Brucella melitensis

I. Organism Information

A. Taxonomy Information
  1. Species:
    1. Brucella melitensis :
      1. GenBank Taxonomy No.: 29459
      2. Description: Brucellae are Gram-negative, facultative, intracellular bacteria that can infect many species of animals and man (Cloeckaert et al., 2003). Brucella belongs to the (alpha)2 subdivision of the proteobacteria, along with ochrobactrum, rhizobium, rhodobacter, agrobacterium, bartonella, and rickettsia (Pappas et al., 2006). Brucellosis is a bacterial disease of animals that can be transmitted to humans. The primary impact of brucellosis stems from losses due to reproductive failure in food animals and the loss of human productivity. Since brucellosis threatens the food supply and causes undulant fever, a long, debilitating disease in humans, Brucella species are recognized as potential agricultural, civilian, and military bioterrorism agents. Brucellosis in food animals is controlled by vaccination. Human brucellosis is treatable with antibiotics, though the course of antibiotic treatment must be prolonged due to the intracellular nature of Brucella (Halling et al., 2005). The genus Brucella consists of at least six species, designated on the basis of host preference, antigenic and biochemical characteristics as Brucella melitensis (goats and sheep), Brucella abortus (cattle), Brucella suis (pigs), Brucella canis (dogs), Brucella ovis (sheep) and Brucella neotomae (wood rats). B. abortus, B. melitensis and B. suis can all infect humans with similar serious disease consequences. Recently, Brucella spp. have been isolated from marine mammals and are infectious for humans. B. melitensis, originally isolated as a pathogen of goats and sheep, is highly pathogenic and a frequent cause of human brucellosis. In contrast, human infections with B. ovis and B. neotomae have not been reported, and human infection with B. canis is rare. Although Brucella species exhibit host preference and virulence restrictions, they are genetically highly related and therefore have been proposed to be included under one species B. melitensis (Rajashekara et al., 2006). B. melitensis was the first species in the genus Brucella to be described. It was first isolated by Bruce in 1887 from the spleens of soldiers dying of Mediterranean fever on the island of Malta. Bruce called it Micrococcus melitensis. Mediterranean fever did not appear to spread among people, and the origins of the disease remained a mystery for nearly 20 years until it was discovered that goats were affected and were providing the source of infection for the human population (Alton, 1990). Synonyms: Micrococcus melitensis, Streptococcus Miletensis (NCBI Taxonomy).
      3. Variant(s):
        • Brucella melitensis 16M :
          • GenBank Taxonomy No.: 224914
          • Parent: Brucella melitensis
          • Description: B. melitensis strain 16M (Biotype1, ATCC 23456) was isolated from an infected goat and is pathogenic to humans. This strain causes abortion in pregnant goats, pigs, cattle, and sheep and is used to study Brucellosis in the murine animal model (DelVecchio et al., 2002). Synonyms: Brucella melitensis str. ATCC 23456 (NCBI Taxonomy)
        • Brucella melitensis biovar Melitensis :
          • GenBank Taxonomy No.: 274058
          • Parent: Brucella melitensis
          • Description: Controversy has arisen concerning Brucella internal taxonomy, and it has been proposed that the DNA-DNA hybridization-based genomospecies concept be applied to the genus. According to this view, only one species, Brucella melitensis, should be recognized, and the classical species should be considered as biovars (B. melitensis biovar melitensis; B. melitensis biovar abortus; etc.) (Moreno et al., 2002).
B. Lifecycle Information :
  1. Vegetative Cell :
    1. Size: 0.6 to 1.5 um long by 0.5 to 0.8 um wide (Corbel, 1989)
    2. Shape: Small Gram-negative cocci, cocobacilli, or short rods (Corbel, 1989).
    3. Picture(s):
      1. Brucella melitensis (Dennis Kunkel Microscopy, Inc):

        Description: Brucella melitensis - Gram-negative, aerobic, coccobacillus prokaryote; causes brucellosis fever (also known as undulant or Malta fever). This zoonotic microorganism passes from goats and sheep to humans in contaminated dairy products. Magnification: x3,600 Type: SEM (Dennis Kunkel Microscopy, Inc).
    4. Description: They occur singly, in pairs, short chains, or small clusters (Corbel, 1989). Flagella, endospores, and capsules are absent although capsule-like structures have been reported in preparations treated with antiserum (Corbel, 1989). The Brucella cell envelope consists of an outer layer of lipopolysaccharide-protein about 9 nm thick. The polysaccharide chains are exposed, whereas the lipid components are in close proximity to the peptidoglycan layer which forms the major part of the cell wall (Corbel, 1989). Brucella can present itself upon culture with either a smooth or rough colony morphology, with some strains presenting a mucoid phenotype (Schurig et al., 2002). Coupled to the rough versus smooth morphology is the composition of the LPS molecule of Brucella. Smooth organisms have LPS molecules containing a polysaccharide O-chain made from a homopolymer of perosamine (N-formyl-4-amino,4,6-dideoxy mannose), while rough organisms lack this chain on their LPS molecule or possess only a greatly truncated portion of it (Schurig et al., 2002). In addition to the LPS the outer membrane of Brucella contains several major proteins (Diaz and Moriyon, 1989). Matrix and porin proteins penetrate the peptidoglycan layer at irregular intervals and are partially exposed at the cell surface (Corbel, 1989). A zone of low electron density, usually identified as the periplasmic space and ranging from 3 to 6 nm in depth in smooth phase cells, up to 30 nm in depth in rough phase cells, separates the peptidoglycan layer from the cell membrane (Corbel, 1989). The cell membrane has an ultra-structure typical of lipoprotein membranes in general (Corbel, 1989). Polysaccharide granules and small vacuoles are fairly evenly dispersed throughout the cytoplasm (Corbel, 1989).
C. Genome Summary:
  1. Genome of Brucella melitensis 16M
    1. Description: The B. melitensis genome was sequenced by using a shotgun approach. The genome size is 3.29 Mb distributed over two circular chromosomes of 2.12 and 1.18 Mb with a 57% GC content. There are 3,197 predicted ORFs distributed on the two chromosomes. Plasmids were not found (DelVecchio et al., 2002). By using the bioinformatics suite ERGO, 2,487 (78%) ORFs were assigned functions. The origins of replication of the two chromosomes are similar to those of other alpha -proteobacteria. Housekeeping genes, including those involved in DNA replication, transcription, translation, core metabolism, and cell wall biosynthesis, are distributed on both chromosomes. Type I, II, and III secretion systems are absent, but genes encoding sec-dependent, sec-independent, and flagella-specific type III, type IV, and type V secretion systems as well as adhesins, invasins, and hemolysins were identified. Several features of the B. melitensis genome are similar to those of the symbiotic Sinorhizobium meliloti (DelVecchio et al., 2002).
    2. Chromosome I:
      1. GenBank Accession Number: NC_003317
      2. Size: 2,117,144 nt (NCBI Entrez Genome)
      3. Gene Count: 2107 genes, 2059 protein coding, 48 structural RNA, 0 pseudo genes, 8 others (NCBI Entrez Genome)
      4. Description: The chromosome I ori is located between ORFs BME2823 and BME2824 with a DnaA binding box identical to that of Caulobacter and Rickettsia and a characteristic low GC skew (55% A + T). In addition, a second putative ori site with two nucleotide differences in the DnaA binding box is located between ORFs BME1001 and BME12059 (DelVecchio et al., 2002). Two ORFs for the replication initiator protein DnaA are located on chromosome I (DelVecchio et al., 2002). The outer membrane protein (Omp) genes of B. melitensis are located on chromosome I (DelVecchio et al., 2002). TEXT (DelVecchio et al., 2002)
    3. Chromosome II:
      1. GenBank Accession Number: NC_003318
      2. Size: 1,177,787 nt (NCBI Entrez Genome)
      3. Gene Count: 1157 genes, 1139 protein coding, 18 structural RNA, 0 pseudo genes, 2 others (NCBI Entrez Genome)
      4. Description: The ori of chromosome II is located between ORFs BME2001 and BME2118 with a low GC skew (64% A + T) and is similar to that on chromosome I (DelVecchio et al., 2002). Genes involved in cell division, such as minC, minD, and topology specificity proteins, are located on chromosome II (DelVecchio et al., 2002).

