coeruleoalba), and less frequently in other cetacean species.Case presentationA juvenile female common bottlenose dolphin (Tursiops truncatus) was found stranded alive in Lanzarote (Canary Islands, Spain) in 2005, but died shortly after. On physical examination, the dolphin showed a moderate body condition and was classified as code 2 (fresh dead) at the time of necropsy. The main gross findings were severe multiorgan parasitism, thickened and congested leptomeninges, and (sero)fibrino-suppurative and proliferative arthritis of the shoulder joint. Histopathological examination revealed the distinct features of a sub-acute systemic disease associated with Cetacean Morbillivirus (CeMV) infection. However, brain lesions diverged from those reported in systemic CeMV infection. This led to suspect that there was a coinfecting pathogen, based on the characteristics of the inflammatory response and the lesion distribution pattern in the central nervous system. Brucella sp. was detected in the brain tissue by PCR and Brucella antigen was demonstrated by immunohistochemistry in the brain and shoulder joint lesions.ConclusionsThe zoonotic potential of marine mammal strains of Brucella has been demonstrated both in natural and laboratory conditions. In this study, PCR detected Brucella sp. in the brain of a common bottlenose dolphin stranded in the Canary Islands; the dolphin was also co-infected with CeMV. This is the first detection of Brucella sp. infection in a stranded cetacean in this archipelago. Therefore, we stress the importance of taking adequate measures during the handling of these species to prevent the transmissions of the infection to humans.

Detection of Brucellae in peripheral blood mononuclear cells for monitoring therapeutic efficacy of brucellosis infection

BackgroundBrucellosis is one of the most severe widespread zoonoses caused by the Gram-negative bacterium Brucella species. The diagnosis and clinical assessment of human brucellosis are very important for the management of patients, while there is a lack of effective methods to detect Brucellae. Classical culture of Brucella species is time consuming and often fails. A simple and sensitive assay is needed for diagnosis of Brucella infection and monitoring of treatment in man.MethodsBlood samples and peripheral blood mononuclear cells (PBMCs) were collected from 154 patients hospitalized for brucellosis. Brucella antibodies were detected by Rose Bengal Plate Test (RBPT), Standard Tube Agglutination Test (SAT) and enzyme-linked immunosorbent assay (ELISA). Intracellular Brucellae were detected by blood culture and immunofluorescence staining (IFS).ResultsAmong 154 brucellosis patients, 59.7% (92/154) were antibody reactive by RBPT, 81.8% (126/154) by SAT and 95.5% (147/154) by ELISA, respectively. Only 3.2% (5/154) of patient blood samples resulted in positive Brucella culture, while 68.8% (106/154) carried IFS detectable Brucella antigens in PBMCs. Gender (P = 0.01) but not age (P > 0.05) was a significant risk factor. The frequency of intracellular Brucella antigens was similar between patients receiving different treatment regimens (P > 0.05). However, a significant decrease of intracellular Brucellae was observed only in patients with acute brucellosis after the third course of treatment (P < 0.05), suggesting that current regimens to treat chronic brucellosis were not effective.ConclusionsIFS appears a sensitive assay for detection of Brucella antigens in PBMCs and could be used for diagnosis and therapeutic monitoring of brucellosis in clinical practice.

A Systematic Detection for Brucellosis at Chronic Stage of Infection in Semen of Sheep and Saanen Goats

The study was conducted in a herd (n: 244) in which goats (n: 206) and sheep (n:38) had a history of brucellosis in Bursa which is located in Northwestern of Turkey between the years 2012-2014. For the detection of Brucella spp. and the other zoonotic bacterial agents, semen samples were taken from Saanen goats (n: 35) and rams (n: 8). Samples were tested by routine diagnostic procedures and PCR. The serum samples of male animals were also tested for Brucellosis by C-ELISA and I-ELISA. The culture results represented Trueperella pyogenes (n:2), Pasteurella pneumotopica (n: 5), Esherichia coli (n: 3), Aeromonas salmonicida subs. Salmonicida (1), Brevundimonas vesicularis (n: 2) and Mycoplasma bovigenitalium (n: 1) and Mycoplasma arginini (n: 1) from semen samples. Rams had no symptoms due to epididymitis or epididymoorchitis in clinical examination, but two bucks showed orchitis and they were serologically positive for brucellosis. Also, one seronegative buck showed epididymitis in a flock. There were no statistically significant differences between the serologically positive and negative animals in an examination of semen samples in terms of their volume, concentration, mass activity, motility and defectivity rate for acrosome. Although 20 of the serum samples were negative for anti-Brucella antibody, 23 of them were serologically positive for brucellosis. As a result of this study, Brucellae were not detected by bacteriologically and molecularly while there were some positive serum samples for brucellosis. This could be attributed that these samples might have been collected from chronically infected animals in which animals generally do not shed the organisms. Therefore, it was thought that sampling with regular intervals might help for the definitive incidence of brucellosis.

