Identification Of Brucella Research Articles

Identification of Brucella spp. in Aborted Fetuses by Guineapig inoculation

Abstract Background: Brucellosis is a zoonotic disease which is endemic in Bangladesh. The prevalence of bovine brucellosis in subsistence management system in Bangladesh is low. However, the prevalence of bovine brucellosis at Central Cattle Breeding and Dairy Farm (CCBDF) is reported to be very high and Brucella abortus DNA has also been detected from cows’ milk of this farm. The principal manifestation of bovine brucellosis is abortion in pregnant cows, which is common at CCBDF. The role of brucellosis in abortion at CCBDF has not been ascertained. Hence, this study was undertaken to confirm Brucella spp. as the etiology of abortion in cows at CCBDF. Materials and Methods: Aborted fetal membranes and vaginal swabs from 3 cows, in which late abortion occurred, were collected aseptically from the CCBDF. The samples were initially stained with modified Zeihl-Neelsen staining method. The stain-positive samples were ground individually using a pestle and mortar, and a homogenized mixture was prepared by adding normal saline. Two milliliters of the homogenate from each sample were inoculated subcutaneously into a guinea pig. The sera of guinea pigs were collected after 3rd week of inoculation to perform rose Bengal test (RBT) and rapid antibody test (RAT). Result: Samples from two cows showed positive staining result in which numerous pink-colored coccobacilli were seen. All the sera collected were tested positive for both RBT and RAT. It is evident from this study that three of two abortions at CCBDF were due to brucellosis. Conclusion: Guineapig inoculation technique could be used as a good alternative of culture for confirming the diagnosis of brucellosis from contaminated clinical samples like placenta.

Delay in the diagnosis of Brucella abortus bacteremia in a nonendemic country: a case report

BackgroundIt is challenging to diagnose brucellosis in nonendemic regions because it is a nonspecific febrile disease. The accurate identification of Brucella spp. in clinical microbiology laboratories (CMLs) continues to pose difficulties. Most reports of misidentification are for B. melitensis, and we report a rare case of misidentified B. abortus.Case presentationA 67-year-old man visited an outpatient clinic complaining of fatigue, fever, and weight loss. The patient had a history of slaughtering cows with brucellosis one year prior, and his Brucella antibody tests were negative twice. After blood culture, the administration of doxycycline and rifampin was initiated. The patient was hospitalized due to a positive blood culture. Gram-negative coccobacilli were detected in aerobic blood culture bottles, but the CML's lack of experience with Brucella prevented appropriate further testing. Inaccurate identification results were obtained for a GN ID card of VITEK 2 (bioMérieux, USA) and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI–TOF MS) using a MALDI Biotyper (Bruker, Germany). The strain showed 100.0% identity with Brucella spp. according to 16S rRNA sequencing. MALDI–TOF MS peaks were reanalyzed using the CDC MicrobeNet database to determine Brucella spp. (score value: 2.023). The patient was discharged after nine days of hospitalization and improved after maintaining only doxycycline for six weeks. The isolate was also identified as Brucella abortus by genomic evidence.ConclusionAutomated identification instruments and MALDI–TOF MS are widely used to identify bacteria in CMLs, but there are limitations in accurately identifying Brucella spp. It is important for CMLs to be aware of the possibility of brucellosis through communication with clinicians. Performing an analysis with an additional well-curated MALDI–TOF MS database such as Bruker security-relevant (SR) database or CDC MicrobeNet database is helpful for quickly identifying the genus Brucella.

Phylogenetic Analysis of Brucella melitensis Strains Isolated from Humans Using 16S rRNA Sequencing and Multiple Locus Variable Number of Tandem Repeats Analysis-16.

