Brucella

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is a rare syndrome that is often fatal despite treatment. It is caused by a dysregulation in natural killer T-cell function, resulting in activation and proliferation of histiocytes with uncontrolled hemophagocytosis and cytokines overproduction. The syndrome is characterized by fever, hepatosplenomegaly, cytopenias, liver dysfunction, and hyperferritinemia. HLH can be either primary, with a genetic aetiology, or secondary, associated with malignancies, autoimmune diseases, or infections.

A serologic survey of anti-Brucella spp. antibodies was undertaken on 2,470 samples of 14 North American marine mammal species collected between 1984-97. Serum or blood from eight species of cetaceans and six species of pinnipeds was sampled from Pacific, Atlantic, and Arctic oceans. Two competitive enzyme-linked immunosorbent assays (C-ELISA's), using specific monoclonal antibodies to Brucella abortus cell wall components, were used to detect anti-Brucella spp. antibodies in the samples. Sera from 33 cetaceans and 61 pinnipeds gave inhibition values, in one or both of the tests, which exceeded the threshold that indicates Brucella spp. exposure in cattle. Seropositive animals were identified from Pacific, Atlantic, and Arctic oceans. While Brucella spp. was not isolated, differences in the response of seropositive cetacean and pinniped sera in the two assays suggest that two antigenically distinct species or biovars of Brucella spp. are present. No pathology consistent with clinical brucellosis was noted in any of the animals tested although detailed examination was not conducted on all carcasses.

Raw or inadequately pasteurized milk from infected animals and cheese made with such milk are a frequent vehicle for human brucellosis infection. Also, biological terrorism is a concern with certain Brucella spp. Due to matrix-associated real-time polymerase chain reaction (qPCR) inhibitors, robust sample preparations are crucial. We compared six commercial nucleic acid extraction kits using nine Mexican and Central American-style soft cheeses or creams and three liquid milk products inoculated with Brucella neotomae, a surrogate for pathogenic Brucella spp. Kits were evaluated by purity and quantity of DNA as determined by qPCR Ct values, reproducibility across cheese and milk types, and cost. At 10 7 CFU/g in four different cheeses, Qiagen statistically outperformed all other kits. When two cheese styles were inoculated at dual levels, Qiagen and High Pure kit extracted samples at 1.5 Â 10 5 CFU/g produced average Ct values of 34e39, while PrepSEQ and MagMAX kit extracted samples exhibited higher or no Ct values. High Pure and Qiagen kits excelled also with liquid milk products. Considering matrices, inoculation levels, and kits evaluated, High Pure and Qiagen products produced Brucella DNA of high quality and quantity indicated by the lowest Ct values and were the least expensive.

We surveyed 13 carcasses of marine mammals (12 Trichechus manatus and one Stenella clymene) that had stranded in northeastern Brazil during 1990-2013 for infectious diseases by screening tissues from the collection of the Brazilian National Center of Research and Conservation of Aquatic Mammal, Chico Mendes Institute for Biodiversity Conservation. Brucella spp. and Mycobacterium spp. were investigated by culturing and PCR of tissue samples, whereas Sarcocystidae parasites, Leptospira spp., and Morbillivirus were surveyed for using specific PCR assays. Brucella spp. and Mycobacterium spp. were not isolated through microbiologic culturing, and all animals were negative for detection of Sarcocystidae parasites, Leptospira spp., Mycobacterium spp., and Morbillivirus by PCR assays. All manatees were negative for Brucella spp. infection, but Brucella ceti was detected in the brain tissue of an S. clymene calf by using a PCR assay.

This review of Brucella-host interactions and immunobiology discusses recent discoveries as the basis for pathogenesis-informed rationales to prevent or treat brucellosis. Brucella spp., as animal pathogens, cause human brucellosis, a zoonosis that results in worldwide economic losses, human morbidity, and poverty. Although Brucella spp. infect humans as an incidental host, 500,000 new human infections occur annually, and no patient-friendly treatments or approved human vaccines are reported. Brucellae display strong tissue tropism for lymphoreticular and reproductive systems with an intracellular lifestyle that limits exposure to innate and adaptive immune responses, sequesters the organism from the effects of antibiotics, and drives clinical disease manifestations and pathology. Stealthy brucellae exploit strategies to establish infection, including i) evasion of intracellular destruction by restricting fusion of type IV secretion system-dependent Brucella-containing vacuoles with...

Bacteria rely on two-component signaling systems in their adaptive responses to environmental changes. Typically, the two-component system consists of a sensory histidine kinase that signals by transferring a phosphoryl group to a secondary response regulator that ultimately relays the signal to the cell. Some of these sensors use PAS (Per-Arnt-Sin) domains. A new member of the PAS super family is the LOV (light, oxygen, voltage) domain, a 10-kDa flavoprotein that functions as a light-sensory module in plant, algal, fungal, and bacterial blue-light receptors. Putative LOV domains have been identified in the genomes of many higher and lower eukaryotes, plants, eubacteria, archaebacteria, and particularly in genes coding for histidine kinases (LOV-histidine kinases, LOV-HKs) of plant and animal pathogenic bacteria, including Brucella. We describe here biochemical, photochemical, and biophysical methodology to purify these enzymes and to characterize their light-activation process.

