Brucella as a Potential Agent of Bioterrorism - IngentaConnect

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Brucella as a Potential Agent of Bioterrorism Gizem D. Doganay1 and Mehmet Doganay2* 1

Department of Moleculary Biology and Genetics, Istanbul Technical University, Istanbul, Turkey and 2Department of Infectious Diseases, Faculty of Medicine, Erciyes University, Kayseri, Turkey

Received: May 16, 2012; Revised: July 9, 2012; Accepted: July 11, 2012

Abstract: Perception on bioterrorism has changed after the deliberate release of anthrax by the postal system in the United States of America in 2001. Potential bioterrorism agents have been reclassified based on their dissemination, expected rate of mortality, availability, stability, and ability to lead a public panic. Brucella species can be easily cultured from infected animals and human materials. Also, it can be transferred, stored and disseminated easily. An intentional contamination of food with Brucella species could pose a threat with low mortality rate. Brucella spp. is highly infectious through aerosol route, making it an attractive pathogen to be used as a potential agent for biological warfare purposes. Recently, many studies have been concentrated on appropriate sampling of Brucella spp. from environment including finding ways for its early detection and development of new decontamination procedures such as new drugs and vaccines. There are many ongoing vaccine development studies; some of which recently received patents for detection and therapy of Brucella spp. However, there is still no available vaccine for humans. In this paper, recent developments and recent patents on brucellosis are reviewed and discussed.

Keywords: Biotechnology, bioterrorism, brucellosis, decontamination, diagnosis, patents. INTRODUCTION Bioterrorism is a form of terrorism caused by intentional release of biological agents resulting in social distress, economic burden, and heavy loss to humans, animals and plants. Bioterrorism is caused by biological agents (bacteria, viruses and other germs) or derived toxins which can be divided into three categories: anti-personal, anti-animal and anti-plant. The use of biological agents is also classified according to the way they are used: biological warfare and bioterrorism. Recently, bioterrorism has been used as a threat or as means of biological agents by individuals or groups for political, religious, ecological or other ideological purpose. In 1972, the United Nations held a Biological Weapons Convention. As a result of this conference, International Leaders signed a Treaty that completely prohibits the development, production and stockpiling of bacteriological and toxin weapons in any part of the world [1, 2]. Regardless of discrimination by the international community on biological weapons, deliberate release of anthrax was recognized in the postal system of United Stated of America (USA) in 2001, changing the public’s perception on bioterrorism. These types of bioterrorist events have worldwide effects on regional security, public health and other related sectors. The Center for Disease Control and Prevention (CDC) and National Institute of Allergy and Infectious Disease (NIAID) have categorized biological agents into three groups; A, B and C, based on their potential for dissemination, expected rate of mortality, public panic and social *Address correspondence to this author at the Professor in Infectious Diseases, Department of Infectious Diseases, Faculty of Medicine, Erciyes University, 38039 Kayseri, Turkey; Tel: +905333682042; Fax: +90 352 437 5273; E-mail: [email protected] 2212-4071/13 $100.00+.00

disruption. One of the potential biological agents is Brucella species, categorized in group B, infects both humans and animals [3, 4]. Brucella infection is distributed throughout the world including Mediterranean countries, Middle East, Central Asia, African countries, Central America and Latin America [5]. In the last two decades, many bioterrosist events have occurred: One was carried out by the Rajneeshees, a religious cult, against inhabitants in Dallas, Oregan in 1984. They used Salmonella typhimurium in this attack which affected 751 people. Another religious cult, The Aum Shinrinkyo, disseminated sarin gas in a Tokyo subway between 19901995. The cult who attempted to obtain Ebola virus from Zaire was also involved in biological warfare activity of botulinum neurotoxins Bacillus anthracis and Coxiella burnettii [2, 6]. Their last attempt was to obtain Ebolavirus from Zaire. The last event was the postal mailing of anthrax spore in USA [7]. In the literature, it is noted that Brucella species has not been used in bioterrorism until today. Brucella species can easily infect both human and animals by aerosols or contaminated animal food products during preparation, packing, transportation and storage. Recently, Pappas et al. [8] reclassified 15 potential biological agents with bioterrorism attack scenarios and awarded lowest score, that is 6 points, to Brucella species. MICROBIOLOGICAL CHARACTERISTICS Up to 1985, the genus Brucella which belongs to the Brucellaceae family was classified into 6 species: B. melitensis, B. abortus, B. suis, B. canis, B. neotomae and B. ovis. Recently, new species were discovered; Brucella ceti, Brucella pinnipedialis, Brucella microti and Brucella inopinata. Brucellosis is principally a zoonotic disease and © 2013 Bentham Science Publishers