Background: Brucellosis is the most important public health problem worldwide, and the annual incidence of the disease in humans is 2.1 million. The Brucella genome is highly conserved, with over 90% similarity among species. The aim of this study was to perform species-level identification of Brucella spp. strains isolated from humans diagnosed with brucellosis and to further investigate the phylogenetic relationships using multiple locus variable number of tandem repeats analysis (MLVA)-16 and 16S rRNA sequencing analysis. Materials and Methods: Brucella spp. was isolated from the blood cultures of 54 patients who tested positive for brucellosis through serological examinations. Real-time PCR was used to identify the isolates in species, and the genus level of Brucella was confirmed with 16S rRNA. All isolates were subjected to phylogenetic analysis using variable number of tandem repeat analysis with multiple loci. Results: Subsequent analysis via real-time PCR confirmed these isolates to be of the Brucella melitensis species. The 16S rRNA sequence analysis showed 100% homogeneity among the isolates. MLVA revealed the formation of five different genotypic groups. While two groups were formed based on the 16S rRNA sequence analysis, five groups were formed in the MLVA. Conclusions: The study concluded that 16S rRNA sequence analysis alone did not provide sufficient discrimination for phylogenetic analysis but served as a supportive method for identification. MLVA exhibited higher phylogenetic power. The widespread isolation of B. melitensis from human brucellosis cases highlights the importance of controlling brucellosis in small ruminants to prevent human infections.

First Argentine database for the accurate identification of Brucella to species level by MALDI-TOF MS

MALDI-TOF mass spectrometry (MS) has proven to be a fast and reliable method for the identification of a large number of taxonomic groups. It offers the advantage of being able to incorporate protein spectra of microorganisms that are absent or poorly represented in commercial databases, such as the genus Brucella. The aim of the study was to build the first database of protein spectra of local biological variants of Brucella in Argentina and of standard strains. First, the identification performance of a panel of 135 strains was evaluated with the Swedish database ¨Folkhälsomyndigheten¨ (containing protein spectra of several international standards of the genus Brucella) imported from the open access site https://spectra.folkhalsomyndigheten.se/spectra/. With this library 100 % of the strains were correctly identified by mass spectrometry to genus level, but not to species level.Due to the limitation found, an in-house database was designed with local Brucella isolates from Argentina and standard strains used in routine bacteriological diagnosis. For its validation, a panel of strains, different from those used to develop the extended local database (n: 177), was used to, simultaneously, challenge both libraries. The samples were processed by triplicate and the results obtained were: 177 strains correctly identified to genus and species level compared to the gold standard method (phenotypic typing), meeting the criteria accepted by the literature and the manufacturer as reliable identification. Only 2 of these isolates had score values lower than 2 (1.862) and were therefore not included in the calculation of results.According to these results, MALDI-TOF MS is a fast and reliable method for the routine identification of the different Brucella species, and even has the advantage of reducing the time of exposure to pathogenic microorganisms for laboratorians. It could be considered a valuable technique to replace, in the near future, the current conventional techniques due to the ease of transferring protein spectra, avoiding the use of reference strains that are difficult to find commercially available and commonly used in phenotypic typing.

First record of the human infection of Brucella melitensis in Kyrgyzstan: evidence from whole-genome sequencing-based analysis.