II. Epidemiology Information

The natural home of B. melitensis appears to be the Mediterranean, especially along its northern and eastern shores, stretching through Central Asia as far south as the Arabian peninsula and as far east as the two Mongolias. Parts of Latin America are also seriously affected, especially Mexico, Peru, and northern Argentina, presumable following the migration of people from the Mediterranean region. Sporadic occurrence is common to Africa and India. Here the available data derive mainly from serological surveys and are imprecise. In the U.S. in recent years, B. melitensis caused sporadic outbreaks in goats near the Mexican border. The country is now believed to be free, as are Canada, Northern Europe (except for sporadic incursions from the south), Southeast Asia, Australia, and New Zealand (Alton, 1990). In recent years some of the countries of West Asia have sought to increase supplies of meat and milk by encouraging intensive sheep farming. In some of the very large flocks established for this purpose severe outbreaks of B. melitensis infection have occurred, leading to epidemics of human brucellosis (Alton, 1990).

A. Outbreak Locations:
  1. MALTA: The catastrophic effect of drinking untreated, infected goat milk was dramatically demonstrated among military personnel in Malta. In 1905 they experiences 913 cases of brucellosis (Mediterranean fever), but in 1907, after the drinking of goat's milk had been prohibited, the number had fallen to 21 (Alton, 1990). Little was done to protect the civilian population during the first four decades of this century. Measures were taken forbidding the entry of goats into the towns for milking, and 1939 the government established a dairy to collect, pasteurize, and market milk. These efforts were nullified when Malta was fiercely attacked during World War II, and the highest number of human cases ever recorded there was in 1946 when 2400 were reported, about 80 per 10,000. After the war, the prohibition against the sale of nonpasteurized milk was gradually extended to the whole of Malta and its sister island Gozo, and by 1959 the annual human incidence had fallen to 220 cases (Alton, 1990).
  2. RUSSIA: In Russia, brucellosis was first diagnosed in 1913 in goats at Tashkent. Nine years later, six cases of human brucellosis were diagnosed in Azerbaijan and the source of infection traced to sheep and goats. However, B. melitensis was not isolated in Russia until 1930, after it had become the Soviet Union (Alton, 1990).
  3. MANCHURIA: In the mid-1930s Japanese veterinarians working in Manchuria had the opportunity to observe acute outbreaks of B. melitensis infection in two large flocks of sheep over successive lambing periods, and in their attendants (Alton, 1990).
  4. MONGOLIA: In the 1960s a high incidence of human infection with B. melitensis was reported from both Mongolias. In each case the disease was brought under control by vaccination of small ruminants (Alton, 1990).
  5. PERSIAN GULF REGION: A most dramatic increase in the incidence of B. melitensis infection occurred in the countries bordering the Persian Gulf, where, during the 1980s, a large and spreading epidemic developed. Long prevalent in Iran, B. melitensis has only recently become a serious problem in other oil-rich states where increasing affluence has stimulated the introduction of massive sheep farming. In Saudi Arabia, Radwan et al. described an outbreak in a flock of 38,000 sheep, kept under intensive conditions, where the serological positivity had reached 24% and where some of the sheep handlers had contracted burcellosis. In nearby Kuwait, the available figures show 32 cases of human brucellosis in 1980, 77 in 1982 and an estimated average of 2000 in both 1985 and 1986 (Alton, 1990).
  6. SOUTH AFRICA: Three outbreaks have been recorded in goats and sheep in South Africa; the first outbreak occurred in sheep in 1965 in the Mpumalanga and Northern Provinces (then both part of the Transvaal Province), the second occurred in sheep in 1989 near Pretoria, Gauteng Province, and the third and current outbreak was diagnosed in a flock of goats in northern KwaZulu-Natal in September 1994 (Emslie et al., 2002).
  7. UNITED STATES: In 1983 an outbreak of human brucellosis caused by Brucella melitensis occurred among residents of a predominantly Hispanic neighborhood in Houston, TX. The source of the infections was traced to unpasteurized goats' milk cheese imported from Mexico (Thapar et al., 1986). From May to September 1988, eight employees of a microbiology laboratory developed acute brucellosis (attack rate, 31%). Seven of the eight affected employees had clinical illness ranging from a nonspecific, flu like illness to severe hepatitis. Blood cultures obtained from five of the affected employees (63%) were positive for Brucella melitensis, biotype 3. Comparison of cases and controls showed that there were no risk factors besides employment in the laboratory. Based on work locations, assignments, and interviews, it was found that person-to-person, droplet, food-borne, and waterborne spread were unlikely. Our investigation disclosed that 6 weeks before the outbreak began, a frozen brucella isolate from a patient hospitalized 3 years earlier had been thawed and subcultured without the use of a biologic safety cabinet. This clinical isolate was subsequently identified as B. melitensis, biotype 3, identical to the employee isolates. It is presumed that transmission occurred via the airborne route. This outbreak reemphasized that all work on Brucella species, an established biosafety level 3 organism, must be conducted under a biologic safety hood (Staszkiewicz et al., 1991).
  8. SPAIN: An outbreak of acute brucellosis infection was detected among the employees of a biologicals manufacturing laboratory located in Girona, Spain. The first cases appeared six weeks after a vaccine with attenuated Brucella melitensis, Rev-1 had been produced for one week. A clinical and epidemiologic investigation conducted among the 164 employees found 22 patients with clinical symptoms and positive serology, and six patients detected by serology only (attack rate: 17.1 per cent). Blood cultures were obtained from two patients and Brucella melitensis was isolated (Olle-Goig et al., 1987). A total of 81 cases of brucellosis were recorded in a period of 25 weeks. All the cases occurred in the same borough or were in some way linked to it. In the case and controls study no differences were found with regard to age, sex, contact with livestock or the consumption of pasteurised milk or cheese. A strong link was established between the illness and the consumption of home-made cottage cheese prepared by a small-scale producer in the borough, whose livestock turned out to be infected with Brucella Mellitensis. This outbreak showed that in Spain, there is a risk of contracting brucellosis by consuming non-pasteurised dairy products, particularly cheese, when these are not subjected to the normal sanitary and health controls (Castell et al., 1996). Eleven brucellosis cases were identified in three municipalities of Cordoba (Andalucia, Spain). A case-control study was conducted, selecting three cases per control. Persons having eaten unpasteurized raw goat cheese produced in a farmhouse located in the epidemic territory, were at higher risk for presenting brucellosis. Brucella melitensis serovar 3 was identified in clinical specimens and in goat tissue and milk samples from the herd's farmhouse. Preventive measures were implemented and the outbreak was stopped after the withdrawal of all suspicious cheeses from the market, additional sanitation of the farmhouse and health promotion activities (Mendez et al., 2003).
