OM

ICS

Gro

up e

Boo

ks

001

Bacterial and Mycotic Infections in Immunocompromised Hosts: Clinical and Microbiological Aspects

Edited byMaria Teresa Mascellino

www.esciencecentral.org/ebooks

OM

ICS

Gro

up e

Boo

ks

002

Brucellosis: A Global Re-emerging Zoonosis History, Epidemiology, Microbiology, Immunology and Genetics

Abbreviations: SB.: Brucella; WHO: world health organization; FAO: food and agriculture organization; WOAH: world organization for animal health; MME: mediterranean and middle east; CDC: centers for disease control and prevention; LPS: lipopolysaccharide; TGF: transforming growth factor; MHC: major histocompatibility complex; IFN: interferon; IL: interleukin; TNF: tumor necrosis factor; NK cells: natural killer cells; GM-CSF: granulocyte monocyte-colony stimulating factor; MLVA: multiple-locus variable- number tandem-repeat analysis; MALDI-FOS-MS: mixed-assisted laser desorption/ionization time-of-flight mass spectrometry; HOOF-Prints: hypervariable octameric oligonucleotide finger-prints; BBP: Brucella bioinformatics portal; STM: signature-tagged transposon mutagenesis.

IntroductionBrucellosis is an infectious disease of animals (zoonosis) that is transmittable to humans. Wild and domestic animals are

the only source of brucellosis and humans are always accidental hosts [1]. At least 6 Brucella species have been identified, 4 of which are human pathogens (Table 1) [1,2]. Brucella does not bear classic virulence factors and they tend to invade and persist in human hosts [2].

The infection is global in distribution and it is endemic in many geographic locations including the Mediterranean and Middle East (MME) regions, Indian subcontinent, Mexico and parts of Central and South America [3-5]. Approximately 60% of emerging human pathogens are zoonotic and brucellosis is considered the commonest zoonotic infection worldwide [4,6-10]. The global map of human brucellosis has significantly changed over the past decade as the infection keeps re-emerging in many foci worldwide [3,4,7,9]. In developing countries, brucellosis is an important public health problem that is associated with minimal mortality and substantial residual morbidity [7,10]. However in the era of globalization and international tourism, brucellosis has become a common imported disease in the developed world [7,9].

Landmarks in The History of BrucellosisIn the year 1853, Jeffery Allen Marston made the first accurate description of the disease in British army troops serving

in Malta during the Crimean war [1]. In 1887, David Bruce isolated Gram negative coccobacilli, named later Brucella melitensis (B. melitensis), from spleens of fatal cases. In 1895, Bernard Bang isolated B. abortus from placental tissues of cattle. In 1897, M.L. Hughes published a review on the clinical and pathological features of the disease and suggested the name undulant fever (Table 1) [1-3]. In 1897, Wright and Semple succeeded in applying serum agglutination method for differentiating brucellosis from other febrile illnesses. In 1904, the Commission of Mediterranean Fever was established [1]. Between 1904 and 1907, several reports on epidemiology, bacteriology and pathology of brucellosis were published.

Al-Anazi KA* and Al-Jasser AMConsultant Hemato-Oncologist, Department of Adult Hematology and Hematopoietic Stem Cell Transplant, Oncology Centre, King Fahad Specialist Hospital, P.O. Box: 15215, Dammam 31444, Saudi Arabia

*Corresponding author: Khalid Ahmed Al-Anazi, Consultant Hemato-Oncologist, Department of Adult Hematology and Hematopoietic Stem Cell Transplant, Oncology Centre, King Fahad Specialist Hospital, P.O. Box: 15215, Dammam 31444, Saudi Arabia, Tel: 966–03–8431111; Fax: 966–03–8427420; E-mail: kaa_alanazi@yahoo.com

Species Biotype Animal Hosts First Description Human VirulenceB. melitensis 1-3 Goats, sheep and camels Bruce, 1887 HighB. abortus 1-6, 9 Cows, camels, yaks and buffalos Bang, 1897 Intermediate

B. suis 1-5 Pigs, wild hares, caribou, reindeer, wild rodents Traum, 1914 Low B. canis - Dogs Carmichael and Bruner, 1968 LowB. ovis - Sheep Van Drimmelen, 1953 None

B. neotomae - Rodents Stoenner and Lackman, 1957 NoneB. pinnipediae and

B. cetaceae (provisional) - Minke whales, dolphins, porpoises and seals Ewart and Ross 1994 None

Table 1: Basic Microbiological Details of Brucella Species.

OM

ICS

Gro

up e

Boo

ks

003

In 1905, Themistocles Zammit identified a Maltese goat as the animal host of brucellosis. In 1918, Alice Evans published data on the antigenic relatedness between B. melitensis and B. abortus. Subsequently, the genus was named Brucella to honor David Bruce. In 1924, human infection with B. abortus was documented by Orpen in the U.K. Similar studies on B. abortus were performed by Morales-Otero in Puerto Rico [1]. In 1914, Jacob Traum isolated B. suis from an aborted swine fetus (Table 1) [1-3]. In 1909, Hutyra and Marek might have recovered the organism in Hungary [1]. In 1953, van Drimmelen made identification of B. ovis in sheep. In 1957, Stoenner and Lackman identified B. neotomae in rodents (Table 1) [2]. In 1964, Carmichael and Bruner identified B. canis in the canines [1,2]. In 1994, Ewalt, Ross and colleagues identified B. pinnediae and B. cetacear (provisionally) in Minke whales, dolphins, porpoises as well as seals (Table 1) [1,2]. In 1979, the WHO (world health organization) established a specialized program with a unit coordinating and managing activities (The Mediterranean Zoonoses Control Centre) operating from Athens in Greece [4]. In 1986, the WHO and FAO (food and agriculture organization) recommended treatment for acute brucellosis in adults with a combination regimen composed of rifampicin and doxycycline orally for 6 weeks [5-7]. In 2004, WHO/FAO/WOAH (world organization for animal health) joint consultation on emerging zoonotic diseases held in Geneva defined an emerging zoonosis as a pathogen that is newly recognized or newly evolved or that has occurred previously but shows an increase in incidence or expansion in geographical, host or vector range [8]. In November 2006, a panel of international experts met in Ioannina in Greece and they made a number of therapeutic recommendations that included treatment of uncomplicated brucellosis using a combination of oral doxycycline for 6 weeks and parenteral streptomycin for 2 to 3 weeks or oral rifampicin for 6 weeks [7].