Brucellosis, a zoonosis mainly transmitted by consumption of unpasteurized dairy products as well as direct contact with infected animals, is endemic in Kyrgyzstan. However, Brucella species in humans have not been investigated and the origin of the disease remains poorly known in wide parts of Сentral Asia. Thus, molecular characterization of the circulating strains is a critical first step in understanding Brucella diversity in the country. In this study, isolates were collected from patients with suspected brucellosis from different regions in Kyrgyzstan between 2019 and 2020. The detection and identification of Brucella was carried out by Bruce-ladder PCR. Next generation sequencing was used to sequence the 89 Brucella isolates, which were genotyped by cgSNP and cgMLST to identify epidemiological connection between Brucella isolates as well as placing them in the context of the global Brucella phylogeny. The Brucella strains isolated from all regions of Kyrgyzstan were identified as B. melitensis. Based on cgSNP analysis, 18 sequence types were differentiated. The highest numbers of different sequence types were found in Batken (n = 8), Osh (n = 8) and Jalal-Abad (n = 6) oblasts. According to cgSNP and cgMLST analyses, different B. melitensis lineages circulate in Kyrgyzstan, all of them belonging to the Eastern Mediterranean group of the global Brucella phylogeny with the highest similarity to strains from Turkmenistan, Iran and Turkey. In the present study, B. melitensis was identified as a causative agent of human brucellosis in Kyrgyzstan and different lineages could be identified. Since this study focused on isolates of human origin, the identity of Brucella species and lineages circulating among animal populations remains elusive. Implementing culture techniques and use of most recent molecular, bioinformatic and epidemiological tools are needed to set up a One Health approach to combat brucellosis in Kyrgyzstan. Further, other Сentral Asian countries need to take part in this effort as brucellosis is a transboundary disease in these regions.

Minimum Core Genome Sequence Typing of Brucella From China

Abstract The Gram-negative bacterial genus Brucella includes six classic species based on host specificity, pathogenicity and phenotypic differences. Four more Brucella species were identified in 2007. Although many Brucella genomes have been sequenced, genome sequences and analysis of Brucella strains isolated in China are still scarce. An efficient genome-based Brucella typing method is also needed. In this study, we used the minimum core genome (MCG) typing method to identify and type Brucella strains. Twenty Brucella isolates from China were newly sequenced. The genome sequences of 55 representative Brucella strains were downloaded. Among the 75 genomes, 1089 genes and 52,030 single nucleotide polymorphisms (SNPs) shared by all isolates were considered as the MCG genes and MCG SNPs. Using these 52,030 MCG SNPs, Brucella was divided into six MCG groups. In addition, average nucleotide identity (ANI) values and the distributions of 184 virulence genes were all computed. The proportions of virulence genes were 90.96%, 93.56%, 95.89%, 86.04%, 85.78% and 91.87% for MCG groups 1 to 6, respectively. The intragroup ANI values were higher than the intergroup values, further confirming the validity of the MCG taxonomy classification. Brucella melitensis and Brucella abortus, the two main Brucella species pathogenic to humans, were well separated from other species. With the development and cost reduction of next-generation sequencing, the MCG typing method can be used for rapid identification of Brucella, which can contribute to the rapid diagnosis of brucellosis and ensure timely and effective treatment.

Brucellosis in pyrexia of unknown origin: Reminiscing the forgotten entity

PurposeBrucellosis is one of the substantial yet forgotten entity of pyrexia of unknown origin, transmitted via contact with animals or animal products. Despite being an important etiological agent of PUO, it is under-reported owing to its non-specific and protean clinical presentations. Thereby, the present study intended to describe clinical profile and outcome of patients of brucellosis from a tertiary care center in North India. MethodsThis was a retrospective study and included all the patients that were reported positive for Brucella in automated blood culture system (BACTEC) and identified using MALDI-TOF MS over a period of 6 years (2014–2020). ResultsA total of nine cases were reported positive for Brucella during the study period. Fever was noted to be the most common symptom present in all the patients (PUO), followed by fatigue. The patients were managed with monotherapy using doxycycline or combination of cotrimoxazole, rifampicin and doxycycline. All the patients responded well to treatment. ConclusionThe present study highlights the significance of automated blood culture system in conjunction with MALDI-TOF MS for early and reliable identification of Brucella. In the present study, all cultures were reported positive within five days of incubation in automated blood culture system, underlining the significance of using the same which lessens the turnaround time and increases the sensitivity. Owing to the protean manifestations and low sensitivity of the available tests, the clinician needs to have a high level of suspicion to precisely diagnose the illness to prevent undue delay in management.