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commonly causes abortions in sheep, goats (B. melitensis; B. ovis), cows (B. abortus), pigs (B. suis) and canines (B. canis). Recently, other species were also isolated from wild life and marine mammals [9, 10].

would affect 125.000 people resulting in 500 deaths [2]. An economical cost had been estimated with an aerosol attack model scenario on an urban population in 1997 which was calculated as $ 477.7 million per 100.000 exposed people [15].

Brucella is a small, nonmotile, nonsporulating, facultative, gram negative coccobacilli bacteria that grow both extracellularly and intracellularly within the macrophages. The principal virulence factor of Brucella lies within the structure of its cell-wall lipopolysaccharide. Strains with smooth lipopolysaccharide are more virulent and more resistant to intracellular destruction by polymorphonuclear leukocytes [9, 10].

In humans, brucellosis is a debilitating and protracted disease with acute, subacute or chronic forms. Although the disease requires prolonged antibiotic treatment, there are a limited number of antibiotics available. Moreover, therapeutic failure and relapse may be possible with various antibiotic regimens. Currently, there is no safe and effective Brucella vaccine available for humans [10, 13, 14]. Many industrialized nations control brucellosis in animals by requiring vaccination, quarantine and slaughter programs. Although Brucella spp. is not considered as a priority for bioterrorism, an attack with Brucella spp. may be associated with severe outbreaks in human population and farm animals. Countries being protected from animal brucellosis may consider agroterrorism as a public health precaution against bioterrorism [16].

Brucella organisms can survive up to two days in milk at 0 8 C, three weeks in frozen meat and three months in goat cheese. Brucella that is present in animal excretions may remain viable for more than 40 days if the soil is damp. These organisms are sensitive to heat, ionizing radiation, commonly used disinfectants and pasteurization [11-13]. BRUCELLA AS AN AGENT OF BIOTERRORISM At the beginning of the 21st century, many countries initiated biological weapon program determining the utility of a biological agent according to its virulence, infectiousness, stability and production. Several key characteristics of Brucella species make it more or less suitable for a biological weapon such as the availability of virulent strains, storage stability, and dissemination into the environment. Aerosol transmission and dissemination is considered to be the most effective means of delivery. In addition, Brucella can be easily cultured from infected animal or human materials. The transfer, storage and dissemination of the bacteria are also manageable. An intentional contamination of food with Brucella species could also pose a threat with low mortality rate [1, 2, 10, 14]. From 1932 to until the end of World War II, Japan developed an extensive biological weapon program. This program included experimentation with various biological agents on human subjects in the infamous Manchurian Units 731 and 100 [1, 14]. Later in 1942, B. suis was weaponized in USA and formulated to maintain its long-term viability. It was placed into bombs and tested with animal targets in field trials between 1944 and 1945. However, the USA declared that the offensive Brucella program has been terminated, and all biological weapon munitions were destroyed in 1969 [10]. During of former the Soviet Union, Brucella was extensively formulated as one of the agents developed for offensive purposes. As mentioned by Pappas et al., according to Ken Alibek antibiotic resistant strains of Brucella were developed and weaponized both in dry and liquid forms with production capability ranging up to 100 tons. It is stated that extensive field testing was performed in the midst of The Aral Sea [14]. Several biological and pathological properties of the Brucella species renders it to be an attractive pathogen that can be used as a potential agent in biological warfare. These bacteria are highly infectious through aerosol route with a dose of approximately 10-100 microorganisms. It has been estimated that release of 50 kg of B. suis from a plane along 2 km line and 10 km upwind on a city with 500.000 people