A novel approach for detection of brucella using a real-time recombinase polymerase amplification assay

Brucella, the etiological agent of brucellosis, is an important zoonosis pathogen worldwide. Brucella infects humans and various domestic and wild animals, and represents a great threat to public health and animal husbandry. In the present study, we developed a real-time recombinase polymerase amplification (RPA) assay for the detection of Brucella. The assay targeted the bcsp31 gene of Brucella, and an RPA exo probe and a pair of primers were selected for assay validation. RPA sensitivity and specificity were evaluated using plasmid standards, Brucella representative strains, and non-Brucella strains. The RPA assay achieved a detection limit of 17 molecules in 95% of cases based on probit analysis, and could successfully distinguish 18 representative Brucella strains (B. abortus biovars 1, 2, 3, 4, 5, 6, 7 and 9, B. melitensis biovars 1, 2 and 3, B. suis biovars 1, 2, 3 and 4, B. canis, B. neotomae and B. ovis), and four Brucella vaccine strains (A19, S19, S2 and M5). A total of 52 Brucella field strains were detected by real-time PCR and RPA in parallel, and compared with real-time PCR, the sensitivity of the RPA assay was 94% (49/52). Thus, this RPA assay may be a rapid, sensitive, and specific tool for the prevention and control of Brucellosis.

Detection of Brucella spp. in artisan cheese commercialized in Parnaíba, Piauí state, Brazil

The aim of this study was to detect Brucella spp. in artisanal cheese commercialized in Parnaiba city, Piaui state, Brazil. For this study, 30 samples of curd cheese (500g) were randomly collected from different points in the commercialization process. In the laboratory, 25g aliquots of the samples were suspended in Brucella broth; after this procedure, 10µL aliquots of this suspension were sown in Thayer Martin agar plates that were supplemented with 10% defibrinated sheep blood and VCNT antimicrobials. After inoculation, the samples were incubated at 37°C in microaerophilic conditions for 14 days. The suspected morphological colonies were identified and confirmed by polymerase chain reaction (PCR). From thirty samples of microbiologically analyzed cheeses, six samples (20%) were confirmed by PCR as bacteria from the Brucella genus. The detection of Brucella spp. was confirmed in cheese commercialized in markets or a public square (3.33%), bakery (3.33%) and small market (13.33%). Brucella spp. was detected in artisanal cheese commercialized in different points from Parnaiba city. Guidelines for the establishment of good practices for the production of artisanal cheese should be determined by the competent authorities. However, for the better control of human brucellosis it is necessary to control or eradicate bovine brucellosis in the herds.

A Cellular Model of Infection with Brucella melitensis in Ovine Macrophages: Novel Insights for Intracellular Bacterial Detection

Intracellular bacteria provoking zoonoses, such as those of the genus Brucella, present a host cell tropism mostly limited to the monocyte/macrophage lineage, leading to chronic inflammatory reactions, difficult-to-eradicate-infections, and widespread prevalence among ruminants. Eradication of brucellosis has been based on programs that translate into a substantial financial burden for both the authorities and stockbreeders, if not strictly followed. To this end, we sought to create an in vitro cell model that could be utilized as future reference for adequately measuring the number of engulfed brucellae/cell, using peripheral blood-derived sheep macrophages infected with B. melitensis at decimal multiplicities of infection (MOI = 5000-5), to simulate the host cell/microorganism interaction and monitor bacterial loads up to 6 days post-infection. We show that the MOI = 5000 leads to high numbers of engulfed bacteria without affecting macrophages’ viability and that the minimum detection limit of our Real-Time PCR assay was 3.97 ± 5.58 brucellae/cell. Moreover, we observed a time-associated, significant gradual reduction in bacterial loads from Day 2 to Day 6 post-infection (p = 0.0013), as part of the natural bactericidal properties of macrophages. Overall, the work presented here constitutes a reliable in vitro cell model of Brucella melitensis for research purposes that can be utilized to adequately measure the number of engulfed brucellae/cell and provides insights towards future utilization of molecular biology-based methods for detection of Brucella.