Development of a Method for Determination of brucella suis Biovars Using Multilocus Real-time PCR

The aim of the study was to develop a methodological approach to determination of Brucella suis biovars through multilocus PCR with real-time registration of results.Materials and methods. We used 16 strains of B. suis of various biovars, B. neotomae and B. canis – 2 strains of each. Determination of the taxonomic affiliation of Brucella strains was carried out according to the Bruce-ladder, Suis-ladder, BRU-DIF protocols. The selection of primers and probes was performed using the software on the website www.genscript.com and the GeneRanner 6.5.52 program. Fragment sequencing according to Sanger was performed on a 3500 XL genetic analyzer in accordance with the manufacturer’s recommendations. Nucleotide sequence homology was assessed using the BLAST algorithm and the GenBank NCBI database.Results and discussion. An analysis of the structural organization of IncP and GI-3 genomic islands has been carried out in B. suis strains of various biovars. It has been established that in strains of B. suis II, IV biovars and B. canis, the terminal part of the BRA0368 gene, comprising 21 nucleotides (repeated in the BRA0367 gene) and the “TAA” stop codon, as well as almost the entire sequence of the BRA0367 gene were lost, owing to homologous recombination in the IncP genome island. A 21-nucleotide direct repeat and the “TGA” stop codon of the BRA0367 gene replaced the analogous region of the BRA0368 gene which resulted in the deletion the size of 185 bp. No differences have been noted in the structure of GI-3 in biovars. The evidence obtained made it possible to develop the approach (SuisDIF) for differentiating B. suis biovars, based on the amplification of genes located in the IncP and GI-3 genomic islands using real-time PCR. Its specificity was confirmed in the study of B. suis strains from the fund of the State Collection of Pathogenic Bacteria of the Russian Research Anti-Plague Institute “Microbe”. The conducted studies expand and supplement the data on the genetic heterogeneity of Brucella species and biovars. The proposed method for differentiating biovars of B. suis using multilocus PCR with real-time registration of results enhances the capacities for Brucella identification using molecular-genetic methods.

High-Resolution Melting PCR as Rapid Genotyping Tool for Brucella Species.

Brucella sp. are the causative agents of brucellosis. One of the main characteristics of the Brucella genus concerns its very high genetic homogeneity. To date, classical bacteriology typing is still considered as the gold standard assay for direct diagnosis of Brucella. Molecular approaches are routinely used for the identification of Brucella at the genus level. However, genotyping is more complex, and to date, no method exists to quickly assign a strain into species and biovar levels, and new approaches are required. Next generation sequencing (NGS) opened a new era into the diagnosis of bacterial diseases. In this study, we designed a high-resolution melting (HRM) method for the rapid screening of DNA and direct assignment into one of the 12 species of the Brucella genus. This method is based on 17 relevant single nucleotide polymorphisms (SNPs), identified and selected from a whole genome SNP (wgSNP) analysis based on 988 genomes (complete and drafts). These markers were tested against the collection of the European Reference Laboratory (EU-RL) for brucellosis (1440 DNAs extracted from Brucella strains). The results confirmed the reliability of the panel of 17 SNP markers, allowing the differentiation of each species of Brucella together with biovars 1, 2, and 3 of B. suis and vaccine strain Rev1 (B. melitensis) within 3 h, which is a considerable gain of time for brucellosis diagnosis. Therefore, this genotyping tool provides a new and quick alternative for Brucella identification based on SNPs with the HRM-PCR assay.