TRANSMISSION TO HUMANS The main sources of brucellosis are found in infected animals or their products, such as milk, cream, butter, fresh cheese, ice cream, urine, blood, carcasses and abortion products. The route of transmission to infect humans include direct contact with infected animals and their secretions through cuts and skin abrasions, by means of infected aerosols inhaled or inoculated into the conjunctival eye sack, or via the ingestion of unpasteurized dairy products [11, 12]. Brucellosis is rarely transmitted from person to person. Consumption of contaminated foods and occupational contact are the major risks of infection. Infection may also result from the entry of bacteria from inhalation of contaminated dust or aerosols present in slaughterhouse, barn and the place of contaminated with aborted materials [17, 18]. However, the disease is also a laboratory transmitted infection. It is easily acquired by sniffing cultures and by generating aerosols while working with cultures or bacterial suspension on the open bench. It is noted that 2% of all human brucellosis are laboratory acquired. The microorganism may enter the body in various ways relevant to laboratory procedures through the respiratory mucosa, conjunctivae, gastrointestinal tract and abraded skin [19, 20]. As seen above, Brucella spp. can be easily aerosolized and sprayed. These are very important bioterrorism-related characteristics for Brucella spp. Using Brucella spp. as a biologic agent for bioterrorism through the food chain could be possible but bioterrorism-related cases may remain as localized clusters. This kind of intentional food contamination would be effective after industrial level since pasteurization kills the pathogen. Not many people are expected to be affected by this type of potential attack with intentional food contaminated [14, 16]. Falenski et al. showed that Brucella spp. survived for 87 days in UHT milk, for 60 days in water and less than a week in yogurt [21]. This study is a good example for intentional food contamination with Brucella spp. BRUCELLA AND BIOTECHNOLOGY Genetic engineering and information could easily be abused for the purpose of development and improvement of infective agents as bioweapons. Misuse of biotechnology in

Brucella as a Potential Agent of Bioterrorism

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genetics enables the development of an antibiotic and/or vaccine resistant microorganisms which can become more lethal, easier to handle, harder to detect, or more stable in the environment. There are various examples of this misuse as described in the literature. For instance, Deinococcus radiodurans is one of such bacterium that is resistant to environmental factors including radiation [22].

method has to be used before the application of any PCR analysis. Brucella genus is classified into six recognised species: B. melitensis, B. abortus, B. suis, B. canis, B. ovis and B. neotomae, some of which have been recorded as different biovars. All genus have a high degree of DNA homology [27]. Thus specific targets on the DNA sequence have to be chosen correctly. One way to design a specific strain-typing method is through a multi-locus variable number tandem repeat analysis (MLVA) [28]. Variable number tandem repeats (VNTRs) are polymorphic genomic elements containing variable numbers of direct head-to-tail repeat sequences. At a specific VNTR locus, variation of the repeat numbers causes allelic distribution among Brucella species [29]. In one study, when a pool of B. melitensis (epidemiologically-linked samples, sporadic, laboratory acquired), B. abortus, B. suis, B. canis and Brucella spp. isolates were screened using MLVA. With the VNTR allelic targeting in a PCR reaction, sufficient discriminatory power to distinguish isolates associated with epidemiologically-linked clusters of brucellosis from isolates representing sporadic cases of brucellosis has been achieved [28]. This Brucella MLVA assay is described as a rapid, single-tube PCR assay accurately differentiating Brucella species from each other. Furthermore, this method could be applied to practical usage for diagnosis by the practitioners [28]. Another diagnostic method for detection and differentiation of Brucella spp is based on PCR which involves the design of a microarray using gene targets for genus-specific sequences and species-specific chromosomal regions [30]. Even though, the developed microarray assay is an easy-to-handle molecular test for high-throughput analysis, it is limited to detection of certain Brucella species. One other Brucella spp. identification method is based on 16S rRNA sequencing of bacteria, which is considered as an appropriate method for species identification and can be routinely adopted as a rapid identification method in any molecular biology laboratory at moderate cost [31, 32]. However, this method can not discriminate between closely related Brucella species since 16S rRNA regions are highly conserved sequences of DNA [33]. To obtain better specificity with 16S rRNA sequencing method, it has to be supported further by additional sequencing of the 23S rRNA gene or 16S-23S rRNA intergenic spacer regions, where variations among species are observed [33].