Epidemiology of BrucellosisIt is estimated that 60% of emerging human pathogens are zoonotic [8]. Brucellosis is the commonest zoonotic infection

worldwide as it has been reported in 56 countries [4]. Also, more than 500,000 new cases of brucellosis are reported annually [9]. However, the reported annual incidence rate of human brucellosis in endemic areas worldwide varies from <0.01 to >200 cases per 100,000 population. In the year 2005, the annual incidence rate per 100,000 population was: 160.30 in Syria, 60.60 in Mongolia, 26.20 in Turkey and 21.40 in Saudi Arabia [10].

The epidemiology of human brucellosis has dramatically changed over the last 25 years, because of various sanitary, socioeconomic and political reasons in addition to the evolution of international travel in the era of globalization. Several areas that had been traditionally considered to be endemic such as France and Latin America, have achieved control of the disease [9]. In certain countries such as Portugal, Spain, Tunisia and Jordan, the incidence of brucellosis has decreased significantly. On the contrary, the incidence is on the rise in countries like Turkey, Algeria in addition to several areas in South Europe and Africa, while the situation in other countries such as Syria has been rapidly worsening. Despite the adopted control measures, the disease is still present, in varying trends, both in Europe and in the USA [9]. MME regions are considered the most important areas for the historic development and the concentration of zoonoses. Twenty five countries in the Middle East keep record of the disease and 6 of them report 90,000 cases per year [4].

Brucellosis is a notifiable disease in most countries [11]. However, brucellosis cases remain often unrecognized and underestimation of cases is clearly seen reflecting inadequacy of diagnostic laboratory services in most of the MME countries [4,12]. Social, technological and environmental factors continue to have a dramatic effect on infectious diseases worldwide, facilitating the emergence of new diseases and the re-emergence of old ones sometimes in drug resistant forms. For brucellosis, the geographical pattern is constantly changing with new foci emerging and re-emerging. The causes and explanations for the resurgence and the recent increase in the incidence of brucellosis include: (1) socioeconomic changes, (2) wars and political turbulence in some MME countries, (3) inadequate control programs in some countries, (4) ease of human international travel recently, (5) uncontrolled animal transportation across open borders and (6) brucellosis being a complex disease that has different cycles of expansion and regression [12-15].

The risk factors for Brucella infection include: consumption of raw milk and unpasteurized dairy products, direct contact with animals and their products, male sex and age between 40 and 49 years [10,11,16-20]. Brucellosis is an occupational disease that poses risk to: shepherds, abattoir workers, veterinarians, diary-industry professionals and personnel in microbiology laboratories [18-20].

Brucella strains have been isolated from terrestrial and marine mammals. Experimentally, a marine mammal Brucella species isolated from a pacific harbor seal has induced seroconversion and abortion in cattle. Recently, marine mammals Brucellae have been identified in 2 patients from Peru with neurobrucellosis and intracerebral granulomas [20].

Pathogenicity and ImmunologyThe pathogenicity of human brucellosis is attributed to factors such as lipopolysaccharide (LPS), adenine and guanine

monophosphate, vitamin B, 24 KDa protein and urease enzyme [3]. Brucellae may enter the host via ingestion, inhalation or through conjunctiva or skin abrasion. They colonize in different body organs with predilection for lymphoreticular system [3]. Brucella species that are pathogenic to humans can be arranged in a decreasing order of virulence as follows: (1) B. melitensis (2) B. suis (3) B. abortus (4) B. canis (Table 1). The first three species are classified as smooth organisms based on the length of their LPS O-side chains, while B. canis is a naturally occurring rough strain [21]. The severity of brucellosis is variable, ranging from lethal to subclinical in humans as in most cases Brucellae establish long-term parasitic relationships with their human hosts. Survival and replication of Brucellae in macrophages is critical for maintenance of such chronic infections [21]. Survival of Brucellae within monocytes is the single most important aspect of pathogenesis contributing to persistence of bacteria in host tissues [22].

OM

ICS

Gro

up e

Boo

ks

004

Virulence of Brucella species depends on survival and replication properties in the host cells. Brucella has developed specific strategies to influence antigen presentation mediated by cells and an evolutionary stealthy strategy to escape recognition by the innate immunity. It has also modulated not only the adaptive immunity of the host but also the signaling events during host adaptive immune response [23]. Brucella modulates both (the innate and the adaptive) functional arms of the human immune system leading to T-cell anergy and chronic infection [24]. Brucella periplasmic cyclic β-1,2-glucan plays an important role during bacterium-host interaction [25]. Cyclic β-1,2-glucan must be transported into the periplasmic space to exert its action as a virulence factor [26]. However, cyclic β-1,2-glucan succinylation is not required for virulence and no low-osmotic stress conditions must be overcome during infection [25].

In the past decade, the mechanisms of Brucella pathogenesis and host protective immunity against Brucella infections have been extensively investigated using the cutting edge systems biology and bioinformatics approaches [27]. Integrative experimental Omics and computational bioinformatics analyses have dramatically advanced our understanding of how Brucella species infect different host species, how Brucella gene expressions are regulated in cell cultures or inside host cells and how host cells respond to Brucella infections [27]. Acquired immunity to brucellosis has been studied through observations of naturally infected hosts e.g. cattle and goats, mouse models and human infection. New systems biology analyses of antigens recognized by human innate responses in brucellosis have identified large numbers of protein antigens with the potential of understanding mechanisms of pathogenesis and immune evasion and may point the way toward novel vaccines and diagnostic approaches [28].