Although a "One Health" approach has been successfully implemented for emerging infectious zoonotic diseases with pandemic potential, we still lack a conceptual framework to address enzootic diseases like brucellosis. The vast majority of published brucellosis studies in the developing world rely solely on serology. An important shortcoming of brucellosis serology is the impossibility to infer which (smooth) Brucella spp. induced antibodies in the host. In this respect, mixed farming and especially raising small ruminants along with cattle, a common practice in the developing world, is reported to be a risk factor and a central question that has to be answered is whether cattle are infected with B. melitensis or with B. abortus or with both Brucella species. Therefore the isolation, identification and molecular characterization of Brucella spp. in human and the different livestock species needs to be undertaken to define a sound conceptual framework, identify the source of infection and plan appropriate control measures.

The epidemiological link between brucellosis in wildlife and brucellosis in livestock and people is widely recognised. When studying brucellosis in wildlife, three questions arise: (i) Is this the result of a spillover from livestock or a sustainable infection in one or more host species of wildlife? (ii) Does wildlife brucellosis represent a reservoir of Brucella strains for livestock? (iii) Is it of zoonotic concern? Despite their different host preferences, B. abortus and B. suis have been isolated from a variety of wildlife species, whereas B. melitensis is rarely reported in wildlife. The pathogenesis of Brucella spp. in wildlife reservoirs is not yet fully defined. The prevalence of brucellosis in some wildlife species is very low and thus the behaviour of individual animals, and interactions between wildlife and livestock, may be the most important drivers for transmission. Since signs of the disease are non-pathognomonic, definitive diagnosis depends on laboratory testing, i...

A cross-sectional investigation was made into the seroprevalence of brucellosis in camels in three arid and semi-arid camel-rearing regions of Ethiopia (Afar, Somali and Borena) between November 2000 and April 2001. When sera collected from 1442 accessible camels were screened with the Rose Bengal plate test (RBPT). 82 (5.7%) of them reacted. The results of a complement fixation test (CFT) on those sera that had given a positive reaction to the screening test then indicated a 4.2% prevalence of brucellosis in the tested camels. There was a significant difference in the prevalence of brucellosis (chi2 = 7.91, p < 0.05), which was highest in Afar (5.2%) followed by Somali (2.8%) and Borena (1.2%) regions. Camels in Afar had a four times higher risk of brucellosis with an odds ratio (OR) of 4.34 (confidence interval, CI = 1.76-10.72, p < 0.001) compared to the risk in Borena. Likewise, Afar had higher risk (OR = 1.76, 1.13-2.74, p < 0.05) than that in Somali. There was no sign...

Human brucellosis still presents scientists and clinicians with several challenges, such as the understanding of pathogenic mechanisms of Brucella spp, the identifi cation of markers for disease severity, progression, and treatment response, and the development of improved treatment regimens. Molecular studies have shed new light on the pathogenesis of Brucella spp, and new technologies have permitted the development of diagnostic tools that will be useful in developing countries, where brucellosis is still a very common but often neglected disease. However, further studies are needed to establish optimum treatment regimens and local and international control programmes. This Review summarises current knowledge of the pathogenic mechanisms, new diagnostic advances, therapeutic options, and the situation of developing countries in regard to human brucellosis.

Brucellosis control and eradication requires serological tests and vaccines. Effective classical vaccines (S19 in cattle and Rev 1 in small ruminants), however, induce antibodies to the O-polysaccharide of the lipopolysaccharide which may be difficult to distinguish from those resulting from infection and may thus complicate diagnosis. Rough attenuated mutants lack the O-polysaccharide and would solve this problem if eliciting protective immunity; the empirically obtained rough mutants 45/20 and RB51 have been used as vaccines. Strain 45/20 is reportedly unstable and it is not presently used. RB51 is increasingly used instead of S19 in some countries but it is rifampicin resistant and its effectiveness is controversial. Some controlled experiments have found good or absolute protection in adult cattle vaccinated orally (full dose) or subcutaneously (reduced dose) and in one field experiment, RB51 was reported to afford absolute protection to calves and to perform better than S19. Controlled experiments in calves, however, have shown reduced doses of RB51 to be ineffective, full doses only partially effective, and RB51 less effective than S19 against severe challenges. Moreover, other observations suggest that RB51 is ineffective when prevalence is high. RB51 is not useful in sheep and evidence in goats is preliminary and contradictory. Rough mutants obtained by molecular biology methods on the knowledge of the genetics and structure of Brucella lipopolysaccharide may offer alternatives. The B. abortus manB core (rfbK) mutant seems promising in cattle, and analyses in mice suggest that mutations affecting only the O-polysaccharide result in better vaccines than those affecting both core and O-polysaccharide. Possible uses of rough vaccines also include boosting immunity by revaccination but solid evidence on its effectiveness, safety and practicality is not available.

Aim: The study was carried out to determine the seroprevalence of Brucella antibody in slaughter cattle and goats; and the role of slaughterhouse workers (SHWs) in spread of Brucella infection during slaughterhouse operations in Enugu State, Southeast Nigeria.

Materials and Methods: Rose Bengal plate test was used to screen for Brucella antibody in 484 cattle and 340 goats slaughtered for human consumption in the state. Structured and pretested questionnaire was used to elicit information from randomly selected SHWs, on socioeconomic characteristics, awareness of brucellosis and involvement in practices that aid dissemination of Brucella infection during slaughterhouse operations.