  9. ISRAEL: In 1997, 7 cases of laboratory-acquired Brucella melitensis infections were detected among the hospital personnel of a medical centre serving an endemic area in southern Israel. Although the onset of symptoms in 6 of the 7 patients occurred during a 2-week period, suggesting a point source exposure, biotype analysis showed that the outbreak was caused by 3 different B. melitensis serovars, indicating multiple exposures. Review of the laboratory records showed that during 1997, the microorganism was recovered from 146 blood and synovial fluid cultures, and that during the 2 months in which the laboratory-acquired cases occurred (April and June), 53 of 530 positive aerobic blood culture bottles (10.0%) grew B. melitensis. The epidemiological investigation did not reveal the source of the outbreak, and no noticeable breaches in laboratory safety practices could be demonstrated (Yagupsky et al., 2000).
  10. ARGENTINA: An outbreak of human brucellosis among farm workers of Argentina was studied and revealed a close relationship with an epidemic of caprine abortions which occurred shortly before on the same farm. High rates of B. melitensis infection were found among goats. Active brucellosis was diagnosed in 33 subjects (14 with positive blood culture for B. melitensis), while other 27 did not show evidence of illness. While 25 of the brucellosis active patients were rural workers, only 5 of the healthy subjects were engaged in rural labour. Active brucellosis was diagnosed in 91.3% of the subjects in continuous contact with goats and in 32% of those having an occasional contact with the animals. All the 60 subjects denied consumption of goat cheese or milk. As shown here, epidemic human infections by B. melitensis may develop among people frequently in contact with infected goat herds or goat manure (Wallach et al., 1997).
B. Transmission Information:
  1. From: Animal To: Human
    Mechanism: Brucellosis is a zoonosis that is nearly always transmitted from animal reservoirs directly to man via three routes: (1) direct contact through the conjunctivae, or through abraded or broken skin with excretions, secretions, or tissues of infected animals or contaminated fomites; (2) inhalation of infectious aerosols with invasion occurring through the mucosa of the upper respiratory tract or the lung; and (3) ingestion of tissues, foodstuff, or fluids containing organisms (Nicoletti, 1989). Infection may also result from the entry of the bacteria from diseased animals through skin lesions or mucous membranes or from inhalation of contaminated dust or aerosols. Inhalation is often responsible for a significant percentage of cases in abattoir employees. Contamination of skin wounds may be a problem for persons working in slaughterhouses or meat packing plants or for veterinarians (Godfroid et al., 2005). Brucella spp. persist for several days in milk (even when it turns sour). It may also persist for weeks in ice cream and months in butter (Godfroid et al., 2005). Therefore, these products always have to be made from pasteurized milk (Godfroid et al., 2005). Sale of improperly prepared cheese and other dairy products by commercial vendors results in additional cases in urban populations in many countries and in travelers who visit areas where brucellosis is endemic (Nicoletti, 1989). Meat of animals with brucellosis may also be a source of infection if eaten when insufficiently cooked. Some particular food habits, such as eating aborted foetuses as seen in Ecuador, may be responsible for human brucellosis (Godfroid et al., 2005). B. melitensis is transmitted more readily from animals to man than other members of the genus (Alton, 1990). Most cases of B. melitensis infection can be related to direct or indirect exposure to infected sheep or goats or their products; other host species, including cattle, other bovidae and camelids, are locally important sources in some areas but probably account for only a small number of infections (Corbel, 1989).

  2. From: Animal To: Animal
    Mechanism: Only when they are excreting the bacteria do sheep and goats present any danger to other animals or to man. Abortion, accompanied as it is by the emission of huge numbers of brucellae, is the chief source of infection for the onward transmission of the disease. If it is accepted that kids and lambs are resistant, excretion in the milk is likely to play a lesser part in transmission to other animals, and then only where the animals are milked (Alton, 1990). The chief danger of introducing the disease into a previously clean country or region is by the purchase of infected animals (Alton, 1990). Intermingling of flocks may occur under nomadic and seminomadic conditions of husbandry and also in static village flocks where animals are taken daily for grazing on common pasture (Alton, 1990). The self-limiting nature of the disease in sheep, which is seldom accompanied by prolonged excretion of the bacteria, means that the disease does not easily maintain itself in small flocks except where replacement animals are purchased, where there is contact with other flocks, and where milking is practiced. The infection is more evident in large flocks even when milking is not practiced, and abortion rates of 10 to 60% have been reported (Alton, 1990). Goats: Infection is generally introduced in the same way as in sheep. The greater susceptibility of the goat, with more copious and prolonged excretion of the bacteria, results more often in abortion storms in goat herds. Nevertheless, a chronic state may soon become established within the herd, with only rare abortions, but the disease is liable to flare up if susceptible animals are introduced (Alton, 1990). It is not though that male sheep or goats play any significant role in spreading the infection to other animals, except, perhaps, mechanically. Neither are wild animals nor insect vectors thought to play any significant part in the epidemiology of sheep and goat brucellosis (Alton, 1990).