Detection and molecular identification of Brucella species infecting Camels in Darfur, Sudan

-Brucellosis is a wide spread zoonotic disease which is characterized by reduced fertility and abortion in animals and in humans. Although not the primary hosts, camels are highly susceptible to Brucella abortus and B. melitensis. Camel meat and milk are consumed raw some times in Sudan, which may increase the prevalence of brucellosis. The aim of this study was to screen camels from three districts of Nyala in South Darfour, Sudan for brucellosis. A sensitive specied-specific PCR assay was adopted for the detection and identification of Brucella spp. in 100 blood samples and 30 milk samples.41 camels were positive for Brucella spp. Out of which, 44% were positive for Brucella abortus, 43% were positive for B. melitensis, and 22% were positive for a co-infection of B. abortus and B. melitensis.The majority of infected samples were collected at the slaughterhouse from camels to be consumed.The finding of Brucella spp in camels' meat and milk is alarming as Sudanese consumers eat raw camels' liver and drink unpasteurized milk. Therefore the main recommendation of this study is to adopt a sensitive and species-specific PCR protocol for routine detection of brucellosis especially in animals to be slaughtered.

Genetic identification of the causative agent of Brucellosis

The development of animal husbandry suffers various kinds of losses due to the spread of infectious diseases among animals, in particular Brucellosis. A challenge faced by Brucella researchers has been the accurate identification of new isolates within the genus while preserving sufficient, and not excessive, biosafety and biosecurity requirements. The availability of discriminatory molecular typing tools to inform and assist conventional epidemiological approaches would be invaluable in controlling these infections, but efforts have been hampered by the genetic homogeneity of the genus. In this work, for better identification of infection, for control and monitor the source of outbreaks in prosperous areas was carried out identification of Brucella spp. strains which circulating in the Kostanay region. For this was used using multilocus analysis of a variable number of tandem repeats sequenced by 16 s – PNK on a genetic analyzer (sequencer). According to the results of a study of cattle, cultures of microorganisms were infected: No. 4, 5, 7, 8. Comparison of the obtained results with similar results of domestic and foreign works by A. Shevtsov, G. Borrello, P. Le Fleche, G. Garofolo suggest that the genotyping of local strains has an importance in the molecular epizootology of the Republic of Kazakhstan.

Molecular detection of brucellosis in dromedary camels of Qatar by real-time PCR technique

Background and methodologyBrucellosis in livestock causes huge economic loses of developing countries and demonstrates a serious health risks to the patrons of infected dairy goods and meat. Prevention of brucellosis in cattle relies on the trustworthiness of the techniques utilized to detect the causative pathogen. In the current investigation, we described the exploration of the well-known real-time PCR technique based on the Brucella-specific IS711 with different tissue samples of dromedary camels in Qatar. ResultsThe findings of the real-time PCR unveiled the occurrence of Brucella spp in 60 % of lung tissues, 71.42 % of liver tissues, 72 % of spleen tissues, 25 % of kidney tissues, and 42.42 % of placental fluid samples. Among them, the liver tissues and spleen tissues possessed the highest number of positive results, whereas kidney tissues displayed the lowest number of positive results for Brucella spp. ConclusionThe findings of this investigation discloses that PCR technique is a sensitive and specific technique for the identification of Brucella spp. in dromedary camels. Most of the tissues samples showed the presence of Brucella spp. and also we concluded that there is a need for further studies to be conducted in order to identify specific species of Brucella.

Risk factors, outcomes and time to detect positive blood culture among cases with acute brucellosis.

Brucellosis causes a disabling human disease and loss of animals' lives. The clinical significance of Brucella bacteremia is still unclear and Brucella identification in blood culture is suboptimal. This was a retrospective study conducted in Medina in Saudi Arabia from August 2016 to May 2019. We included cases with brucellosis symptoms and a positive culture or serological evidence for brucellosis, comparing bacteremic with non-bacteremic brucellosis cases for the rates of complications, infection relapses and brucellosis development. Also, we estimated blood culture positivity rates and the time to detect Brucella in an automatic blood culture instrument. Of the total number of 147 cases, 62 (42%) had a positive blood culture for Brucella, and the blood culture instrument (BACT/ALERT 3D) detected all positive blood cultures within 3 d of incubation. We found higher rates of chronic brucellosis in bacteremia than non-bacteremia cases (OR 7.25, 95% CI 1.41 to 37.23; p=0.018). Patients aged <15 y developed a higher rate of bacteremia than those aged ≥15 yr (OR 11.93 95% CI 1.37 to 103.75; p=0.025). Brucella bacteremia is an independent predictor for the development of chronic infection. Brucella bacteremia cases may need long follow-up periods and a more thorough evaluation to exclude deep-seated infection.