Results: Suspected seroprevalence of 2.5% and 4.1% were recorded for Brucella antibody in cattle and goats respectively. There was poor awareness of brucellosis (32.1%) among the workers surveyed. Slaughterhouse practices that aid acquisition or spread of Brucella infection and percentage of SHWs engaged in the practices are: non-use of personal protective clothing during slaughterhouse operations (70.8%), discharge of eviscerated fetuses or pregnant uterine contents by open-air dump method of refuse disposal (64.9%) and illegal sell of eviscerated fetuses or gravid uterine contents for human consumption (59.9%) or preparation of dog food (71.5%).

Conclusion: The 4.1% suspected seroprevalence of Brucella antibodies in goats represents 128% increase from 1.8% seroprevalence earlier reported in the same species and study area in 2009. Significant amounts of Brucella antibody was detected in the food animals screened. Slaughterhouse workers played significant roles in spread of Brucella infection by their involvement in risk practices and behaviours that facilitate pathogen transmission. Therefore, massive awareness campaign and coordinated brucellosis control program in Enugu State are imperative to forestall the zoonotic and economic consequences associated with brucellosis.

In the present study 62 Aeromonas strains, isolated from different sources, were characterised by biochemical tests (Biolog system) and amplified 16S rDNA restriction analysis (ARDRA), previously proposed as a useful tool to identify Aeromonas species. Fifteen type strains and one reference strain were also analysed. The Biolog system ascribed most of the local isolates to A. media and A. veronii bv. sobria. Sixteen isolates were recorded as Aeromonas spp. ARDRA subdivided all analysed strains in 13 clusters. None of our isolates were attributed to A. sobria, A. schubertii, or to Aeromonas unnamed HG11 with both approaches. However, discrepancies were found between Biolog and 16S ARDRA species attribution of isolates. Moreover, total DNA restriction profile analysis on a selected group of isolates showed a low similarity among strains with identical ARDRA profiles. Therefore, sets of our data suggest that a single approach cannot be adequate for the identification of Aeromonas new isolates at the species rank and point out the need to apply a polyphasic approach, integrating phenotypic, phylogenetic (16S rDNA sequencing) and genomic (i.e. DNA/DNA hybridisation values) data, to define the taxonomic status of the genus Aeromonas.

Background and Aim: Brucellosis is a bacterial disease notorious for its ability to infect a wide range of domestic and wildlife animals, as well as humans. This study aimed to determine the seroprevalence of ovine and caprine brucellosis and the associated risk factors in the Province of Bam in Burkina Faso.

Materials and Methods: The individual serological status of 300 unvaccinated sheep and 300 unvaccinated goats was determined by Rose Bengal and indirect enzyme-linked immunosorbent assay (iELISA) serological tests used in parallel. The frequency of behaviors conferring risk of developing this zoonotic disease was determined through two epidemiological questionnaires, which identified known risk factors for the transmission of brucellosis between animals and humans.

Results: Individual seroprevalence was estimated at 6.0% (18/300) in sheep and 4.3% (13/300) in goats. The "herd" prevalence of brucellosis was estimated at 60% in sheep while 40% in goats. Positivity in the iELISA serological test was significantly associated with age, sex, and husbandry system in sheep and goats.

Conclusion: These results indicate that Brucella melitensis circulates in sheep and goat farms in the Province of Bam in Burkina Faso. As B. melitensis is highly pathogenic to humans, adequate measures must be taken to protect the population against this zoonotic disease.

Nowadays most of people buy milk in the markets. Prepared milk (in the market) ; the application of the pasteurization and UHT method. Raw milk should be kept in pasteurized after heating(boiling) at home as our mothers does. On the other hand, pasteurization at home by boiling can cause to be lost of vitamins. Therefore , we need to know how long can we keep raw milk and pasteurized milk at home paying sufficient attention being lack in having refrigerator or electricity.

Brucellosis is one of the major bacterial zoonoses worldwide. In the past decade, an increasing number of atypical Brucella strains and species have been described. Brucella microti in particular has attracted attention, because this species not only infects mammalian hosts but also persists in soil. An environmental reservoir may pose a new public health risk, leading to the reemergence of brucellosis. In a polyphasic approach, comprising conventional microbiological techniques and extensive biochemical and molecular techniques, all currently available Brucella microti strains were characterized. While differing in their natural habitats and host preferences, B. microti isolates were found to possess identical 16S rRNA, recA, omp2a, and omp2b gene sequences and identical multilocus sequence analysis (MLSA) profiles at 21 different genomic loci. Only highly variable microsatellite markers of multiple-locus variable-number tandem repeat (VNTR) analysis comprising 16 loci (MLVA-16) showed intraspecies discriminatory power. In contrast, biotyping demonstrated striking differences within the genetically homologous species. The majority of the mammalian isolates agglutinated only with monospecific anti-M serum, whereas soil isolates agglutinated with anti-A, anti-M, and anti-R sera. Bacteria isolated from animal sources were lysed by phages F1, F25, Tb, BK2, Iz, and Wb, whereas soil isolates usually were not. Rough strains of environmental origin were lysed only by phage R/C. B. microti exhibited high metabolic activities similar to those of closely related soil organisms, such as Ochrobactrum spp. Each strain was tested with 93 different substrates and showed an individual metabolic profile. In summary, the adaptation of Brucella microti to a specific habitat or host seems to be a matter of gene regulation rather than a matter of gene configuration.