Outbreak of any bioterrorist attack first results in an illness upon which medical intervention is undertaken. During causative agent identification of the bioweapon, clinicians and microbiologists share great responsibility in the diagnosis of the disease and work together to undertake appropriate experiments, respectively. Since bioweapons are the products of genetically modified organisms, it can be difficult to find out the source of the microorganism. In order to correctly identify the bacteria responsible for bacterial infections, laboratory confirmation by serologic or bacteriologic methods is often required. Due to the diversity of clinical manifestations for brucellosis, clinical diagnosis should be confirmed with a laboratory test. Conventional laboratory methods for the detection of bacteria involve some basic steps; pre-enrichment, selective enrichment, molecular/biochemical analysis and serological confirmation [23, 24]. Bacterial detection assays must be sensitive and specific, capable of detecting low concentrations of targets without interfering with any other agents. Also, a rapid detection of bacteria in a variety of specimen types should be assured in the designed diagnostic test. Each method used for the identification of bacteria has its own pitfalls as the results of an analysis obtained through any method usually requires additional confirmations through the application of other available tests. In practice, serological testing is the most applied screening method for the diagnosis of Brucella infection; however, in the early stages of the infection, crossreactivity with other gram-negative pathogens is widely observed with this method, decreasing its specificity and sensitivity and resulting in false-negative or only weak positive reactions (i.e. in immunoassay type serologic testing, the specificity of immunoassays is limited by antibody quality and sensitivity). Also, serologic tests require isolation and cultivation of the organism from clinical specimens; laboratory personnel are at significant risk for the Brucella infection, which is one of the most commonly reported laboratory-acquired infections [25, 26]. The methods that are used to minimize the risks for accidental exposure to brucellosis include molecular and bioanalytical methods: Polymerase Chain Reaction (PCR)-based technologies, application of instrumental methods and biosensor-based detections. PCR-based methods are heavily used to amplify small quantities of DNA for the screening of bacterial species. Nowadays, in any molecular biology laboratory, simple PCR or real-time quantitative PCR assays can be easily applied. For diagnosis of Brucella spp., PCR-based identification methods are also frequently preferred. PCR is a very sensitive method yielding higher sensitivity than that of immunoassays. However, in Brucella spp. screening, isolation of genetic material is an issue that affects the recovery of Brucella DNA from suspensions and spiked swabs influencing the sensitivity of real-time PCR assays [25]. A study performed by Dauphin and coworkers [25], showed that a well-optimized DNA extraction

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Brucella spp. is also being detected using instrumental methods; such as Fourier Transform Infrared Spectroscopy (FTIR) [34], which is based on the study of absorption bands corresponding to different chemical groups of the structural components of bacteria (proteins, lipids, carbohydrates, amino acids, etc.). Unique absorption bands can be detected for bacteria in an FTIR spectrum. Using FTIR, MiguelGomez and coworkers [34] were able to differentiate the three bacterial groups from each other: B. melitensis, other Brucella species and other microorganisms. This method is specific to the genus of Brucella, but it lacks species specification due to the similarity of chemical composition of bacteria at the molecular level. Another method used to distinguish Brucella species is flow cytometry which is usually used as a complementary method to ELISA tests. Flow cytometry enables quantification of bacterial cells by particularly labeling their certain cellular constituents such as proteins, carbohydrates, DNA, RNA and enzymes. For Brucella detection, flow cytometry is used to measure the surface ex-