  3. From: Human To: Human
    Mechanism: Human brucellosis is not considered a contagious disease (Godfroid et al., 2005). Human-to-human transmission by tissue transplantation or sexual contact have occasionally been reported but are, in epidemiological terms, insignificant (Godfroid et al., 2005). An asymptomatic donor infected with Brucella was shown to be the likely source of infection of a recipient of bone marrow cells. Transmission by shared needles and coitus have been suggested but not proven (Nicoletti, 1989). Infection in man is a dead-end process and further transmission to other persons or to other host species occurs so rarely that it can make no contribution to the natural history of the disease (Corbel, 1989).

C. Environmental Reservoir:
  1. Goats and Sheep (Alton, 1990):
    1. Description: The natural hosts of Brucella melitensis are adult goats and certain breeds of sheep, among which it spreads readily, causing abortion (Alton, 1990). The goat was originally considered the principal host of B. melitensis, and this continues to be the case in certain areas, notably Malta and Latin America, where sheep are not significantly infected even when kept in close contact with goats. In many other areas the disease is more important in sheep, despite the fact that goats are always more susceptible. There are two principal reasons for this difference. First, there is great variation in the susceptibility of different breeds of sheep: the Maltense sheep, and presumable those of Latin America are almost totally resistant, while in areas bordering the Mediterranean and through West and Central Asia, the principal breeds of sheep are almost as susceptible to B. melitensis as goats. Second, in these latter areas sheep are the predominant species of small ruminant, being kept for the production of much-favored dairy products and meat, often in large flocks, in conditions that encourage the spread of infection (Alton, 1990).
    2. Survival Information: B. melitensis infection in female sheep and goats causes disease in sexually mature animals (Alton, 1990). In goats the infection may vary in duration from a very short period in slight infections rapidly overcome (especially in vaccinated animals) to persistence for years, where excretion of the organism in the milk may continue for at least two lactations and perhaps many more. The situation in sheep is very different, though this varies with the susceptibility of the breed. Stiedter et al. infected 101 sheep subcutaneously, and after the injection of small doses they were only able to recover the organism from local lymph nodes between 15 and 30 d post inoculation. Larger doses produced abortion and more prolonged infection (45 to 90 d), but in 54 sheep experimentally and 35 naturally infected, all were found to be bacteriologically negative between 4 and 13 months after infection (Alton, 1990). Viable kids or lambs from an infected dam may themselves be infected with B. melitensis but generally lose the infection by the time they are 2 months old (Alton, 1990).
  2. Cattle (Alton, 1990):
    1. Description: It is the least species specific of the brucellae and from the reservoir of infection in goats and sheep transmits to many other species (Alton, 1990). In regions where B. melitensis is prevalent in sheep and goats, cattle are liable to catch the infection from them (Alton, 1990).
    2. Survival Information: It has not yet been established whether B. melitensis can maintain in it self indefinitely in a cattle population in the absence of infected sheep or goats, but an extensive outbreak has been reported in a herd of cows where no sheep or goats were present. B. melitensis infection in cattle sometimes causes them to abort, but less frequently than B. abortus, and a focus of infection in cattle may die out spontaneously. Unfortunately, colonization of the udder is frequent, and excretion of the organism in the milk may be prolonged for months or years and has frequently led to epidemics of brucellosis in people working with the cattle or drinking the milk (Alton, 1990).
  3. Other animals (Alton, 1990):
    1. Description: Sheep dogs working with infected flocks frequently become infected with B. melitensis but rid themselves of the infection relatively quickly (Alton, 1990). Dogs and wolves may be more important as mechanical disseminators of infection by carrying away infected materials such as fetuses or fetal membranes (Alton, 1990). The isolation of B. melitensis from camels has been reported only rarely (Alton, 1990). Saigas are sheep-like antelopes with strange inflatable pouch-like nostrils (Alton, 1990). Saigas often share watering places with domestic sheep. B. melitensis is said to spread naturally among Saigas and to cause abortion. The position seems somewhat obscure, but they are considered a possible source of Brucella infection for domestic animals (Alton, 1990). Saigas often share watering places with domestic sheep. B. melitensis is said to spread naturally among Saigas and to cause abortion. The position seems somewhat obscure, but they are considered a possible source of Brucella infection for domestic animals (Alton, 1990). Any of the small mammals that come in contact directly or indirectly with infected sheep or goats seem liable to temporary infection with B. melitensis. Rats, mice, rabbits, hares, wild guinea pigs, and even domestic poultry have all been found infected, but there is no evidence of significant disease being caused or of any ability to transmit infection. Wild birds, especially vultures, have the ability to transmit the infection mechanically, but we have no information on whether they play a part in the epidemiology of the disease. Outbreaks of brucellosis in fur-bearing animals such as mink have been traced to the feeding of infected meat, e.g., dead lambs containing B. melitensis (Alton, 1990). Ticks and biting insects have been found to harbor B. melitensis, but none of these creatures have been incriminated in the epidemiology of the disease (Alton, 1990).
D. Intentional Releases:
  1. Intentional Release information :
    1. Description: Brucella has traditionally been considered a biological weapon. It was the subject of extensive offensive research in the past, and still belongs to category B pathogens on most lists (Pappas et al., 2006). Its significance as a potential agent of bioterrorism was acknowledged early, and the pathogen remains on the category B biodefense research list of both the Centers for Disease Control and Prevention (CDC) and the National Institute of Allergy and Infectious Diseases (NIAID) (Pappas et al., 2006). Brucella was one of the agents with which Japan experimented in the infamous 731 Manchurian Unit before and during World War II (Pappas et al., 2006). In the former Soviet Union, Brucella was one of the agents developed for offensive purposes by Biopreparat, the extensive Soviet biological weapons program (Pappas et al., 2006). As with other agents developed by the Soviet Union, extended field testing was performed on the island of Vozroshdeniye, in the midst of the Aral Sea (Pappas et al., 2006).
    2. Emergency contact: Immediately notify state public health laboratory director (or designate) and state public health department epidemiologist/health officer if Brucella cannot be ruled out and a bioterrorist event is suspected. The state public health laboratory/state public health department will notify local FBI agents as appropriate (American Society for Microbiology).
    3. Delivery mechanism: Brucella can be easily aerosolized, and when in air, can be easily transmitted through the airways and induce disease, while staying for a protracted period in this virulent form (Pappas et al., 2006). In a hypothetical attack scenario, it was estimated that release of an aerosolized form of brucella under optimal circumstances for dispersion would cause 82,500 cases of brucellosis and 413 fatalities (Pappas et al., 2006). Using Brucella as a biological weapon through the food chain could be feasible, but would result in localized clusters of cases: one would have to intervene at a post-industrial level, since pasteurization kills the pathogen (Pappas et al., 2006).
    4. Containment: Because of ethical considerations, the heterogeneous nature of the events leading to Brucella exposure, difficulties in determining the actual risk for individual workers, late recognition of outbreaks, and the small number of persons involved in each outbreak, no controlled studies have been performed to assess the value of administering postexposure prophylaxis to persons at risk. However, anecdotal evidence suggests that administering prophylactic antimicrobial drug therapy may reduce the risk of developing clinical disease (Yagupsky et al., 2005). Isolation of patients is not necessary (Bossi et al., 2004).