Mongolia implemented a brucellosis livestock mass vaccination campaign from 2000 to 2009. However, the number of human cases did not decline since 2004 and the current epidemiological situation in Mongolia was uncertain. The objective of this study was to estimate the representative seroprevalences of humans and livestock in two provinces in view of their comparison with officially reported data. A representative cross-sectional study using cluster sampling proportional to size in humans, sheep, goats, cattle, yaks, horses, camels and dogs was undertaken to assess the apparent seroprevalence in humans and animals. A total of 8054 livestock and dog sera and 574 human sera were collected in Sukhbaatar and Zavkhan provinces. Human and animal sera were tested with the Rose Bengal and ELISA tests. The overall apparent seroprevalence of brucellosis was 27.3% in humans (95% CI 23.7-31.2%), 6.2% (95% CI 5.5-7.1%) in sheep, 5.2% (95% CI 4.4-5.9%) in goats, 16.0% (95% CI 13.7-18.7%) in cattle, 2.5% (95% CI 0.8-7.6%) in camels, 8.3 (95% CI 6.0-11.6%) in horses and 36.4% (95% CI 26.3-48.0%) in dogs. More women than men were seropositive (OR = 1.7; P < 0.0014). Human seroprevalence was not associated with small ruminant and cattle seroprevalence at the nomadic camp (hot ail) level. Annual incidence of clinical brucellosis, inferred from the seroprevalence using a catalytic model, was by a factor of 4.6 (1307/280) in Sukhbaatar and by a factor of 59 (1188/20) in Zavkhan. This represents a 15-fold underreporting of human brucellosis in Mongolia. The lack of access to brucellosis diagnostic testing at the village level hinders rural people from receiving appropriate treatment. In conclusion, this study confirms the high seroprevalence of human and livestock brucellosis in Mongolia. Stringent monitoring and quality control of operational management of a nationwide mass vaccination of small and large ruminants is warranted to assure its effectiveness. More research is needed to understand the complex animal-human interface of brucellosis transmission at different scales from farm to provincial level.

Brucella are facultative intracellular pathogens which have developed the capacity to survive and multiply in professional and nonprofessional phagocytes. The genetic basis of this aspect of Brucella virulence is still poorly understood. To identify new virulence factors, we have adapted signaturetagged transposon mutagenesis, which has been used essentially in animal models, to an in vitro human macrophage infection model. A library of 1,152 Brucella suis 1330 tagged mini-Tn5 Km2 mutants, in 12 pools, was screened for intracellular survival and multiplication in vitamin D 3 -differentiated THP1 cells. Eighteen mutants were identified, and their attenuation was confirmed in THP1 macrophages and HeLa cells. For each avirulent mutant, a genomic fragment containing the transposon was cloned. The genomic DNA sequence flanking the transposon allowed us to assign functions to all of the inactivated genes. Transposon integration had occurred in 14 different genes, some of which were known virulence genes involved in intracellular survival or biosynthesis of smooth lipopolysaccharide (the virB operon and manB), thus validating the model. Other genes identified encoded factors involved in the regulation of gene expression and enzymes involved in biosynthetic or metabolic pathways. Possible roles in the virulence of Brucella for the different factors identified are discussed.

A prospective study was carried out to elucidate the clinical, epidemiological and laboratory features of human brucellosis. A total of 26 948 blood samples (from adults aged 15 years and above) were screened for serological evidence of brucellosis over a period of 16 years. The slide agglutination/Rose Bengal plate agglutination test gave positive results in 517 patients, of which 509 had detectable titres by the standard tube agglutination test (SAT). The diagnosis of brucellosis was documented in 495 (1?8 %) patients based on diagnostic titres (¢1 : 160, 490 cases) and rising titres from insignificant titres (four cases) by serology and for one case by blood-culture isolation alone. Blood cultures were carried out in 345 cases, of which 191 cases (55?3 %) yielded Brucella melitensis. In 77/79 cases undertaken for follow up, there was a steady fall in 2-mercaptoethanol (2ME) agglutination titres along with clinical improvement (P <0?01). SAT titres remained detectable in most cases for a longer period in spite of an effective antimicrobial therapy and clinical recovery. A substantial number of patients (84?2 %) presented with fever, this being the only complaint in 51?1 % of the cases. Complications were present in 8?8 % of the patients (arthritis excluded): this included the unusual complications of hydrocele (two cases), Stevens-Johnson syndrome (one case) and urinary tract infection (one case). Brucella agglutinins were demonstrated in synovial, testicular, hydrocele and cerebrospinal fluids. There was no clinical suspicion of brucellosis in 439 cases (88?7 %) and the diagnosis was made only by routine serology. A two-drug regimen for 42-84 days with a follow-up 2ME test resulted in lower levels of relapse. These results suggest that, in endemic areas of the world, it should be mandatory to screen routinely for brucellosis due to protean clinical manifestations.