CD80/CD28 costimulation enhances the interaction of antigen / major histocompatibility complex (MHC) and is critical for adequate induction and maintenance of the Th1 response [24,29]. In humans, brucellosis is characterized by an intense Th1cytokine production with strikingly high serum levels of interferon (IFN)-γ and evidence of defective monocyte function [30]. Although CD4 and CD8 cells are closely involved in the production of IFN-γ and despite that CD8 T cells may be cytotoxic, the role of natural killer (NK) cells and cytotoxicity in protective immunity to brucellosis have not been substantiated experimentally. Also, antibodies have been shown to have a limited role in passive transfer studies [31]. Although infected macrophages may persist in the presence of Brucella-specific T cells, CD8 T cells have been shown to have an important role in clearance of Brucellae following the peak of infection and may act by lysing chronically infected macrophages [32]. NK cells may be capable of modulating the development of Brucella infection in human beings by lysing infected host cells. Chronicity or elimination of Brucella infection depends upon the balance between the contradictory effects induced by the bacteria that favor either the host or the pathogen [31,33]. CD4 (+) invariant NK T cells have antibacterial activity and participate directly in the elimination of bacteria and/or in the control of bacterial growth by killing infected cells. These cells inhibit intramacrophagic growth of Brucellae by different mechanisms [34].

Human Vγ9Vδ2 T cells play a crucial role in the early immune response to intracellular pathogens. In brucellosis, these cells are drastically increased in the peripheral blood during the acute phase of infection. They are able to use a combination of mechanisms that reduce the total numbers of B. suis thus they may benefit the host by limiting the spread of Brucella species [35]. Interleukin (IL)-37 is a soluble factor responsible for part of the bactericidal activity of Vγ9Vδ2 T cells [36]. Murine and bovine γ/δ T cells are important for early protection against B. abortus infections [37]. These cells increase considerably in acute brucellosis and decrease significantly following efficient treatment. Hence, γ/δ T cell receptor bearing cell counts may be used as a supplementary marker for monitoring brucellosis [38].

An important feature of brucellosis is the persistence of bacterial colonization in cells of the reticuloendothelial system [39]. Chronicity of brucellosis results from the ability of some Brucellae to survive the reactive oxygen intermediate and the nitric oxide killing in the host phagocytes, following which they activate bacterial genes in response to the acidic phagosome environment, prevent fusion of phagolysosomes by remodeling the intracellular compartment and subsequently replicate intracellularly. The crucial component of immunity that results in survival of the host and thus maintenance of the chronic infective state is IFN-γ [31]. The interrelationship between different cells must be taken into consideration in the analysis of bacterial virulence and in the development of in vitro models of human macrophage infection [40].

The proper use of animal models, particularly mice, has recently allowed accumulation of valuable data regarding pathogenicity, immunology and antibiotic susceptibility of Brucella species in vivo. New technologies in mouse genetics are likely to bring about even greater insights into the interactions between Brucella species and the immune system that lead to evolution of the disease in humans [41,42]. The shut-down of response to IFN-γ may be necessary for survival of B. abortus in the short term and the lack of endogenous IFN-γ is more important to control brucellosis than CD8+ T cells and IL-12-dependent IFN-γ deficiencies [43,44]. Production of IL-12 and tumor necrosis factor (TNF)-α usually occurs early in intracellular bacterial infection. These cytokines contribute to resistance of the intracellular bacterium B. abortus by different mechanisms, while IL-10 may downregulate the immune response to B. abortus by affecting both the macrophage effector function and the production of protective Th1cytokine IFN-γ [45,46]. Once the infection has been established, B. abortus 2308 selectively limits IL-18 secretion without affecting endogenous IFN-γ production [47]. However, the virulent smooth strain B. abortus S2308 causes more apoptosis and necrotic dendritic cell death than the live-attenuated vaccine strain RB51 [48]. In murine models having chronic brucellosis, cytokine production is characterized by: prominent IL-6 production, increased IFN-γ production and substantial production of granulocyte monocyte-colony stimulating factor (GM-CSF) [49].

Genetics and Genomics in BrucellosisBrucella species are highly monomorphic with minimal genetic variation among species thus hindering the development

OM

ICS

Gro

up e

Boo

ks

005

of reliable tools for epidemiologic and phylogenetic analyses. Current microbiological and low-resolution molecular typing assays are useful for identifying Brucella isolates and determining species and biovar designations, but have limited value for epidemiological trace-back investigations [50]. Multiple-locus variable-number tandem-repeat analysis (MLVA) targets multiple repeat regions with higher mutation rates than other genomic markers and demonstrates higher genetic resolutions when applied to Brucella species than assays targeting other more monomorphic molecular markers such as the outer membrane proteins, the rpoβ gene and the insertion sequence regions [50]. Thus, MLVA is a useful tool for identifying and genotyping Brucella strains and the resultant data can be used for epidemiological trace-back investigations [51]. MLVA-16 has a good discriminatory power for species determination, typing of B. melitensis isolates and inferring their geographic origin [52]. It can be considered a valid alternative to standard genotyping techniques and is particularly useful in dealing with a large number of samples in the short term [53]. The correlation found among B. melitensis groups and rpoβ types suggests that study of the single nucleotide polymorphisms (SNPs) of rpoβ as an initial marker of brucellosis may be of use in epidemiological surveillance [54]. Brucella hypervariable octameric oligonucleotide finger-prints (HOOF-Prints) technique is highly discriminatory among Brucella species, among previously characterized Brucella strains and among unrelated field isolates that cannot be differentiated by classical methods [55]. For human Brucella isolates, both MLVA and HOOF-Print assays are rapid, highly discriminatory and reproducible. They significantly contribute to Brucella epidemiology and may advance surveillance and control of human brucellosis [55,56]. Also, mixed-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-FOS-MS) is a rapid method for the analysis of biological samples. The accurate identification of Brucella species can be achieved with MALDI-FOS-MS by constructing a Brucella reference library based on the genetic relationships according to MLVA data [57].