Investigation of Mutations in the Rifampin-Resistance-Determining Region of the rpoB Gene of Brucella melitensis by Gene Analysis

Background: RNA polymerase beta subunit (rpoB) gene analysis in bacterial communities is known as a method for determining rifampin sensitivity and genetic diversity among Brucella spp. Detection of antibiotic resistance among Brucella isolates can be a critical approach to control brucellosis. However, rpoB gene analysis of Brucella melitensis for assessing rifampicin resistance has not yet been performed in Iran, which is considered an endemic area for brucellosis. Objectives: The aim of this study was to analyze the whole sequence of rpoB genes of different B. melitensis isolates from humans to identify the single-nucleotide polymorphisms (SNPs) and mutations related to rifampin resistance and to analyze the genetic diversity of these bacteria in Iran. Methods: Between 2017 and 2019, a total of 156 blood samples along with 12 synovial fluid specimens were collected from brucellosis patients in different Iranian provinces and subjected to bacterial culture in Brucella selective media. Brucella identification was carried out using classical biotyping and molecular examinations. Polymerase chain reaction (PCR)-based amplification of the rpoB gene was performed by specific rpoB primers for whole gene sequencing. The antimicrobial susceptibility of Brucella isolates was assessed using disk diffusion susceptibility tests and minimal inhibitory concentration (MIC) methods. The presence of rifampin-binding sites and SNPs were investigated through rpoB whole gene sequencing. Results: Clinical B. melitensis isolates were obtained from blood (13) and synovial fluid (1) samples of patients from different regions of Iran. The results of MIC and disk diffusion susceptibility tests showed that all the isolates were sensitive to rifampin except for one isolate showing intermediate rifampin resistance based on the standards defined for slow-growing bacteria by the Clinical and Laboratory Standards Institute (CLSI). Gene analysis for identifying the mutations related to rifampin resistance and investigating genetic diversity showed that none of the B. melitensis isolates had missense mutations, confirming the susceptibility of all the studied isolates to rifampin. Conclusions: The present study revealed that rpoB gene analysis could be used for the efficient and precise identifying of the mutations related to rifampin resistance, investigating rifampin binding sites, and genotyping Brucella species. Furthermore, the identification of B. melitensis isolates with intermediate resistance to rifampicin highlighted the importance of periodically carrying out antibiotic susceptibility testing. The molecular detection of rpoB mutations in different Brucella isolates may help to prevent the spread of rifampin-resistant Brucella spp. among humans and livestock.

Clinical and laboratory features of brucellosis in a university hospital in Tunisia

Aim of the studyThe increase in the number of brucellosis cases between 2014 and 2017 (14 and 90 cases respectively) led us to study the biological and clinical-epidemiologic characteristics patients hospitalized in Rabta hospital of Tunis. Material and methodsThis retrospective study was conducted in Rabta Hospital in Tunis between 2016 and 2017. It includes 131 patients who had a positive bacteriological diagnosis of Brucella between 2016 and 2017. Diagnosis of brucellosis was made in blood culture by using Bactalert (Biomerieux®). Identification of Brucella was realized by Gram staining, catalase, oxydase. Serological diagnosis was made by testing sera for brucellosis agglutinins with Rose Bengale and the standard agglutination test. The collected data were analyzed by SPSS softcare version 24. ResultsThe prevalence of Brucellosis in Rabta hospital increases from 14 cases in 2014 to 90 cases in 2017. The mean age was 45 years and ages range from 16 to 84 years. Rural origin was found in 75 cases (68%). Ninety-seven patients (89%) were hospitalized in the infectious diseases department. The average length of hospitalization was 17.25 days. Fifty-seven patients (52%) had a history of consuming unpasteurized dairy products and 45 (41%) were farmers. Fever was the predominant symptom in 104 cases (95%). Osteoarticular involvement is the most common complication of brucellosis and it occurred in 28% of patients. Blood cultures were 73 cases and 42 (57%) were positive for Brucella spp. Rose Bengale was positive in 100% of cases. High titles of the standard agglutination test (superior to 1/1280) were noted in 24 cases (22%). ConclusionBrucellosis is still endemic In Tunisia. Contact with domestic animals and consumption of raw milk and milk products seems to be the major mode of transmission. Control of animal infection by vaccination, occupational and personal hygiene, farm sanitation and preventive measures can reduce disease incidence.