posure of outer membrane protein epitopes since Brucella species (and strains designed for vaccine) can express different levels of either smooth or rough lipopolysaccharides, namely S-LPS and R-LPS, respectively, as the major surface antigen. Flow cytometry offers an effective means for rapid analysis of specific targets, as mentioned for the analysis of Brucella epitopes, allowing quantitative differentiation of homogeneous or heterogeneous binding distributions in a cell population. But due to small size of bacteria and variations of bacterial growth conditions more sensitive instrumentation is often required [13, 14]. Biosensor-based detections of bacteria generally involve a biological recognition component such as receptors, nucleic acids, or antibodies to initiate contact with an appropriate transducer. There are mainly two different ways to detect the target in the design of biosensors: direct detection methods target the analyte directly whereas indirect detection based biosensors measure the products of a biochemical reaction that takes place in bacteria [35]. In addition, signal transduction method is important for biosensor design, and can be divided into four basic groups: optical, mass, electrochemical, and thermal transduction [36-38]. To our knowledge, there are few biosensors designed for Brucella genus [35, 39-41]. Lee and coworkers [39] used biosensors based on indirect measurement of fluorescence and potentiometric detection. They have used B. melitensis as the target species, compared the biosensors’ ability to detect bacteria with each other, and reported the limits of detection for this bacteria were 0.5 ng per well for the potentiometric sensor and 10 ng per well for the fluorescence detection. Their work revealed that sensitivity of detecting filtration-captured bacteria via a potentiometric sensor assay is higher compared to that of fluorescence detection derived from fluorescein-labelled antibodies; but they commented that once the optical configuration of the fluorescence assay is optimized, fluorescence-based biosensor design sensitivity could be improved. Another indirect biosensor designed to detect Brucella spp. infection and to quantify anti-Brucella spp. antibodies in serum was developed as an immunosensor which measures the fluorescence changes due to the interactions of Cyanine-5 conjugated to the antibody with the target antigen [40]. Quantified anti-Brucella spp. antibodies in ovine serum samples were in the range from 0.005 to 0.11 mg ml1 [40], which was comparable to the formerly described fluorescence-based measurement. One other biosensor, The Bead Array Counter (BARC), for the detection and identification of biological warfare was developed through the application of a multi-analyte biosensor that uses DNA hybridization, magnetic microbeads, and giant magnetoresistive (GMR) sensors [41]. This sensor could be used to differentiate Bacillus anthracis, Yersinia pestis, Brucella suis, Francisella tularensis, Vibrio cholerae, Clostridium botulinum, Campylobacter jejuni, and Vaccinia virus from each other [41]. This biosensor has not been used in the identifying of Brucella species yet, as it is limited to certain Brucella strain typing. However, it is useful for identifying biological warfare agents. RECENT PATENTS FOR BRUCELLA AS A BIOTERRORISM AGENT The events of September 11, 2001 changed the way scientists view the use of biotechnology in biodefense research. Many researchers have initiated their studies on the early

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detection of agents, appropriate sampling, new decontamination procedures, and new drug and vaccine developments for potential bioterrorism agents. During the last decade, there have been many improvements in molecular biology. As, B. anthracis is one of the most attractive potential bioterrorism agent, there have been many studies in recent years, that have focused on the development of early detection methods for the presence of B. anthracis together with other bioterrorism agents including Brucella species and others on different sources. Molecular techniques like immunoassay, gene probe assay, nucleic acid amplification and enzyme inhibition using a silicon-based biosensor are now available to detect biologic agents as elaborated above [42, 43]. When an attack occurs, appropriate sampling is very important for the early detection of microorganisms. Edward and coworkers [44] received a patent in 2010 for their surface sampler design for bioterrorism particle detection. This device was developed primarily for obtaining biological contaminated samples from environmental surfaces. Biological particle such as bacteria, virus, other microorganisms or other particles of biological origin including nucleic acids, proteins and toxins can be removed by this device. It can also capture particles from liquid samples. Srinagesh and Sulatha [45] invented a novel apparatus for detection of very small quantities (a few hundred molecules) of bioparticles. Dielectrophoresis is used to concentrate materials and the detection process uses the fluorescence of nanoparticles. A new patent was also given to a device of olfactory receptor-functionalized transistor device for highly selective bioelectronic nose and biosensor. This invention is useful for a bioelectronic nose which can detect specific odorants and may be used to detect anti-bioterrorism, diseases diagnostics and food safety [46]. Peter et al. [47] invented a device to monitor a variety of airborne or surface pathogens, not limited to anthrax, and also received a patent. The inventors claimed that bioamplification-coupled proteomics assay provides a reliable result for detection of pathogens, and this device offers a more compact, rapid and cost effective means compared to that of other anthrax detectors, thus is suggested as an effective weapon against bioterrorism. Sampath et al. [48] developed a new oligonucleotide primer pair that is useful for identifying and determining characteristics of bacteria in a sample. Grote [49] also invented a test kit for detecting B. melitensis and B. abortus in environmental samples by PCR amplification, using two primers to generate detectable amplicons from a specific DNA sequence. Ecker et al. [50] developed a new method for identification of unknown bioagents including bacteria and viruses. The method was based on a combination of nucleic acid amplification and molecular weight determination using primers which hybridize to conserve sequence regions of nucleic acids derived from a bioagent. Another new method has been developed for rapid identification of pathogens in humans and animals. This method [51] is based on the differentiation of two or more bioagents by contacting nucleic acid from bioagents with oligonucleotide primers to produce amplification products and determination of base compositions of the amplified products. The current invention provides methods of identifying pathogens in biological samples from humans,