The Brucella bioinformatics portal (BBP) is a gateway for Brucella researchers to search, analyze and curate Brucella genome data originating from public databases and medical literature. Brucella gene mutations and genetic interactions are annotated using Limix leading to better understanding of Brucella pathogenesis [58]. The sequence database provides a powerful dataset for addressing ongoing controversies in Brucella taxonomy and a tool for unambiguously placing atypical, phenotypically discordant and newly emerging Brucella isolates [59]. The identification of novel genes within previously described groups has added insight with regard to the regulatory elements, nutritional demands and mechanisms required for efficient intracellular growth and survival of the organism [60]. Four genes of B. suis that are necessary to resist specifically the action of γ9δ2 T cells have been identified. B. suis that induces chronic disease in humans might have developed specific strategies to subvert the immune system at the level of the innate secondary response [61].

Transposon mutagenesis is the most frequently used approach in the identification of genes involved in the virulence of bacterial pathogens [62]. A recent modification of the classical transposon mutagenesis technique, signature-tagged transposon mutagenesis (STM), allows the detection of a given mutant within a complex pool of mutants by hybridization with a probe obtained by polymerase chain reaction (PCR) with primers based on constant regions [62]. Very little is known about the genetic basis of Brucella virulence. However, stress response proteins and smooth LPS may be required for virulence in vitro and in animal models. A two-component system (Bvr AS) and a type IV secretion system (VirB) have recently been identified as essential virulence factors. More recently, the application of STM has allowed identification of B. suis genes affecting intracellular survival in an in vitro human macrophage infection model [62]. The Brucella genome contains an IS 711 transposon element that is often used in finger printing Brucella species samples. While B. suis 1330 and B. suis ATCC 23445 contain 7 and 13 IS71 copies respectively, B. suis VBI 22 has 8 copies. All 7 IS 711 loci in B. suis 1330 genome are observed in the genomes of ATCC 23445 and VBI 22 strains. B. suis VBI 22 has an additional IS 711 locus right after the stop codon of the BSB 122_A1627 gene, which has not yet been previously observed in any sequenced Brucella species [63]. Obtaining the complete Brucella genome and identification of the global expression genetic profile of Brucella species will be a great step in understanding: biology and evolution of the pathogen, virulence of the organism and the interaction between Brucella and its host at the molecular level in order to improve the development of vaccines and new antimicrobial therapies [64,65]. The STM technique is a powerful method that allows a large number of mutants to be screened for attenuation. The isolation of attenuated mutants in virB operon and manB, that are known Brucella virulence factors, has validated the application of STM in Brucella virulence studies [62].

The rough strains RB51, RB115 and B18 which are characterized by different antigenic and immunological properties show differences in genes involved in LPS synthesis. Specific genes affected by such mutations have been identified [66]. Brucella global expression profile studies can provide novel information on growth phase-specific gene expression. Further characterization of some genes that have been found to be differentially expressed in most invasive cultures will likely bring new insights into the initial molecular interactions between Brucella and its host [65]. However, limited genome diversity exists among Brucella species. Comparison of Brucella species whole genome is likely to identify factors responsible for differences in host preference and virulence restriction. Deletions of genetic content identified in Brucella species are conserved in multiple strains of the same species and genomic islands missing in a given species are often restricted to that particular species [67].

The genome of B. melitensis strain 16M has been sequenced and found to contain 2,294,935 bp distributed over 2 circular chromosomes of 2,117,144 and 1,177,787 encoding 3,197 ORFs [68]. Housekeeping genes including those involved in DNA replication, transcription, core metabolism and cell wall biosynthesis are distributed on both chromosomes [68]. To date, no genome-wide study has scanned genes related to host specificity of Brucella species [69]. Comparative whole-genome microarray analysis has revealed genomic islands, limited genome diversity in Brucella species and alterations as well as deletions of genes responsible for virulence [70]. Pathogenicity islands, specialized secretion systems, virulence plasmids, fimbriae, pili, adhesions and toxins are all classical virulence factors [71]. Genomic islands contribute to Brucella

OM

ICS

Gro

up e

Boo

ks

006

pathogenicity by helping to establish Brucella infection and survival strategies. Biophotonic imaging suggests that Brucella dissemination in mice parallels acute and chronic infections of humans [72,73].

Brucella enters macrophages through lipid raft microdomains, avoids their bactericidal attacks and phagolysosome fusion, expressing a set of virulence genes and inhibiting TNF-α secretion and apoptosis by persistence in microorganisms [70]. The first Brucella species was isolated and characterized almost 120 years ago but only recently the complete nucleotide sequences of the genomes of a number of well-characterized Brucella strains have been determined [74]. A feature that distinguishes Brucella species is that they do not express classical virulence factors. Disruption of putative virulence genes and studying their effect on attenuation in cell lines of mouse models is a widely used method [74]. The genus Brucella consists of 6 species, 3 of them including several biovars display a high degree of DNA homology. DNA polymorphism is able to differentiate the 6 Brucella species and some of their biovars [75]. DNA polymorphism within the genus Brucella might be involved in the differences in pathogenicity and host preference displayed by Brucella species [76].

Rifampicin is one of the most potent and broad-spectrum antibiotics against bacterial pathogens [77]. Mutations of the rpoβ gene have been characterized in rifampicin-resistant (Rifs) strains of Escherichia coli and Mycobacterium tuberculosis. Recent molecular studies have shown an association between rpoβ gene mutations and the development of rifampicin resistant Brucella species [77]. Efflux plays an important role in Brucella sensitivity to erythromycin. Polymorphisms among ribosomal loci from the reference Brucella species correlate with their highly differential susceptibility to erythromycin [78].

Microbiological Aspects of BrucellosisBrucellae belong to α-2 subdivision of proteobacteria [3]. The organism is a Gram negative, non-spore-forming,

coccobacillus [18]. It is aerobic, partially acid fast and has short rods. It is oxidase, catalase, nitrate reductase and urease positive [3]. It is localized predominantly in organs with numerous macrophages e.g. lungs, liver, spleen, bone marrow and synovium. At least 6 species have been recognized, 4 of them are pathogenic to humans: B. melitensis, B. abortus, B. suis and B. canis [18]. The organism is shed in: milk, fetal membrane, semen and uterine discharges. Reservoirs of Brucella infection include: goats, sheep, camels, cattle, dogs, pigs and deers (Table 1) [19].