Mass spectrometry analysis of protein blood extracts of animals with experimental brucellos

Aim. The aim of the present research was to study the possibility of direct detection of the causative agent of brucellosis in a biomaterial under experimental conditions via the MALDI-TOF MS method using Mass-Up program resources and a set of packages for open-source statistical software R. Materials and methods. We used laboratory mice infected with the causative agents of Brucellosis (strains B. melitensis 548, B. abortus 544, B. suis 1330) as models. Protein profiling was performed on a MALDI-TOF Microflex «Bruker Daltonics» mass spectrometer. Results. The bioinformatic-statistical approach used for analyzing MALDI-TOF mass spectra allows to carry out a direct detection of Brucella in the biomaterial; besides, it is possible to determinate their species via the identification of a group of biomarkers. Conclusion. It was experimentally confirmed that the protein profiles of the blood extracts of infected animals contain 11 markers, including 6 genus specific for Brucella spp., which can be associated with Brucella infection.

Designing an immunosensor for detection of Brucella abortus based on coloured silica nanoparticles

Brucellosis has always been a threat to the health and economics of societies. We report a new colorimetric immunoassay based on colored silica nanoparticles for detection of Brucella abortus. An immunosensor was designed based on blue-SiNPs and paramagnetic nanoparticles (PMNPs). The synthesized immunosensor was conjugated with a polyclonal antibody against B. abortus, which was activated by 1-Ethyl-3–(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to form detection and capture probes, respectively. After adding the conjugates to the bacterial suspension, sandwich structure of PMNPs B. abortus-blue-SiNPs was formed and then separated by a magnet. The blue dye was released from the silica structure and its absorbance was measured at 670 nm with a spectrophotometer. Under optimal conditions, results showed a wide dynamic range from 1.5 × 103 to 1.5 × 108 cfu mL−1 with a detection limit of 450 cfu mL−1. The specificity of the sensor was confirmed in comparison with 5 other bacteria. Also, during the 120-days period, the complex was stable. The results suggested that it can be used in real samples (R 2 = .9865). This designed colorimetric immunoassay strategy can be used as an alternative, user-friendly and on-site tool for the rapid detection of Brucella spp. compared to other common methods with high sensitivity and specificity in a short time.

Serological and Molecular Diagnosis of Bovine Brucellosis in Menoufia Province

Brucellosis represents a serious problem in cattle relaying huge economic losses. Brucella meletensis on of brucella strains that can infect human which its usual host is sheep, might isolated from cattle. The aim of present study was detection of brucellosis among cattle population in Menoufia governorate, Egypt and analyze risk factors. Serologically brucella were detected with RBPT. Evaluation of the specificity and sensitivity of RBPT, IC assay and cELISA against CFT test were done. Molecular detection of brucella using PCR and bacteriological isolation followed by phenotypic and molecular typing of the isolated bacteria. The overall prevalence using RBT was 1.44%. Holstein Friesian cattle have highest prevalence (1.57%) while native cattle were (1.28%). Cattle over 3 years old have the highest seroprevalence (2.77%). The positive result for RBPT, cELISA, IC Assay and CFT were 91%, 60%, 91% and 88% respectively after examination 100 serum sample. Brucella melitensis biovar 3 was isolated from the tissue specimens (uterus and/or lymph nodes). PCR targeting (Immuno-dominant antigen, gene bp26) generated product of 450 bp from (16/20) tissues specimens. These findings assisting in future planning pragmatic control strategies against bovine brucellosis in Egypt and for herd health fertility.

Evaluation of Different PCR Techniques in Diagnosis of Camel (Camellus Dromedary) Brucellosis in Sudan

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.

Detection of Brucella abortus by a platform functionalized with protein A and specific antibodies IgG.