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animals and environmental. Marc L [52] developed a method for detecting one or more bioterrorism target agents. This invention provides methods and compositions for early diagnosis of exposure to or infection by a chemical or biological weapon by rapid and specific detection of one or more bioterrorism target agents in a sample.

profuse sweating, fatigue, anorexia, weight loss, headache, arthralgia and generalized aching. Localized organ involvement (osteoarticular, gastrointestinal, hepatobiliary, nervous system, genitourinary, cardiovascular system etc.) can be seen in brucellosis as a complication. A case having symptoms and findings clinically compatible with Brucella infection is confirmed with the isolation of Brucella spp. from blood or other clinical specimens or demonstration of a specific antibody response (> 160 or 4-fold rise in Brucella agglutination titer between acute and convalescent phase) [12, 13, 56].

Recent advances are seen in the biosensing technologies that use electrochemical, piezoelectric, optical, acoustic and thermal biosensors for the detection of pathogenic bacteria. These technologies can be used in clinical diagnosis, food analysis and environmental monitoring [35, 43]. Gadner et al. [53] invented an Integrated Detector Sample Cell for detecting pathogenic microorganisms including B. anthracis, Brucella species and other bioterror agents. The system is based on spectroscopic imaging, employing Raman, fluorescence, UV-visible reflectance/absorption and/or nearinfrared reflectance/absorption spectroscopic techniques for the characterization of bioweapon agents. A membrane strip biosensor device which uses a fluid mobile conductive composition of ferromagnetic particles bound to a conductive polymer and capture reagent received a patent [54]. It is claimed that this device can be used to detect pathogens, proteins and other biological materials of interest in food, water and environmental samples. The device can also be used for on-site diagnosis and against potential bioterrorism. Wang et al. [55] invented a genetic liquid phase chip for rapid detection of five maliciouspathogenic bacteria of B. anthracis, Yersinia pestis, Brucella species, Francisella tularensis and Burkholderia pseudomallei. The inventers claim that the liquid phase chip has high sensitivity and specificity, high efficacy and requires less sample capacity. DIAGNOSIS AND TREATMENT It is assumed that the incubation period varies between 960 days (ranging from few days to 6 months), and relatively prolonged, when compared with other pathogens that are considered as potential bioterrorism agents [14, 56]. An intentional release of Brucella spp. would not lead to an epidemic that suddenly arouses. The outbreak could induce a smooth curve of gradual increase followed by a decrease over a period of 1-2 months [8, 14]. For this character of released brucellosis, primary care physicians, emergency service physicians, infectious disease physicians, and hospital epidemiologist need to be aware of clustered cases of brucellosis, particularly from patients that have flown prior to any human and animal brucellosis cases being reported. Veterinarians should also be aware of increasing animal cases in their regions. Unexpected and clustered human and animal cases with brucellosis must be analyzed epidemiologically for the source of infection. The differentiation of this situation is very difficult in endemic countries for Brucella infection. On the other hand, some physicians working in industrial countries are not familiar with clinical presentation of brucellosis. For this reason, the diagnosis of human cases may be delayed. Released brucellosis and naturally occurring brucellosis show same clinical presentation. Clinical picture may be acute or insidious onset with continued, intermittent or irregular fever of variable duration. The symptoms consist of