The genome contains 2 circular chromosomes of 2.1 Mb and 1.5 Mb except for B. suis biovar which has a single chromosome of 3.1 Mb. There are 2 types of smooth LPS surface antigens, designated A and M. The A antigen predominates in B. abortus and B. suis, while the M antigen is the major antigen in B. melitensis. Numerous outer and inner membrane, cytoplasmic and periplasmic proteins have also been characterized [3].

Transmission of Brucellosis and Means of InfectionBrucellosis is commonly transmitted by: (1) consumption of unpasteurized, contaminated animal dairy products, (2)

direct contact with infected animal parts and (3) inhalation of infected aerosolized particles. Person to person transmission is less common, but has been reported [16-19]. The disease can be transmitted by blood transfusion. Reports of blood transfusion as a means of transmission of brucellosis in recipients of blood product transfusion existed as early as the year 1950. Screening of blood donors for brucellosis has revealed presence of Brucella antibodies in serum samples of 0.057 to 3.19% of blood donors [79-81]. In Saudi Arabia, the national seroprevalence of brucellosis is 15% [82]. Brucellosis can also be transmitted by transfusion of harvested bone marrow in recipients of hematopoietic stem cell transplantation [83]. Sexual transmission, although rare, has been reported in humans. In the reported cases, Brucella was either cultured from semen or its presence in serum was demonstrated by PCR [84,85].

Laboratory Exposure to Brucellosis and BioterrorismThe potential use of Brucella as a bioweapon derives from its great infectivity (virulence), ability to incapacitate infected

individuals (potential lethality), the stubborn persistent nature of human disease (ability to develop resistance to known antimicrobials) and the absence of a safe and an effective vaccine for use in humans. Both the USA and the former Soviet Union weaponized Brucella in 1945 [86-88]. Brucella is classified by the centers for disease control and prevention (CDC) as a category B pathogen that has potential for development as a bioweapon. Moreover, Brucella species are considered as the most common laboratory-acquired pathogen [13,20,89]. However, it is crucial to discriminate between true brucellosis and Y 09 infections that cause false positive serological reactions in testing for brucellosis [20]. In the management of bioterrorism, with the potential use of Brucella as a bioweapon, doxycycline should be considered a first-line antibiotic [87]. Discovery of a laboratory exposure to Brucellae should prompt an exhaustive investigation of the event and its circumstances, definition of the population at risk, enforcement of safe laboratory practices and administration of antimicrobial prophylaxis for exposed individuals [19,89,90].

ConclussionsBrucellosis is a common global re-emerging zoonosis that constitutes a major health and economic problem in many

parts of the world. Brucellae are Gram negative, intracellular coccobacilli that predominantly affect organs with rich macrophage content. At least 6 species have been recognized species, 4 of them are human pathogens. Infection can be acquired by: consumption of unpasteurized dairy products, direct contact with animals, blood product transfusion and sexual transmission. In the era of globalization, international tourism and travel across free borders, brucellosis has become a common imported disease in developed countries. Laboratory exposure to the organism and possible utilization as a

OM

ICS

Gro

up e

Boo

ks

007

biological weapon add more to the pathogenic potential of the organism. The recent immunologic, genetic and genomic advances have translated into better understanding of the pathogenesis of brucellosis and are likely to be utilized well in the vaccination, prevention and therapy of this global infection.

References1. Evans AS, Brachman PS (2009) Bacterial Infections of Humans Epidemiology and Control. (4thedn), Springer Science + Business

Media, LLC, 233, New York, NY 10013, USA.

2. Pappas G, Akritidis N, Bosilkovski M, Tsianos E (2005) Brucellosis. N Engl J Med 352: 2325-2336.

3. Mantur BG, Amarnath SK (2008) Brucellosis in India-a review. J Biosci 33: 539-547.

4. Seimenis A, Morelli D, Mantovani A (2006) Zoonones in the Mediterranean region. Ann Ist Super Sanita 42: 437-445.

5. Hasanjani Roushan MR, Mohraz M, Hajihamdi M, Ramzani A, Valayati AA (2006) Efficacy of gentamicin plus doxycycline versus streptomycin plus doxycycline in the treatment of brucellosis in humans. Clin Infect Dis 42: 1075-1080.

6. Skalsky K, Yahav D, Bishara J, Pitlik S, Leibovici L, et al. (2008) Treatment of human brucellosis: systematic review and meta-analysis of randomized controlled trials. BMJ 336: 701-704.

7. Ariza J, Bosilkovski M, Cascio A, Colmenero JD, Corbel MJ, et al. (2007) Perspectives for the treatment of human brucellosis in the 21st Century: the Ioannina Recommendations. PLoS Med 4: 317.

8. Cutler SJ, Fooks AR, van der Poel WH (2010) Public health threat of new, reemerging and neglected zoonoses in the industrialized world. Emerg Infect Dis 16: 1-7.

9. Pappas G, Papadimitriou P, Akritidis N, Christou L, Tsianos EV (2006) The new global map of human brucellosis. Lancet Infect Dis 6: 91-99.

10. Donev D, Karadzovski A, Kasapinov B, Lazarevik V (2010) Epidemiological and public health aspects of brucellosis in the republic of Macedonia. Prilozi 31: 33-54.

11. Vassalo CM, Economou V, Vassalou E, Papadopaulor C (2009) Brucellosis in humans: why is it so elusive? Rev Med Microbiol 20: 63-73.

12. Gwida M, Al Dahouk S, Melzer F, Rosler U, Neubauer H, et al. (2010) Brucellosis-regionally emerging zoonotic disease? Croat Med J 51: 289-295.

13. Seleem MN, Boyle SM, Srivanganathan N (2010) Brucellosis: a re-emerging zoonosis. Vet Microbiol 140: 392-398.

14. Russo G, Pasquali P, Nenova R, Alexandrov T, Ralchev S, et al. (2009) Reemergence of human and animal brucellosis, Bulgaria. Emerg Infect Dis 15: 314-316.

15. Farina F, Fuser R, Rossi M, Scotton PG (2008) Brucellosis outbreak in Treviso province caused by infected cheese from an endemic area. Infez Med 16: 154-157.