Oriented immobilization of antibodies on a sensor surface is critical for enhancing both the antigen-binding capacity and the sensitivity of immunosensors. In this study, we describe a strategy to adsorb immunoglobulin G (IgG) anti-Brucella antibodies onto a silicon surface, oriented by protein A obtained from Staphylococcus aureus (SpA). X-ray photoelectron spectroscopy and atomic force microscopy were used to characterize topographically, morphologically, and chemical changes of the sensor functionalization. The activity of the biosensor was assessed by confocal microscopy, scanning electronic microscopy, and bacteria capture assays (BCA). According to the BCA, the efficiency of Brucella abortus detection with the SpA-IgG anti Brucella biosensor was three-fold higher than that of the random orientated IgG anti Brucella biosensor. The limit of detection was 1 × 106 CFU/ml. These data show that the orientation of antibodies immobilization is crucial to developing immunosensors for bacterial antigen detection as Brucella spp and improve its sensibility level. Functionalization with protein A increases Brucella detection by an antibody-coated surface. Functionalized silicon surface for Brucella detection was characterized by atomic force microscopy, X-ray photoelectron spectroscopy and confocal microscopy.

Comparative Ecology of Bartonella and Brucella Infections in Wild Carnivores.

Phylogenetic sister clades Bartonella and Brucella within the order Rhizobiales present some common biological characteristics as well as evident differences in adaptations to their mammalian reservoirs. We reviewed published data on Bartonella and Brucella infections in wild carnivores to compare the ecology of these bacteria in relatively similar host environments. Arthropod vectors are the main mechanism for Bartonella species transmission between mammalian hosts. The role of arthropods in transmission of Brucella remains disputed, however experimental studies and reported detection of Brucella in arthropods indicate potential vector transmission. More commonly, transmission of Brucella occurs via contact exposure to infected animals or the environment contaminated with their discharges. Of 26 species of carnivores tested for both Bartonella and Brucella, 58% harbored either. Among them were bobcats, African lions, golden jackals, coyotes, wolves, foxes, striped skunks, sea otters, raccoons, and harbor seals. The most common species of Bartonella in wild carnivores was B. henselae, found in 23 species, followed by B. rochalimae in 12, B. clarridgeiae in ten, and B. vinsonii subsp. berkhoffii in seven. Among Brucella species, Br. abortus was reported in over 30 terrestrial carnivore species, followed by Br. canis in seven. Marine carnivores, such as seals and sea lions, can host Br. pinnipedialis. In contrast, there is no evidence of a Bartonella strain specific for marine mammals. Bartonella species are present practically in every sampled species of wild felids, but of 14 Brucella studies of felids, only five reported Brucella and those were limited to detection of antibodies. We found no reports of Bartonella in bears while Brucella was detected in these animals. There is evident host-specificity of Bartonella species in wild carnivores (e.g., B. henselae in felids and B. vinsonii subsp. berkhoffii in canids). A co-adaptation of Brucella with terrestrial wild carnivore hosts is not as straightforward as in domestic animals. Wild carnivores often carry the same pathogens as their domesticated relatives (cats and dogs), but the risk of exposure varies widely because of differences in biology, distribution, and historical interactions.

Simultaneous Detection of Brucella, Leptospira, Mycoplasma and Listeria Species by Multiplex-PCR

Reproductive disorders in farm animals represent a great importance due to economic losses for breeder and country economy. The main cause of reproductive failure in cattle is infectious abortion. Brucella, Leptospira, Mycoplasma and Listeria are important zoonosis and leading to sever economic losses in animals around the world, especially in Mediterranean countries and Egypt. Since early detection is crucial in control and treatment, molecular techniques proved to be a more rapid, sensitive and specific diagnostic tool. The present study is aimed to detect these four important pathogens using multiplex- PCR. The multiplex PCR has been standardized by using 4-pairs of primers to amplify 31kDa gene encoding protein in Brucella spp., lig gene in pathogenic Leptospira, hlyA gene in Listeria monocytogenes and 16S rDNA in Mycoplasma spp. A total of 161 different clinical samples were used, an expected band at 223bp, 468p, 370 bp and 270bp were obtained from Brucella spp., pathogenic Leptospira, Listeria monocytogenes and Mycoplasma bovis, respectively. In conclusion, the results of this conventional m-PCR assay revealed that it can be a valuable tool for simultaneous, rapid, cost saving and reliable detection of these microbial agents causing bovine abortion.