Seroprevalence and Molecular Identification of Brucella spp. in Bovines in Pakistan-Investigating Association With Risk Factors Using Machine Learning.

Bovine brucellosis is a global zoonosis of public health importance. It is an endemic disease in many developing countries including Pakistan. This study aimed to estimate the seroprevalence and molecular detection of bovine brucellosis and to assess the association of potential risk factors with test results. A total of 176 milk and 402 serum samples were collected from cattle and buffaloes in three districts of upper Punjab, Pakistan. Milk samples were investigated using milk ring test (MRT), while sera were tested by Rose–Bengal plate agglutination test (RBPT) and indirect enzyme-linked immunosorbent assay (i-ELISA). Real-time PCR was used for detection of Brucella DNA in investigated samples. Anti-Brucella antibodies were detected in 37 (21.02%) bovine milk samples using MRT and in 66 (16.4%) and 71 (17.7%) bovine sera using RBPT and i-ELISA, respectively. Real-time PCR detected Brucella DNA in 31 (7.71%) from a total of 402 bovine sera and identified as Brucella abortus. Seroprevalence and molecular identification of bovine brucellosis varied in some regions in Pakistan. With the use of machine learning, the association of test results with risk factors including age, animal species/type, herd size, history of abortion, pregnancy status, lactation status, and geographical location was analyzed. Machine learning confirmed a real observation that lactation status was found to be the highest significant factor, while abortion, age, and pregnancy came second in terms of significance. To the authors' best knowledge, this is the first time to use machine learning to assess brucellosis in Pakistan; this is a model that can be applied for other developing countries in the future. The development of control strategies for bovine brucellosis through the implementation of uninterrupted surveillance and interactive extension programs in Pakistan is highly recommended.

Isolation and molecular identification of Brucella spp. in bovine herds kept at householders in the Delta region of Egypt by MALDI-TOF and AMOS-PCR.

Brucellosis is a widespread disease in Egypt which cause huge economic losses in the dairy industry. The present study aims at isolating and identifying Brucella (B.) spp. circulating in bovine and buffalo dairy herds kept at farmers houses in four districts of the Delta region of Egypt. One hundred and five tissue specimens were collected from seropositive cattle and buffaloes. The samples included 10 vaginal swabs, 3 placentas, 3 uteri and 86 supra-mammary lymph nodes from dams, as well as 3 stomach contents from aborted fetuses. Matrix-assisted laser desorption ionization (MALDI) and the conventional biotyping techniques were used for preliminary identification of isolates into the genus level. AMOS-PCR was applied to differentiate Brucella isolates into species level. Nineteen Brucella strains have been identified, four B. abortus strains were recovered from cattle and 15 B. melitensis strains were isolated from both cattle (n = 8) and buffaloes (n = 7). The predominant occurrence of B. melitensis in bovines raises the fact that B. melitensis clone can cross species barriers and can establish a permanent reservoir in cattle and buffaloes. Presence of culture-positive animals at householders represent a high-risk factor for human infection. This knowledge is of significant importance in the control of brucellosis in bovines.

Seroprevalence and Molecular Identification of Brucella spp. in Camels in Egypt.