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Patients confirmed with the diagnosis of brucellosis by isolation of Brucella spp. or serologically proven should receive therapy with doxycycline and rifampin for 6 weeks. Alternatively, a combination of doxycycline for 6 weeks and an intramuscular aminoglycoside (gentamicin or streptomycin) for the first 2 weeks is recommended. Various combinations of drugs therapy are suggested for complicated or lifethreatening clinic forms, such as meningitis or endocarditis. It should not be forgotten that the administration of tetracyclines is contraindicated during pregnancy [12, 13, 56] Although, vaccination is very important in post exposure prophylaxis or at the risk of Brucella infection, currently, no safe and effective human brucellosis vaccine exists. On the other hand, live and attenuated strains of B. abortus, B. melitensis and B. suis have been used effectively as vaccines in livestock. For this reason, antibiotics would be the only option in the prophylaxis of deliberate released brucellosis. Antibiotic prophylaxis in asymptomatic persons exposed to Brucella microorganisms is not studied. The current suggestions for the cases exposed to deliberate released brucellosis are derived from the data of accidental laboratory exposure [2, 14, 20, 56]. If suspension of living Brucella bacteria is spilled and as soon as the organism is detected in the laboratory, the current suggestion is that the entire laboratory workers should be immediately evacuated, and doors should be closed. After, effective disinfectants such as 3% phenol or 10% hypochlorite solution should be applied by a trained person wearing a safe mask, goggles, an impermeable laboratory gown and gloves [2, 10]. Laboratory accidents and control measurements are a good example for intentional released brucellosis. In the event of a biological attack, the N 95 mask may be an adequate protection for the personnel from airborne Brucella infection in the event location. Brucella spp. is unable to penetrate from intact skin. All victims should be evacuated from the attack area. After take of clothing, skin and other surface can be decontaminated with standard disinfectants to minimize risk of infection by accidental ingestion or conjunctival inoculation of viable organisms. Recently, a low odor chemical decontaminant has received a patent for the decontamination of hard surfaces. This liquid disinfectant is composed of multiple components such as peracetic acid and hydrogen peroxide [58]. On the other hand, portable chlorine generator [59] and air disinfectant devices [60] also recently received a patent for disinfection and preventing the spread of toxic or infectious agents after any intentional attack. Although there is little evidence to support for post exposure prophylaxis, the current suggestion is to use chemopro-


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phylaxis for the treatment of exposed people after an event occurred. A combination regimen includes oral doxycycline twice a day plus rifampicin 600 mg once a day and this therapy is recommended for 3 to 6 weeks. Trimethoprimsulfamethoxazole (160 mg/ 800mg) should be considered for patients having contraindications to doxycycline [2, 14, 20, 57]. The authors’ opinion is that an exposure is confirmed with Brucella organism, an antibiotic susceptibility test should be performed to the first isolate from index case in a casualty. Because, genetically modified and resistant strains should be considered for the planning postexposure prophylaxis.

[8]

There is no record showing transmission from a person to person. For this reason, a strict isolation or quarantine in the hospital is not required. The victims are recommended to have a follow up at least 3 months; in some cases may be scheduled from 3 to 9 months [14, 20, 57].

[13]

In conclusion; Brucella species might be used as an agent of bioterrorism, but priority is very low. The agent is stable and could be transmitted by the airborne route and infectious dose is also low. The disease is associated with low mortality and transmission does not occur from person to person, but infects both human and animals. Food may be intentionally easily contaminated with Brucella spp. and further used as a biohazard. Training of physicians, particularly, in industrial countries is mandatory, and adequate awareness might help in limiting unnecessary interventions. CONFLICT OF INTEREST The authors confirm that this article content has no conflicts of interest. ACKNOWLEDGEMENTS The authors would like to thank to Dr. Duygu Yucel and Mrs. Donna Sue for proofreading of the paper.

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