16. Al-Tawfiq JA, Abukhamsin A (2009) A 24-year study of the epidemiology of human brucellosis in a health-care system in Eastern Saudi Arabia. J Infect Public Health 2: 81-85.

17. Kadanali A, Ozden K, Altoparlak U, Erturk A, Parlack M (2009) Bacteremic and nonbacteremic brucellosis: clinical and laboratory observations. Infection 37: 67-69.

18. Akhvlediani T, Clark DV, Chubabria G, Zenaishvili O, Hepburn MJ (2010) The changing pattern of human brucellosis: clinical manifestations, epidemiology and treatment outcomes over three decades in Georgia. BMC Infect Dis 10: 346.

19. Arizona Department of Health Services (2004) Brucellosis: Bioterrorism Agent Profiles for Health Care Workers, Office of Public Health Emergency Preparedness and Response, 5.1-5.4.

20. Godfroid J, Cloeckaert A, Liautard JP, Kohler S, Fretin D, et al. (2005) From the discovery of Malta fever’s agent to the discovery of a marine mammal reservoir, brucellosis has continuously been a re-emerging zoonosis. Vet Res 36: 313-326.

21. Paradise LJ, Bendinelli M, Friedman H (2002) Opportunistic Intracellular Bacteria and Immunity. Kluwer Academic Publishers, USA.

22. Ritchie JA, Rupper A, Cardelli JA, Bellaire BH (2012) Host interferon-γ inducible protein contributes to Brucella survival. Front Cell Infect Microbiol 2: 55.

23. Martirosyan A, Moreno E, Gorvel JP (2011) An evolutionary strategy for a stealthy intraceullar Brucella pathogen. Immunol Rev 240: 211-234.

24. Skendros P, Sarantopoulos A, Tselios K, Boura P (2008) Chronic brucellosis patients retain low frequency of CD4+ T-lymphocytes expressing CD25 and CD28 after Escherichia coli LPS stimulation of PHA-cultured PBMCs. Clin Dev Immunol 2008: 327346.

25. Roset MS, Ciocchini AE, Ugalde RA, de Iannino NI (2006) The Brucella abortus cyclic β-1,2 glucan virulence factor is substituted with O-ester-linked succinyl residues. J Bacteriol 188: 5003-5013.

26. Roset MS, Ciocchini AE, Ugalde RA, Inon de Iannino N (2004) Molecular cloning and characterization of cgt, the Brucella abortus cyclic β 1,2 glucan transporter gene and its role in virulence. Infect Immun 72: 2263-2271.

27. He Y (2012) Analyses of Brucella pathogenesis, host immunity and vaccine targets using systems biology and bioinformatics. Front Cell Infect Microbiol 2: 2.

28. Cannela AP, Tsolis RM, Liang L, Felgner PL, Saito M, et al. (2012) Antigen-specific acquired immunity in human brucellosis: implications for diagnosis, prognosis and vaccine development. Front Cell Infect Microbiol 2: 1.

29. Skendros P, Boura P, Kamaria F, Raptopoulou-Gigi M (2006) CD80/CD28 co-stimulation in human brucellosis. Clin Exp Immunol 146: 400-408.

30. Rodriguez-Zapata M, Matias MJ, Prieto A, Jonde MA, Monserrat J, et al. (2010) Human brucellosis is characterized by an intense Th1 profile associated with a defective monocyte function. Infect Immun 78: 3272-3279.

OM

ICS

Gro

up e

Boo

ks

008

31. Baldwin CL, Goenka R (2006) Host immune responses to the intracellular bacteria Brucella: Does the bacteria instruct the host to facilitate chronic infection? Crit Rev Immunol 26: 407-442.

32. Baldwin CL, Winter AJ (1994) Macrophages and Brucella. Immunol Ser 60: 363-380.

33. Dornard J, Gross A, Lafont V, Liautard J, Oliaro J, et al. (2002) The innate immune response against Brucella in humans. Vet Microbiol 90: 383-394.

34. Bessoles S, Dudal S, Besra GS, Sanchez F, Lafont V (2009) Human CD4+ invariant NKT cells are involved in antibacterial immunity against Brucella suis through CD1d-dependent but CD4-independent mechanisms. Eur J Immunol 39: 1025-1035.

35. Oliaro J, Dudal S, Liautard J, Andrault J-B, Liautard JP, et al. (2005) Vγ9Vδ2 T cells use a combination of mechanisms to limit the spread of the pathogenic bacteria Brucella. J Leukocyte Biol 77: 652-660.

36. Dudal S, Turriere C, Bessoles S, Fontes P, Sanchez F, et al. (2006) Release of LL-37 by activated human Vγ9Vδ2 T cells: a microbicidal weapon against Brucella suis. J Immunol 177: 5533-5539.

37. Skyberg JA, Thornburg T, Rollins M, Huarte E, Jutila MA, et al. (2011) Murine and bovine γδ T cells enhance innate immunity against Brucella abortus infections. PLoS ONE 6: e21978.

38. Kilic SS, Akbulut HH, Ozden M, Bulut V (2009) Gamma/delta T cells in patients with brucellosis. Clin Exp Med 9: 101-104.

39. Atluri VL, Xavier MN, de Jong MF, den Hartigh AB, Tsolis RM (2011) Interactions of the human pathogenic Brucella species with their hosts. Annu Rev Microbiol 65: 523-541.

40. Dornand J, Lafont V, Oliaro J, Terraza A, Castaneda-Roldan E, et al. (2004) Impairement of intramacrophagic Brucella suis multiplication by human natural killer cells through a contact-dependent mechanism. Infect Immun 72: 2303-2311.

41. Grillo M-J, Blasco JM, Gorvel JP, Moriyon I, Moreno E (2012) What have we learned from brucellosis in the mouse model? Vet Res 43: 29.

42. Silva TMA, Costa EA, Paixao TA, Tsolis RM, Santos RL (2011) Laboratory animal models for brucellosis research. J Biomed Biotechol 2011: 518323.