III. Infected Hosts

  1. Human:
    1. Taxonomy Information:
      1. Species:
        1. Human :
          • GenBank Taxonomy No.: 9606
          • Scientific Name: Homo sapiens (NCBI_Taxonomy)
          • Description: B. melitensis is transmitted more readily from animals to man than other members of the genus (Alton, 1990). World-wide, most cases of human brucellosis are caused by B. melitensis which is considered the most invasive and pathogenic of the three classical species of the genus (Nicoletti, 1989). The resulting disease is severe, often long lasting, and rural populations may be unable to support the expense of extended antibiotic treatment (Alton, 1990). The economic wastage resulting from loss of young replacement animals, reduction of milk yield, etc. is keenly felt, even when the cause is not clearly understood, but this is greatly exceeded in terms of human misery by the impact of B. melitensis infection on human health (Alton, 1990).

    2. Infection Process:
      1. Infectious Dose: It has been estimated that 10-100 organisms only are sufficient to constitute an infectious aerosol dose for humans (Bossi et al., 2004).
      2. Description: In humans, ovine/caprine brucellosis caused by B. melitensis is by far the most important clinically apparent disease (Corbel et al., 2002). Communication from animal to man may be direct or indirect. Direct contact with infected animals is dangerous for animal attendants, owners of infected animals and their families (Alton, 1990). The maximum danger is during the lambing or kidding period when infected animals, whether they have aborted or not, may discharge billions of bacteria from the uterus in birth products and the discharges that follow (Alton, 1990). Under dusty conditions aerosols containing dangerous numbers of brucellae may also be responsible for human infection even on public roads (Alton, 1990). Dairy products are the main source of infection for people who do not have direct contact with animals (Alton, 1990). Much of the milk drunk is now rendered safe by pasteurization or boiling, but cheeses made from sheep or goat milk are very popular in most countries where sheep or goats are the main source of milk (Alton, 1990). Such cheeses are a major vehicle for transmission of B. melitensis to the human population, sometimes in areas remote from where the cheese was prepared (Alton, 1990). Abattoir workers handling infected sheep or goats are also at risk, especially from the contents of the uteri and udders (Alton, 1990). B. melitensis is one of the most easily acquired laboratory infections (Alton, 1990).

    3. Disease Information:
      1. Malta fever, Mediterranean fever, Undulant fever (i.e., Human Brucellosis) (CDC):
        1. Pathogenesis Mechanism: Brucella infection occurs through inhalation or ingestion of organisms via the nasal, oral, and pharyngeal cavities. Following penetration of the mucosal epithelium, the bacteria are transported, either free or within phagocytic cells, to the regional lymph nodes (Ko et al., 2003). The spread and multiplication of Brucella in lymph nodes, spleen, liver, bone marrow, mammary glands, and sex organs occurs via macrophages (Ko et al., 2003). Brucella organisms not only resist killing by neutrophils following phagocytosis but also replicate inside macrophages and nonprofessional phagocytes (Ko et al., 2003). They reside in specialized compartments with acidic environments, and multiply using parts of the cytoskeleton, without interrupting cell cycle and function; on the contrary they are apoptosis inhibitors, thus creating a frame for eternal survival and replication (Pappas et al., 2006). Survival in macrophages is considered to be responsible for the establishment of chronic infections, allowing the bacteria to escape the extracellular mechanisms of host defense such as complement and antibodies (Ko et al., 2003). After replication, brucellae are released with the help of hemolysins and induced cell necrosis (Pappas et al., 2006). The host reacts to this infection with innate and adaptive immune responses. Critical aspects in this response include secretion of IL-12 and IFN-gamma involving APCs and Th1 cells. MHCclass I-restricted CTLs are generated and are required to clear the infection (Golding et al., 2001). Interferon-gamma has a central role in the pathogenesis of brucellosis by activating macrophages, producing reactive oxygen species and nitrogen intermediates; by inducing apoptosis, enhancing cell differentiation and cytokine production; by converting immunoglobulin G to immunoglobulin G2a; and by increasing the expression of antigen-presenting molecules (Pappas et al., 2006). It has been long postulated that the outcome of the disease reflects the equilibrium developed between the bacterium and the human immune response, and that relapses and chronic disease should also be viewed in this context (Pappas et al., 2006).

          • Model of intracellular trafficking and survival of Brucella inside macrophages (Cellular traffic):

            Description: After phagocytic uptake, intracellular Brucella resides within a vacuole (BCV) that interacts with early endosomes. These early BCVs avoid fusion with late endocytic compartments, yet acquire LAMP-1, and interact with endoplasmic reticulum (ER) exit sites (ERES) within the first hours after infection (A). Such interactions are sustained over time and lead to limited fusion events with the ER (B), ultimately generating an ER-derived organelle permissive for Brucella replication (C). Biogenesis of the replicative organelle requires the Brucella VirB type IV secretion system and Sar1-dependent functions of the ER exit sites (NIAID).

        2. Incubation Period: The incubation period of brucellosis is highly variable, from one to 60 days, up to several months, with an average of 1-2 months (Bossi et al., 2004).