Following the recent discovery of new Brucella strains from different animal species and from the environment, ten Brucella species are nowadays included in the genus Brucella. Although the intracellular trafficking of Brucella is well described, the strategies developed by Brucella to survive and multiply in phagocytic and non-phagocytic cells, particularly to access nutriments during its intracellular journey, are still largely unknown. Metabolism and virulence of Brucella are now considered to be two sides of the same coin. Mechanisms presiding to the colonization of the pregnant uterus in different animal species are not known. Vaccination is the cornerstone of control programs in livestock and although the S19, RB51 (both in cattle) and Rev 1 (in sheep and goats) vaccines have been successfully used worldwide, they have drawbacks and thus the ideal brucellosis vaccine is still very much awaited. There is no vaccine available for pigs and wildlife. Animal brucellosis control strategies differ in the developed and the developing world. Most emphasis is put on eradication and on risk analysis to avoid the re-introduction of Brucella in the developed world. Information related to the prevalence of brucellosis is still scarce in the developing world and control programs are rarely implemented. Since there is no vaccine available for humans, prevention of human brucellosis relies on its control in the animal reservoir. Brucella is also considered to be an agent to be used in bio-and agroterrorism attacks. At the animal/ecosystem/human interface it is critical to reduce opportunities for Brucella to jump host species as already seen in livestock, wildlife and humans. This task is a challenge for the future in terms of veterinary public health, as for wildlife and ecosystem managers and will need a "One Health" approach to be successful.

Immunological cross-reactions between Brucella spp. and Ochrobactrum anthropi were investigated in ani- mals and humans naturally infected by Brucella spp. and in experimentally infected rams (Brucella ovis infected), rabbits (Brucella melitensis infected), and mice (B. melitensis and Brucella abortus infected). In the animals tested, O. anthropi cytosolic proteins evoked a delayed-type hypersensitivity reaction of a frequency and intensity similar to

Background: Brucella abortus, the major causative agent of abortion in cattle and a zoonotic pathogen, needs to be diagnosed at an early stage. Loop-mediated isothermal amplification (LAMP) test is easy to perform and also promising to be adapted at field level. Objective: To develop a LAMP assay for specific and rapid detection of B. abortus from clinical samples of cattle. Methods: LAMP primers were designed targeting BruAb2_0168 region using specific software tool and LAMP was optimized. The developed LAMP was tested for its specificity with 3 Brucella spp. and 11 other non-Brucella spp. Sensitivity of the developed LAMP was also carried out with known quantity of DNA. Cattle whole blood samples and aborted fetal stomach contents were collected and used for testing with developed LAMP assay and results were compared with polymerase chain reaction (PCR). Results: The developed LAMP assay works at 61 C for 60 min and the detection limit was observed to be 100-fold more than the conventional PCR that is commonly used for diagnosis of B. abortus. Clinical sensitivity and specificity of the developed LAMP assay was 100% when compared with Rose Bengal plate test and standard tube agglutination test. SYB Ò green dye I was used to visualize the result with naked eye. Conclusion: The novelty of the developed LAMP assay for specifically detecting B. abortus infection in cattle along with its inherent rapidness and high sensitivity can be employed for detecting this economically important pathogen of cattle at field level as well be exploited for screening of human infections.

The diagnostic value of enzyme-linked immunosorbent assay (ELISA) was evaluated when blood specimens of 92 patients suspected of brucellosis underwent the ELISA (IgM and IgG), standard tube agglutination (SAT) , and 2-mercaptoethanol (2-ME) tests and blood cultures; 38 sera from non-brucellosis patients and 34 sera from blood donors were also subjected to ELISA, SAT, and 2 -ME tests. SAT was able to pinpoint only 23 (25%), whereas ELISA confirmed the etiology in 56 (60.9%; P < 0.001) patients with brucellosis, including 31 culture-confirmed cases. The sensitivity and specificity of ELISA were 100% and 71.31%, respectively. Because they were confirmed by ELISA, the diagnosis could never be excluded with SAT in 33 cases. ELISA has been found to be more sensitive in acute (28% higher sensitivity; P < 0.02) and chronic (55% higher sensitivity; P < 0.01) cases. For accurate diagnosis in suspected brucellosis cases detection, we recommend both ELISA IgM and IgG tests. ELISA IgG and 2 -ME tests seem to be promising tools in judging prognosis.

One hundred and three milk samples were collected from 52 cows, 21 ewes, 18 goats and 12 camels. The animals tested positive to at least one of the following: (1) standard tube agglutination test (SAT); (2) Rose Bengal plate test (RBPT); (3) milk ring test (MRT). All milk samples were examined by culture and single-step polymerase chain reaction (PCR) techniques for detection of Brucella species. The PCR assay amplified Brucella-DNA from 29 bovine milk samples, 10 from sheep, 13 from goats and one from a camel. The direct culture method detected Brucella organisms from 24 samples of cows' milk, 12 from sheep, 10 from goats and failed to detect any Brucella organisms from camels' milk. PCR detected up to 100 colony forming units (CFU) of B. abortus per millilitre of milk in 100% of diluted milk samples, and 1000 CFU of B. melitensis from 70% of milk samples. Although the overall sensitivity of the PCR was higher than the culture method, it should be possible to increase the sensitivity to detect lower numbers of Brucella organisms in field samples. The speed and sensitivity of the PCR assay suggest that this technique could be useful for detection of Brucella organisms in bovine milk, as well as in sheep, goat, and camels milk.