Brucellosis is one of the most important worldwide zoonoses of many countries including Egypt. Camel brucellosis has not gained much attention in Egypt yet. This study is focused on the three governorates with the highest camel populations and the largest camel markets in the country to determine the disease seroprevalence and identify the Brucella species in local camel holdings. In total, 381 serum samples were collected from male and female camels from Giza, Aswan, and Al-Bahr Al-Ahmar (the Red Sea) governorates. Samples were serologically examined using the Rose–Bengal plate test (RBPT), indirect ELISA (i-ELISA), competitive ELISA (c-ELISA) and complement fixation test (CFT). Brucella antibodies were detected in 59 (15.5%), 87 (22.8%), 77 (20.2%) and 118 (31.0%) of sera by RBPT, i-ELISA, c-ELISA and CFT, respectively. Using real-time PCR, Brucella DNA was amplified in 32 (8.4%) seropositive samples including Brucella abortus (25/32), Brucella suis (5/32) and Brucella melitensis (2/32), defining a complex epidemiological status. To the best of our knowledge, this is the first study reporting Brucella suis DNA in camel serum. The risk-associated factors including age, sex, breed and geographical distribution were statistically analyzed, showing non-significant association with seroprevalence. The results of this study will raise awareness for camel brucellosis and help develop effective control strategies.

Genome-wide unique insertion sequences among five Brucella species and demonstration of differential identification of Brucella by multiplex PCR assay

Brucellosis is a neglected zoonotic disease caused by alpha proteobacterial genus Brucella comprising of facultative intracellular pathogenic species that can infect both animals and humans. In this study, we aimed to identify genome-wide unique insertion sequence (IS) elements among Brucella abortus, B. melitensis, B. ovis, B. suis and B. canis for use in species differentiation by conducting an intensive in silico-based comparative genomic analysis. As a result, 25, 27, 37, 86 and 3 unique ISs were identified respectively and they had a striking pattern of distribution among them. To explain, a particular IS would be present in four species with 100% identity whereas completely absent in the fifth species. However, flanking regions of that IS element would be highly identical and conserved in all five species. Species-specific primers designed on these flanking conserved regions resulted in two different amplicons grouping the species into two: one that possesses IS and the other that lacks it. Seeking for species-specific amplicon size for particular species was sufficient to identify it irrespective of biovar. A multiplex PCR developed using these primers resulted in successful differentiation of the five species irrespective of biovars with significant specificity and sensitivity when examined on clinical samples.

Identification of Brucella spp. isolates and discrimination from the vaccine strain Rev.1 by MALDI-TOF mass spectrometry

Brucellosis' surveillance and control programs require robust laboratory techniques that can reliably identify and biotype Brucella strains and discriminate between vaccine and field infection. In the recent years, Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) has revolutionized the routine identification of several microorganisms in clinical microbiology laboratories. Nevertheless, its application on Brucella spp. identification is limited since there are no reference spectra in the commercial databases, due to the microorganism's potential bioterrorist use.In this study, a custom MALDI-TOF MS reference library was constructed and its performance on identification at species level was evaluated using 75 Brucella spp. isolates. Furthermore, distinct peak biomarkers were detected for biovar assignment and discrimination from vaccine strain Rev.1. Analysis of mass peak profiles allowed Brucella accurate identification at genus and species level (100%) with no misidentifications. Despite the high intrageneric similarity, MALDI-TOF MS database succeeded in classifying at biovar level, 47 out of 62 B. melitensis bv. 3 isolates (75.81%), whereas all B. melitensis strains, except for one, were correctly discriminated from vaccine strain Rev.1.MALDI-TOF MS appeared to be a rapid, cost-effective and reliable method for the routine identification of brucellae which reduces time consumption in pathogen identification and could replace in the near future the current conventional and molecular techniques. Its ability to differentiate vaccine from field infection could facilitate brucellosis’ monitoring systems contributing in the effective control of the disease.