        3. Prognosis: Historically, 2% of untreated B. melitensis-infected patients die, and Brucella-induced deaths are still reported (Ko et al., 2003). It is usually due to B. melitensis endocarditis or meningitis. Nearly all patients respond to antibiotic treatment, with fewer than 10% manifesting relapses. The systemic symptoms may last for weeks or months. Most of the patients recover within a year, even without antibiotic treatment (Bossi et al., 2004).

        4. Diagnosis Overview: In countries where brucellosis is enzootic (i.e. present in animal reservoirs), human confirmed cases are based on clinical symptoms associated with positive serology without attempts to isolate Brucella spp., in the vast majority of suspected or confirmed cases. In countries where animal brucellosis has virtually been eradicated, for every single suspected case of human brucellosis, particularly in the presence of positive serology, the emphasis is put on the epidemiological inquiry in order to confirm the case (Godfroid et al., 2005). Diagnosis: Demonstration of a specific antibody response (>160 or 4-fold rise between acute and convalescent phase); Demonstration by immunofluorescence of Brucella sp. in a clinical specimen; Isolation of Brucella sp. from a clinical specimen (Bossi et al., 2004).

        5. Symptom Information :
          • Syndrome -- Human brucellosis (CDC):
            • Description: Signs and symptoms are similar in patients whatever the route of transmission and are mostly non-specific (Bossi et al., 2004). Physical examination is usually without abnormalities (Bossi et al., 2004). The illness may be mild and self-limited or severe. Onset of the disease may be abrupt or insidious (50%) (Bossi et al., 2004). Brucellosis is a systemic infection that can involve any organ or organ system. It is generally characterised by fever, which may be continuous, intermittent or irregular (Bossi et al., 2004).

            • Symptoms Shown in the Syndrome:

            • General symptoms (Bossi et al., 2004):
              • Description: Fever, which is the most common symptom, may be associated with other symptoms such as weakness, profuse sweating, chills, diffuse or localised arthralgias, malaise, weight loss and generalised pain (Bossi et al., 2004). Fever is invariable and can be spiking and accompanied by rigors, if bacteremia is present, or may be relapsing, mild, or protracted (Pappas et al., 2006).
              • Observed: Fever: 91%; Constitutive symptoms (e.g., malaise, arthralgias): 26% (Pappas et al., 2006). Sweating: 40-90%; Weight loss and generalised pain: 40-70% (Bossi et al., 2004).
            • Cardiovascular symptoms (Bossi et al., 2004):
              • Description: Endocarditis remains the principal cause of mortality in the course of brucellosis. It usually involves the aortic valve and typically requires immediate surgical valve replacement (Pappas et al., 2006). Other cardiovascular complications include myocarditis, pericarditis and aneurysms of the aorta and cerebral vessels (Bossi et al., 2004).
              • Observed: Endocarditis occurs in approximately 2% of patients (Bossi et al., 2004).
            • Cutaneous manifestations (Bossi et al., 2004):
            • Gastrointestinal symptoms (Bossi et al., 2004):
              • Description: Gastrointestinal symptoms such as anorexia, nausea, vomiting, abdominal pain, diarrhoea or constipation are also frequent (Bossi et al., 2004).
              • Observed: Up to 70% of cases (Bossi et al., 2004).
            • Haematological abnormalities (Bossi et al., 2004):
              • Description: Haematological abnormalities may include anaemia, neutropenia and thrombocytopenia (Bossi et al., 2004). The blood count is often characterized by mild leukopenia and relative lymphocytosis, along with mild anemia and thrombocytopenia. Pancytopenia in brucellosis is multifactorial and is attributed to hypersplenism and bone marrow involvement. Rarely, marked pancytopenia or isolated deficits can be attributed to diffuse intravascular coagulation, hemophagocytosis, or immunologically mediated cellular destruction (Pappas et al., 2006).
              • Observed: Relative lymphocytosis: 40%; Isolated thrombocytopenia: 5%; Isolated leukopenia: 2%; Pancytopenia: 2% (Pappas et al., 2006).
            • Liver pathology and functional abnormalities (Bossi et al., 2004):
              • Description: The liver is involved in the great majority of cases as shown by a mild increase of liver function enzymes (Bossi et al., 2004). Non-caseating epithelioid granulomas indistinguishable from the ones seen in sarcoidosis can be found in liver biopsy. Hepatic abscesses and acute cholecystitis have also been reported (Bossi et al., 2004). Hepatitis is common, usually manifesting as mild transaminasemia (Pappas et al., 2006). Ascites is often present, either as a temporary exacerbation of preexisting hepatic disease or as frank peritonitis (Pappas et al., 2006).
              • Observed: Hepatomegaly or hepatosplenomegaly is reported in 60 to 70% of patients infected with B. melitensis (Young, 1989). Transaminasemia: 24% (Pappas et al., 2006).
            • Neurological/neuropsychiatric symptoms (Bossi et al., 2004):
              • Description: The clinical presentation of neurobrucellosis includes meningoencephalitis, meningovascular involvement, parenchymatous dysfunction, peripheral neuropathy/radiculopathy (PNP), and various degrees of behavioral abnormalities. Among the clinical manifestations, meningitis has been the most frequent presentation in clinical series and usually presents in an acute or chronic form (Yetkin et al., 2006). Severe behavioural changes of unknown aetiology occur in some patients (Bossi et al., 2004). Neuropsychiatric complaints, which include headache, depression and irritability, are frequent (Bossi et al., 2004).
              • Observed: Neurologic manifestations of brucellosis occur in 3-5% of patients (Yetkin et al., 2006).
            • Osteoarticular complications (Bossi et al., 2004):
              • Description: Osteoarticular complications of brucellosis are common and include sacroiliitis, which represents the most frequently involved joint, peripheral joint arthritis, bursitis, osteomyelitis and particularly vertebral osteomyelitis or spondylitis. Arthritis of peripheral joints usually involves large joints such as the hips, knees and ankles. Osteoarticular complications are usually due to B. melitensis (Bossi et al., 2004). Brucellosis has also been proposed as a cause of reactive arthritis. A second form, characterized by sacroiliitis, is readily diagnosed, also usually in the context of acute brucellosis. On the other hand, a third form of osteoarticular disease, spondylitis, remains notoriously difficult to treat and often seems to result in residual damage. The lumbar spine is the usual site of involvement. Spondylitis can be easily diagnosed with plain radiography, in which the characteristic Pons sign (a step like erosion of the anterosuperior vertebral margin) can be identified, or with scintigraphy and magnetic resonance imaging. The latter imaging technique is popular and produces impressive scans but is costly and not always available. Osteoarticular complications are sometimes linked to a genetic predisposition, with recent data suggesting an association with HLA-B39 (Pappas et al., 2006).
              • Observed: Up to 40% of cases (Bossi et al., 2004). Spondylitis: 6% (Bossi et al., 2004). Peripheral arthritis: 22% (8% in hips, 7% in knees, 4% in elbows, 4% in wrists, 4% in other locations); Sacroiliitis: 3%; Spondylitis: 19% (15% lumbar, 3% dorsal, 1% cervical) (Pappas et al., 2006).
            • Reproductive symptoms (Pappas et al., 2006):
              • Description: The reproductive system is the second most common site of focal brucellosis. Brucellosis can present as epididymoorchitis in men and is often difficult to differentiate from other local disease. The effect of the local inflammation on subsequent testicular function has not been adequately studied. Brucellosis in pregnancy poses a substantial risk of spontaneous abortion (Pappas et al., 2006).
              • Observed: Epididymoorchitis: 5.7% (Pappas et al., 2006).
            • Respiratory symptoms (Bossi et al., 2004):
              • Description: Cough and chest pain are present in 15%-25% of infected patients with a normal chest radiograph. Lung abscesses, nodules and pleural effusions have been reported (Bossi et al., 2004).
              • Observed: Respiratory complications of brucellosis are considered rare. A recent multinational review of cases with respiratory complications indicated that approximately 16 percent of cases had pulmonary involvement that included lobar pneumonia and pleural effusions (Pappas et al., 2006).