Brucellosis is an occupational hazard of livestock farmers, dairy workers, veterinarians, slaughterhouse workers, and laboratory personnel, all of whom are considered to belong to the high-risk occupational group (HROG). A study was undertaken to determine the seroprevalence of brucellosis, identify risk factors associated with brucellosis seropositivity, and detect Brucella at genus level using real-time polymerase chain reaction (PCR) among people in the HROG in the Dhaka division of Bangladesh. A sample of 500 individuals from the HROG was collected from three districts of Dhaka division of Bangladesh. A multiple random effects logistic regression model was used to identify potential risk factors. Two types of real-time PCR methods were applied to detect Brucella genus-specific DNA using serum from seropositive patients. The prevalence of brucellosis based on the three tests was observed to be 4.4% based on a parallel interpretation. The results of the multiple random effects logistic regression analysis with random intercept for district revealed that the odds of brucellosis seropositivity among individuals who had been in contact with livestock for more than 26 years was about 14 times higher as compared to those who had less than 5 years of contact with livestock. In addition, when the contact was with goats, the odds of brucellosis seropositivity were about 60 times higher as compared to when contact was with cattle only. Noticeable variation in brucellosis seropositivity among humans within the three districts was noted. All of the 13 individuals who tested positive for the serological tests were also positive in two types of real-time PCR using the same serum samples. Livestock farmers of brucellosis positive herds had a significantly higher probability to be seropositive for brucellosis. The study emphasized that contact with livestock, especially goats, is a significant risk factor for the transmission of brucellosis among individuals in the HROG.

Brucella-specific nucleotide sequences encoding the BCSP 31 kDa protein, Omp2 and the 16S rRNA were employed in three independent diagnostic PCR assays. Results of the three PCR assays on six reference strains of Brucella were in complete agreement. The results of PCR assays based on bcsp and omp2 on 19 Indian field isolates (human, bovine and murine tissues) also agreed completely. However, when the 16S rRNA gene was employed as the diagnostic target in the PCR, only 14 out of these 19 isolates and 2 out of 7 bovine milk isolates were identified as the genus Brucella. The bovine blood samples were insensitive to 16S rRNA PCR. The antibody-detecting ELISA results of field samples (n587) from a serologically positive herd in India were compared separately with omp2 and bcsp PCRs of blood (n562). While the bcsp PCR was the most sensitive, the degree of association of ELISA with omp2 blood PCR (k50.37 at P ,0.05) was similar to that with the bcsp blood PCR (k 50.34 at P ,0.05). An improvement in the correlation between ELISA and blood PCR was noticed (k 50.5 at P ,0.05) when a consensus result of omp2 and bcsp blood PCR was considered for comparison with ELISA. The use of more than one marker-based PCR gave increased sensitivity and higher specificity and appears to be a more reliable molecular diagnostic approach for screening of field animals.

A total of 17,359 samples were analysed serologically, of which 1,061, 15,758 and 585 samples were from Makerere, Entebbe and Tororo laboratories, respectively, were used to determine the seroprevalence of brucellosis. The overall seroprevalence of brucellosis was 10% while from individual laboratories was 38%, 32% and 7% for Makerere, Entebbe and Tororo laboratories, respectively. Majority of these positive brucellosis test results were in the cattle corridor with P value=0.399. There were significant differences in brucellosis seroprevalence among species (P value=0.014). The trends of brucellosis seroprevalence among the different species were decreasing with time but were highest in bovine species (P value=0.043). Brucellosis seroprevalence had a bimodal monthly pattern corresponding with rainfall. The study showed that brucellosis was prevalent, though the trend of the disease has declined over years. It was recommended that regular disease surveillance, control programmes and further studies be carried out in the country.

This study aimed to investigate the prevalence of human brucellosis in Najran, southwestern Saudi Arabia, and to assess the performances of ELISA and PCR as diagnostic tools for brucellosis with respect to conventional methods. The study included 340 patients with clinical characteristics of brucellosis. Blood samples from cases and controls were subjected to culture, standard tube agglutination test (SAT), ELISA for IgM and IgG, and brucella PCR. The diagnosis of brucellosis was confirmed in 54 (15.9%) of the 340 provisionally diagnosed brucellosis patients. Blood culture identified only 14 (25.9%) cases. The SAT was positive for 50 (92.6%) cases, whereas ELISA IgM, IgG and PCR were found positive in 46, 52 and 38 cases respectively. The sensitivities of ELISA IgM and IgG were 85.2% and 96.3% respectively and the specificity was 100% for each. For PCR, the sensitivity and specificity were 70.4% and 100% respectively. In conclusion, ELISA offers a significant advantage over conventional serological methods in the diagnosis of brucellosis in endemic areas. The PCR test results can be particularly important in patients with clinical signs and symptoms, and negative serological results, allowing the early and rapid confirmation of the brucellosis.