43. Murphy EA, Sathiyaseelan J, Parent MA, Zou B, Baldwin CL (2001) Interferon-γ is crucial for surviving a Brucella abortus infection in both resistant C57BL/6 and susceptible BALB/c mice. Immunology 103: 511-518.

44. Brandao APMS, Oliveira F, Carvalho NB, Vieira LQ, Azevedo V, et al. (2012) Host susceptibility to Brucella abortus infection is more pronounced in IFN- γ knockout than IL-12/β2 microglobulin double-deficient mice. Clin Dev Immunol 2012: 589494.

45. Zhan Y, Liu Z, Cheers C (1996) Tumor necrosis factor alpha and interleukin-12 contribute to resistance to the intracellular bacterium Brucella abortus by different mechanisms. Infect Immun 64: 2782-2786.

46. Fernandez DM, Baldwin CL (1995) Interleukin-10 downregulates protective immunity to Brucella abortus. Infect Immun 63: 1130-1133.

47. Fernandez-Lago L, Orduna A, Vizcaino N (2005) Reduced interleukin-18 secretion in Brucella abortus 2308-infected murine peritoneal macrophages and in spleen cells obtained from B. abortus 2308-infected mice. J Med Microbiol 54: 527-531.

48. Li X, He Y (2012) Caspase-2-dependent dendritic cell death, maturation and priming of T cells in response to Brucella abortus infection. PLoS ONE 7: 43512.

49. Zhan Y, Kelso A, Cheers C (1993) Cytokine production in the immune response to Brucella infection or immunization with antigenic extracts. Immunology 80: 458-464.

50. Tiller RV, De BK, Boshra M, Huynh LY, Van Ert MN, et al. (2009) Comparison of two multiple-locus variable-number tandem-repeat analysis methods for molecular strain typing of human Brucella melitensis isolates from the Middle East. J Clin Microbiol 47: 2226-2231.

51. Jiang H, Fan M, Chen J, Mi J, Yu R, et al. (2011) MLVA genotyping of Chinese human Brucella melitensis biovar 1,2 and 3 isolates. BMC Microbiol 11: 256.

52. Kattar MM, Jaafar RF, Araj GF, Fleche PL, Matar GM, et al. (2008) Evaluation of a multilocus variable-number tandem-repeat analysis scheme for typing human Brucella isolates in a region of brucellosis endemicity. J Clin Microbiol 46: 3935-3940.

53. De Santis R, Ciammaruconi A, Faggioni G, Fillo S, Gentile B, Di, et al. (2011) High throughput MLVA-16 typing of Brucella based on the microfluidics technology. BMC Microbiol 11: 60.

54. Valdezate S, Navarro A, Villalon P, Carrasco G, Saez-Nieto JA (2010) Epidemiological and phylogenetic analysis of Spanish human Brucella melitensis strains by multiple-locus variable-number tandem-repeat typing, hypervariable octameric oligonucleotide fingerprinting and rpoβ typing. J Clin Micobiol 48: 2734-2740.

55. Bricker BJ, Ewalt DR, Halling SM (2003) Brucella ‘HOOF-Prints’: strain typing by multi-locus analysis of variable number tandem repeats (VNTRs). BMC Microbiol 3:15.

56. Al Dahouk S, Fieche PL, Nockler K, Jacques I, Grayon M, et al. (2007) Evaluation of Brucella MLVA typing for human brucellosis. J Microbiol Methods 69: 137-145.

57. Lista F, Reubsaet FAG, De Santis R, Parchen RR, de Jong AL, et al. (2011) Reliable identification at the species level of Brucella isolates with MALDI-TOF-MS. BMC Microbiol 11: 267.

58. Xiang Z, Zheng W, He Y (2006) BBP: Brucella genome annotation with literature mining and curation. BMC Bioinformatics 7: 347.

59. Whatmore AM, Perrett LL, MacMillan AP (2007) Characterization of the genetic diversity of Brucella by multilocus sequencing. BMC Microbiol 7: 34.

60. Wu M, Pei J, Turse C, Ficht TA (2006) Mariner mutagenesis of Brucella melitensis reveals genes with previously uncharacterized roles in virulence and survival. BMC Microbiol 6: 102.

61. Liautard J, Ouahrani-Bettache S, Jubier-Maurin V, Lafont V, Kohler S, et al. (2007) Identification and isolation of Brucella suis virulence genes involved in resistance to the human innate immune system. Infect Immun 75: 5167-5174.

62. Foulongne V, Bourg G, Cazevielle C, Michaux-Charachon S, O’Callaghan (2000) Identification of Brucella suis genes affecting

OM

ICS

Gro

up e

Boo

ks

009

intracellular survival in an in vitro human macrophage infection model by signature-tagged transposon mutagenesis. Infect Immun 68: 1297-1303.

63. Tae H, Shallom S, Settlage R, Hawkins GN, Adams LG, et al. (2012) Complete genome sequence of Brucella suis VBI 22, isolate from bovine milk. J Bacteriol 194: 910.

64. Boschiroli ML, Foulongne V, O’Callaghan D (2001) Brucellosis: a worldwide zoonosis. Curr Opin Microbiol 4: 58-64.

65. Rossetti CA, Galindo CL, Lawhon SD, Garner HR, Adams LG (2009) Brucella melitensis global gene expression study provides novel information on growth phase-specific gene regulation with potential insights for understanding Brucella: host initial interactions. BMC Microbiol 9: 81.

66. Adone R, Muscillo M, La Rosa G, Francia M, Tarantino M (2011) Antigenic, immunologic and genetic characterization of rough strains of B. abortus RB51, B. melitensis B115 and B. melitensis B18. PLoS ONE 6: 24073.

67. Rajashekara G, Glasner JD, Glover DA, Splitter GA (2004) Comparative whole-genome hybridization reveals genomic islands in Brucella species. J Bacteriol 186: 5040-5051.

68. Del Vecchio VG, Kapartal V, Redkar RJ, Patra J, Mujer C, et al. (2002) The genome sequence of the facultative intracellular pathogen Brucella melitensis. Proc Natl Acad Sci U.S.A 99: 443-448.