        6. Treatment Information:
          • Antibiotic combination regimens (Pappas et al., 2006): The World Health Organization (WHO) endorses regimens that combine doxycycline, 100 mg b.i.d., and rifampin, 600-1200 mg daily, for 6 weeks, or doxycycline for 6 weeks and streptomycin, 15 mg/kg daily, for 2-3 weeks. The latter combination is considered superior, but demands parenteral administration. Gentamicin can adequately replace streptomycin, at a dose of 5 mg/kg for 5-7 days. Alternatives include trimethoprim-sulfamethoxazole in various combinations, and combinations including ofloxacin or ciprofloxacin. Quinolone-containing regimens are generally adequate, but cost-effectiveness and the possibility of community resistance are issues to be considered. Triple or quadruple protracted regimens should be used in serious complications, in conjuction with invasive procedures, as indicated. Rifampicin and trimethoprim-sulfamethoxazole are the mainstays of treatment in pregnancy and pediatric populations, respectively (Pappas et al., 2006).
            • Tetracyclines (AHFS Drug Information 2006): Tetracyclines (doxycycline, tetracycline hydrochloride) generally are considered the drugs of choice for brucellosis. Limited data suggest that combined anti-infective therapy may reduce the likelihood of disease relapse, and some clinicians recommend that another anti-infective (e.g., streptomycin or gentamicin and/or rifampin) be used in conjunction with a tetracycline for the treatment of brucellosis. For treatment of serious brucellosis or when there are complications, including endocarditis, meningitis, or osteomyelitis, some clinicians recommend that an aminoglycoside (streptomycine or gentamicin) be used concomitantly with the tetracycline for the first 7-14 days of therapy; rifampin can also be used in the regimen to reduce the rate of relapse (AHFS Drug Information 2006).
              • Applicable: Doxycyclin -- Minimum inhibitory concentration: 0.06-1ug/ml; Dose -- 100 mg twice daily for 6 wk (Pappas et al., 2006).
              • Contraindicator: Tetracyclines are contraindicated in patients hypersensitive to any of the tetracyclines (AHFS Drug Information 2006). Tetracyclines should not be used in children younger than 8 years of age unless other appropriate drugs are ineffective or are contraindicated (AHFS Drug Information 2006). Doxycycline is contraindicated in nursing women (AHFS Drug Information 2006).
              • Complication: Adverse effects reported in 6% or more of patients receiving doxycycline hyclate tablets (containing 20 mg of doxycycline) include headache, common cold, flu symptoms, toothache or tooth disorder, GI symptoms (diarrhea, nausea, dyspepsia), and joint pain (AHFS Drug Information 2006). Use of tetracyclines may result in overgrowth of nonsusceptible organisms, including fungi (AHFS Drug Information 2006).
              • Success Rate: Tetracyclines are among the most potent antibiotics against Brucella, and they have the advantages of oral administration and low toxicity (Young, 1989).
              • Drug Resistance: Tetracyclines generally are active in vitro and in vivo against the following gram-negative bacteria: Brucella (AHFS Drug Information 2006).
            • Rifampin (AHFS Drug Information 2006): Rifampin is used as an adjunct to other anti-infective agents for the treatment of brucellosis. Tetracyclines generally are considered the drugs of choice for the treatment of brucellosis; however, concomitant use of another anti-infective (e.g., streptomycin or gentamicin and/or rifampin) may reduce the likelihood of disease relapse and usually is recommended in serious infections or when there are complications such as meningitis, endocarditis, or osteomyelitis (AHFS Drug Information 2006).
              • Applicable: Rifampin is the mainstay of treatment in cases of brucellosis during pregnancy, in various combinations (Pappas et al., 2006). Rifampin -- Minimum inhibitory concentration: 0.1-2 ug/ml; Dose: 600-1200 mg/day for 6 wk (Pappas et al., 2006).
              • Contraindicator: Rifampin is contraindicated in patients with a history of hypersensitivity to the drug or any of the rifamycins (AHFS Drug Information 2006).
              • Complication: Because rifampin used alone or in conjunction with other drugs has been associated with adverse hepatic effects (e.g., severe liver injury) and adverse hematologic effects, liver function (hepatic enzymes, bilirubin) and hematologic status (complete blood cell and platelet counts) should be assessed prior to initiation of rifampin therapy (AHFS Drug Information 2006). Another consideration in the use of rifampin is its interaction with other drugs. Rifampin is known to accelerate the metabolism of a variety of drugs, including anticoagulants and digitalis, through the induction of hepatic microsomal enzymes (Young, 1989).
              • Success Rate: Unfortunately, the relapse rate in patients treated with rifampin alone was unacceptable high, and for this reason it was recommended that it was best used in combination with other anti-brucella drugs. In some studies the combination of rifampin-doxycycline was comparable to treatment with tetracycline-streptomycin, with a relapse rate of < 5%. In contrast, others reported a relapse rate with rifampin-doxycycline as high as 38.8% (Young, 1989).
              • Drug Resistance: Rifampin resistance does not appear to be a major cause of relapse in human brucellosis, since it was shown that the antibiotic susceptibility of Brucella strains recovered from patients in relapse were not different from those recovered before therapy was begun. Nevertheless, rifampin resistance among Brucella strains remains a concern. Corbel reported that rifampin-resistant variants occur in vitro, and recently a rifampin-resistant strain of B. melitensis was recovered during relapse from a patient who had been treated for 6 weeks with rifampin-doxycycline (Young, 1989). The use of rifampin in areas in which brucellosis is endemic, where tuberculosis is also usually endemic, raises concern about the development of community resistance to rifampin (Pappas et al., 2006).