Isolation of brucellae from aborted fetuses, hygroma fluids, milk and vaginal swabs obtained from aborting cattle, sheep, goats, pigs, and horses in Nigeria was carried out. A total of 25 isolates, obtained mainly from cattle, sheep and horses, were biotyped. All strains belonged to one species, Brucella abortus biovar 1. The epidemiological significance of this finding is discussed. Some preliminary observations on the zoonotic and public health implications of Brucella infection in Nigerian livestock are presented. A control programme involving improved management, animal movement restrictions, public health education and mass vaccination of animals is suggested. # 2004 Published by Elsevier B.V.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is a rare syndrome that is often fatal despite treatment. It is caused by a dysregulation in natural killer T-cell function, resulting in activation and proliferation of histiocytes with uncontrolled hemophagocytosis and cytokines overproduction. The syndrome is characterized by fever, hepatosplenomegaly, cytopenias, liver dysfunction, and hyperferritinemia. HLH can be either primary, with a genetic aetiology, or secondary, associated with malignancies, autoimmune diseases, or infections.

Brucella melitensis and B. ovis outer membrane blebs contained a protein displaying a temperature-dependent molecular mass upshift from 25 kDa to 30 kDa. A fraction of the protein tightly bound to LPS did not show the molecular mass upshift which was also blocked by exposure of the protein to Zwittergent 314. The B. melitensis heat-modifiable protein and Escherichia coli OmpA shared antigenic determinants. These data indicate that the Brucella group 3 outer membrane proteins belonged to the OmpA-family of proteins.

Following the recent discovery of new Brucella strains from different animal species and from the environment, ten Brucella species are nowadays included in the genus Brucella. Although the intracellular trafficking of Brucella is well described, the strategies developed by Brucella to survive and multiply in phagocytic and non-phagocytic cells, particularly to access nutriments during its intracellular journey, are still largely unknown. Metabolism and virulence of Brucella are now considered to be two sides of the same coin. Mechanisms presiding to the colonization of the pregnant uterus in different animal species are not known. Vaccination is the cornerstone of control programs in livestock and although the S19, RB51 (both in cattle) and Rev 1 (in sheep and goats) vaccines have been successfully used worldwide, they have drawbacks and thus the ideal brucellosis vaccine is still very much awaited. There is no vaccine available for pigs and wildlife. Animal brucellosis control strategies differ in the developed and the developing world. Most emphasis is put on eradication and on risk analysis to avoid the re-introduction of Brucella in the developed world. Information related to the prevalence of brucellosis is still scarce in the developing world and control programs are rarely implemented. Since there is no vaccine available for humans, prevention of human brucellosis relies on its control in the animal reservoir. Brucella is also considered to be an agent to be used in bio-and agroterrorism attacks. At the animal/ecosystem/human interface it is critical to reduce opportunities for Brucella to jump host species as already seen in livestock, wildlife and humans. This task is a challenge for the future in terms of veterinary public health, as for wildlife and ecosystem managers and will need a "One Health" approach to be successful.

Brucellosis is a worldwide zoonosis leading to tremendous economic losses and severe human illness. Fast and reliable laboratory tests are needed to detect disease in both humans and animals and to monitor the production of safe food products and feed. For rapid identification of the genus Brucella and differentiation of its species, a multiplex polymerase chain reaction microarray assay based on 11 signature sequences and redundant oligonucleotide probes was developed. The gene targets included genus-specific sequences in bcsp31, perA, cgs, and omp2b, as well as chromosomal regions displaying species-specific hybridization patterns. Brucella reference strains and a representative panel of 102 field isolates were unambiguously identified by their hybridization patterns. The differentiation of species, however, was limited in members of the groups B. suis bv 3/4/B. canis and B. neotomae/B. microti. In summary, the newly developed Brucella ArrayTube® assay is an easy-to-handle molecular test for high-throughput and parallel analysis.

Brucellosis has been in South East Asia for many generations affecting both man and animals and responsible for massive economic losses. The threat to the economy and to public health from this zoonotic disease is so great to ignore. Over the years there have been many attempts to control and possibly eradicate this infection but these efforts have not fully yielded the desired results due to many factors mentioned in this paper. Eradication, though expensive, actually saves a lot of economic resources when properly implemented. In this review, the past and current situation of brucellosis in South East Asia is explored with particular reference to Malaysia and challenges to the full eradication of the infection are elucidated.

Two slow-growing, Gram-negative, non-motile, non-spore-forming, coccoid bacteria (strains F60 T and F965), isolated in Austria from mandibular lymph nodes of two red foxes (Vulpes vulpes), were subjected to a polyphasic taxonomic analysis. In a recent study, both isolates were assigned to the genus Brucella but could not be attributed to any of the existing species. Hence, we have analysed both strains in further detail to determine their exact taxonomic position and genetic relatedness to other members of the genus Brucella. The genome sizes of F60 T and F965 were 3 236 779 and 3 237 765 bp, respectively. Each genome consisted of two chromosomes, with a DNA G+C content of 57.2 %. A genome-to-genome distance of .80 %, an average nucleotide identity (ANI) of 97 % and an average amino acid identity (AAI) of 98 % compared with the type species Brucella melitensis confirmed affiliation to the genus. Remarkably, 5 % of the entire genetic information of both strains was of non-Brucella origin, including as-yet uncharacterized bacteriophages and insertion sequences as well as ABC transporters and other genes of metabolic function from various soil-living bacteria. Core-genome-based phylogenetic reconstructions placed the novel species well separated from all hitherto-described species of the genus Brucella, forming a long-branched sister clade to the classical species of Brucella. In summary, based on phenotypic and molecular data, we conclude that strains F60 T and F965 are members of a novel species of the genus Brucella, for which the name Brucella vulpis sp. nov. is proposed, with the type strain F60 T (5BCCN 09-2 T 5DSM 101715 T). Abbreviations: AAI, average amino acid identity; ANI, average nucleotide identity. The GenBank/EMBL/DDBJ accession numbers for the genome sequences (chr1 and chr2) of strains F60 T and F965 are LN997863-LN997864 and LN998033-LN998034, respectively, and can be retrieved from the European Nucleotide Archive under