69. Kim KW, Sung S, Kim H (2011) A genomic-wide identification of genes potentially associated with host specificity of Brucella species. J Microbiol 49: 768-775.

70. Kulakov IuK, Zhelodkov MM (2006) Molecular basis of Brucella persistence. Zh Mikrobiol Epidemiol Immunobiol 4: 72-77.

71. Delrue RM, Lestrate P, Tibor A, Letesson JJ, De Bolle X (2004) Brucella pathogenesis, genes identified from random large-scale screens. FEMS Microbiol Lett 231: 1-12.

72. Rajashekara G, Krepps M, Eskra A, Mathison A, Montgomery A, et al. (2005) Unravelling Brucella genomics and pathogenesis in immunocompromised IRF-1-/- mice. Am J Reprod Immunol 54: 358-368.

73. Rajashekara G, Eskra A, Mathison A, Petersen E, Yu Q, et al. (2006) Brucella: functional genomics and host-pathogen interactions. Anim Health Res Rev 7: 1-11.

74. Seleem MN, Boyle SM, Sriranganathan N (2008) Brucella: a pathogen without classic virulence genes. Vet Microbiol 129: 1-14.

75. Vizcaino N, Cloeckaert A, Verger J, Grayon M, Fernandez-Logo L (2000) DNA polymorphism in the genus Brucella. Microbes Infect 2: 1089-1100.

76. Vizcaino N, Caro-Hernandez P, Cloeckaert A, Fernandez-Logo L (2004) DNA polymorphism in the omp25/om31 family of Brucella spp.: identification of a 1.7-kb inversion in Brucella cetaceae and of a 15.1-kb genomic island, absent from Brucella ovis, related to the synthesis of smooth lipopolysaccharide. Microbes Infect 6: 821-834.

77. Marianelli C, Ciuchini F, Tarantino M, Pasuali P, Adone R (2004) Genetic bases of the rifampicin resistance phenotype in Brucella spp. J Clin Microbiol 42: 5439-5443.

78. Halling SM, Jensen AE (2006) Intrinsic and selected resistance to antibiotics binding the ribosome: analysis of Brucella 23Srrn, L4, L22, EF-TuL, EF-Tu2, efflux and phylogenetic implications. BMC Microbiol 6: 84.

79. Economidou J, Kalafatas P, Vatopoulou T, Petropoulou D, Kattamis C (1976) Brucellosis in two thalassaemic patients infected by blood transfusions from the same donor. Acta Haematol 55: 244-249.

80. Akcakus M, Esel D, Cetin N, Kisaarslan AP, Kurtoğlu S (2005) Brucella mellitensis in blood cultures in two newborns due to exchange transfusion. Turk J Pediatr 47: 272-274.

81. Khorasgani MR, Esmaeili H, Pourkarim MR, Mankhian AR, Salehi TZ (2008) Anti-Brucella antibodies in blood donors in Boushehr, Iran. Comparative Clinical Pathology 17: 267-269.

82. Khan MY, Mah MW, Memish ZA (2001) Brucellosis in pregnant women. Clin Infect Dis 32: 1172-1177.

83. Ertem M, Kürekci AE, Aysev D, Unal E, Ikincioğullari A (2000) Brucellosis transmitted by bone marrow transplantation. Bone Marrow Transplant 26: 225-226.

84. Meltzer E, Sidi Y, Smolen G, Banai M, Bardenstein S, et al. (2010) Sexually transmitted brucellosis in humans. Clin Infect Dis 51: 12 - 15.

85. Vandercam B, Zech F, de Cooman S, Bughin C, Gigi J, et al. (1990) Isolation of Brucella melitensis from human semen. Eur J Clin Microbiol Infect Dis 9: 303-304.

86. Luther E. Lindler, Frank J. Lebeda, George W. Korch (2005) Biological Weapons Defence: Infectious Diseases and Counterbioterrorism. Humana Press, Totowa, New Jersey, USA.

87. Brouillard JE, Terriff CM, Tofan A, Garrison MW (2006) Antibiotic selection and resistance issues with flouroquinolones and doxycycline against bioterrorism agents. Pharmacotherapy 26: 3-14.

88. Burt Anderson, Herman Friedman, Mauro Bendinelli (2006) Microorganisms and Bioterrorism, Infectious Agents and Pathogenesis. Springer Science+Business Media, Inc., 233 Spring Street, New York, NY 10013, USA.

89. Centers for Disease Control and Prevention [CDC] (2008) Laboratory-acquired brucellosis---Indiana and Minnesota, 2006, MMWR Morb Mortal Wkly Rep, 57: 39-42.

90. Yagupsky P, Jo Baron E (2005) Laboratory exposures to Brucellae and implications for bioterrorism. Emerg Infect Dis 11: 1180-1185.

List of Publications - ORDER YOUR BOOKS!

Please visit our website athttp://www.thalassaemia.org.cy/list-of-publications

Hard copies and CD-ROM or DVD versions can be ordered directly from TIF and are distributed free of charge. Place your order at thalassaemia@cytanet.org.cy

The translation of TIF’s educational publications into various languages continues in 2013.

All translated publications are or will become available on our website. Check with us to get updated on the latest translations!

NEW! Just Released!

NEW! Just Released!

Free of charge

All our publications are available as PDF files on our website, completely free of charge.

UPCOMING TIF PUBLICATIONS

Community Awareness Booklets on α-thalassaemia, β-thalassaemia & Sickle Cell Disease (Greek)(Eleftheriou A) Sickle Cell Disease: A booklet for parents, patients and the community, 2nd Edition (Inati-Khoriaty A)Guidelines for the Clinical Management of Transfusion Dependent Thalassaemias, 3rd Edition (Cappellini M D, Cohen A, Eleftheriou A, Piga A, Porter J, Taher A)

TIF Publications cater to the needs of readers of all ages and educational backgrounds, and provide concise up-to-date information on every aspect of thalassaemia - from prevention to clinical management. TIF’s publications have been translated into numerous languages in order to cover the needs of the medical, scientific, patients and parents communities and the general community.

TIF Publications

•

•

•

Sponsor Advertisement