brucellosis; Brucella spp.; ancient DNA; cavitating lytic vertebral lesions ABSTRACT Ancient skeletal remains can harbor unique information about past civilizations at both the morphological and molecular levels. For instance, a number of diseases manifest in bone, some of which have been confirmed through DNA analysis, verifying their presence in ancient populations. In this study, anthropological analysis of skeletal remains from the ancient Albanian city of Butrint identified individuals with severe circular lytic lesions on their thoracic and lumbar vertebrae. Differential diagnosis suggested that the lesions resulted from pathologies known to affect these skeletal regions, such as tuberculosis (TB) or brucellosis. Relevant bones of two adolescent males from the 10th to 13th century AD that displayed the lesions, along with unaffected individuals, were collected in the field.

Background: Brucellosis is regarded as one of the major zoonotic infections worldwide. It was first reported in Egypt in 1939 and is now endemic, the predominate species of Brucella in cattle and buffalo in Egypt is B. melitensis. The aim of the study was to estimate seroprevalence of Brucella spp. in cattle and buffalo reared in households in an Egyptian village, identify risk factors for animals testing seropositive and to assess the knowledge, attitudes and practices (KAPs) of livestock owners with regards to brucellosis. Methods: A cross-sectional study was carried out in a village in Menufiya Governorate of Egypt. In June and July 2009, 107 households were selected using systematic sample and all lactating cattle and buffalo present in the household were sampled and tested for antibodies against Brucella spp. In addition, a questionnaire collecting information on potential risk factors for Brucella spp. infection in cattle and buffalo was administered to the household member responsible for rearing the livestock. Between December 2009 and February 2010 households were revisited and a second questionnaire regarding KAPs associated with brucellosis was administered. Results: True individual and household seroprevalence were estimated to be 11.0% (95% CI: 3.06% to 18.4%) and 15.5% (95% CI: 6.61% to 24.7%), respectively. Cattle and buffalo kept in a household with sheep and goats had 6.32 (95% CI: 1.44 to 27.9) times the odds of testing seropositive for Brucella spp., compared to cattle and buffalo that were not. Most participants in the study stated that livestock owners assist in the parturition of ruminants without wearing gloves and that some farmers sell animals which they suspect are Brucella infected to butchers or at market. Many participants made their livestock's milk into cheese and other dairy products without pasteurising it. Conclusions: Brucellosis was endemic at high levels, in the current study. Although livestock owners had good general knowledge of brucellosis, they still appeared to participate in high-risk behaviours, which may contribute to the high seroprevalence in the area. Veterinarians, public health authorities and other community leaders need to collaborate to control the disease in animals and to manage the risk of human exposure.

Members of the genus Brucella are worldwide pathogens of wildlife and livestock and are the most common zoonotic infection in humans. In general, Brucella exhibit a range of hostspecificity in animals that has led to the identification of at least seven Brucella species. The genomes of the various Brucella are highly conserved, which makes differentiation of species highly challenging. However, we found single-nucleotide polymorphisms (SNPs) in housekeeping and other genes that differentiated the seven main Brucella species or clades and thus, enabled us to develop Real-Time PCR assays around these SNPs. Screening of a diverse panel of 338 diverse isolates with these assays correctly identified each isolate into its previously determined Brucella clades. Six of the seven clade-specific assays detected DNA concentrations of less than 10 femtograms, indicating a high level of sensitivity. This SNP-based approach places samples into a phylogenetic framework, allowing reliable comparisons among lineages of clonal bacteria and provides a solid basis for genotyping. These PCR assays provide a rapid and highly sensitive method of differentiating the major Brucella groups that will be valuable for clinical and forensic applications.

Vaccination is the most important approach to counteract infectious diseases. Thus, the development of new and improved vaccines for existing, emerging, and re-emerging diseases is an area of great interest to the scientific community and general public. Traditional approaches to subunit antigen discovery and vaccine development lack consideration for the critical aspects of public safety and activation of relevant protective host immunity. The availability of genomic sequences for pathogenic Brucella spp. and their hosts have led to development of systems-wide analytical tools that have provided a better understanding of host and pathogen physiology while also beginning to unravel the intricacies at the host-pathogen interface. Advances in pathogen biology, host immunology, and host-agent interactions have the potential to serve as a platform for the design and implementation of better-targeted antigen discovery approaches. With emphasis on Brucella spp., we probe the biological as...

Brucellosis is a systemic infectious disease caused by Gram-negative bacilli, the genus Brucella, and clinical features are diverse. Therefore, several infectious and non-infectious diseases are considered in its differential diagnosis. In this study, we aimed to determine the positivity rate of Brucella agglutination tests in the culture-positive brucellosis and in diseases mimicking brucellosis clinically.