            • Streptomycin (AHFS Drug Information 2006): Streptomycin is used in the treatment of brucellosis. Tetracyclines generally are considered the drugs of choice for the treatment of brucellosis; however, concomitant use of another anti-infective (e.g., streptomycin or gentamicin and/or rifampin) may reduce the likelihood of disease relapse and usually is recommended in serious infections or when there are complications such as meningitis, endocarditis, or osteomyelitis. Some experts recommend a 3-drug regimen that includes a tetracycline, an aminoglycoside, and rifampin for the treatment of brucellosis in patients with meningoencephalitis or endocarditis. Although data are limited, alternative regimens that have been suggested for the treatment of brucellosis include co-trimoxazole with or without gentamicin (or streptomycin) or rifampin; ciprofloxacin (or ofloxacin) and rifampin; and chloramphenicol with or without streptomycin (AHFS Drug Information 2006).
              • Applicable: Streptomycin -- Minimum inhibitory concentration: 0.25-16 ug/ml; Dose: 15 mg/kg of body weight intramuscularly for 2-3 wk (Pappas et al., 2006).
              • Contraindicator: A specific aminoglycoside preparation is contraindicated in patients with a history of hypersensitivity to that preparation or any ingredient in the formulation (AHFS Drug Information 2006). Aminoglycosides should be used during pregnancy only in life-threatening situations or severe infections for which safer drugs cannot be used or are ineffective (AHFS Drug Information 2006).
              • Complication: The major adverse effect of streptomycin therapy is vestibular dysfunction, which is especially common in the elderly (Young, 1989). Parenteral administration of streptomycin mandates either hospital admission or the existence of an adequate health care network - both of which are often absent in areas of endemic disease (Pappas et al., 2006).
              • Success Rate: Used alone, streptomycin is less effective than tetracycline, but in combination with other agents appears to lessen the rate of relapse (Young, 1989). When used alone, both streptomycin and tetracycline are reported to have a high rate of clinical relapse (Young, 1989).
            • Co-trimoxazole (Trimethoprim-sulfamethoxazole) (AHFS Drug Information 2006): Oral co-trimoxazole is considered an alternative to tetracyclines for the treatment of brucellosis when tetracyclines are contraindicated, including brucellosis in pediatric patients. To decrease the incidence of relapse, many clinicians recommend that rifampin be used in conjunction with co-trimoxazole or a tetracycline. For treatment of serious brucellosis or when there are complications, including endocarditis, meningitis, or osteomyleitis, some clinicians recommend that an aminoglycoside (streptomycin or gentamicin) be used concomitantly with co-trimoxazole or a tetracycline for the first 7-14 days of therapy; rifampin can also be included in the regimen to reduce the risk of relapse (AHFS Drug Information 2006).
              • Applicable: The drug is administered by mouth and is preferred over tetracycline for children and pregnant women with brucellosis (Young, 1989). Trimethoprim-sulfamethoxazole -- Minimum inhibitory concentration: 0.38-8 ug/ml; Dose: 960 mg twice daily for 6 wk (Pappas et al., 2006).
              • Contraindicator: Co-trimoxazole is contraindicated in patients with known hypersensitivity to trimethoprim or sulfonamides, with marked hepatic damage or severe renal impairment when renal function status cannot be monitored, or with documented megaloblastic anemia secondary to folate deficiency (AHFS Drug Information 2006). The manufacturers of co-trimoxazole recommend that the drug not be used in infants younger than 2 months of age (AHFS Drug Information 2006).
              • Complication: The most frequent adverse effects of co-trimoxazole are adverse GI effects (nausea, vomiting, anorexia) and sensitivity skin reactions (e.g., rash, urticaria), each reportedly occurring in about 3.5% of patients (AHFS Drug Information 2006). Hypersensitivity and hematologic reactions are the most serious adverse effects of co-trimoxazole, reportedly occurring in less than 0.5% of patients (AHFS Drug Information 2006).
              • Success Rate: When used in a fixed combination (80 mg TMP/400mg SMZ), the drug has proved highly effective in treating brucellosis (Young, 1989). Some authors have reported a relapse rate of approximately 40%, even when the drug was used for 45 days, however, this finding has not been universal, especially in children, where the success rate is high (Young, 1989).
            • Ofloxacin (AHFS Drug Information 2006): Ofloxacin has been used alone in a limited number of patients for the treatment of brucellosis caused by Brucella canis, B. abortus, or B. melitensis (AHFS Drug Information 2006). For the treatment of brucellosis, a 6-week regimen of oral ofloxacin in a dosage of 400 mg once daily in conjunction with oral rifampin (600 mg once daily) has been effective in some patients (AHFS Drug Information 2006).
              • Applicable: Ofloxacin -- Minimum inhibitory concentration: 0.1-2 ug/ml; Dose - 400 mg twice daily for 6 wk (Pappas et al., 2006).
              • Contraindicator: Ofloxacin is contraindicated in patients with a history of hypersensitivity to the drug or to other quinolones (AHFS Drug Information 2006). Safety and efficacy of ofloxacin in children younger than 18 years of age have not been established (AHFS Drug Information 2006). There are no adequate and controlled studies to date using ofloxacin in pregnant women (AHFS Drug Information 2006).
              • Complication: As with other anti-infectives, use of ofloxacin may result in overgrowth of nonsusceptible organisms, especially enterococci or Candida. Resistant strains of some organisms (e.g. Pseudomonas aeruginosa, staphylococci) have developed during ofloxacin therapy (AHFS Drug Information 2006).
              • Success Rate: Relapse has occurred occasionally following initial apparent response, and more study is needed to evaluate efficacy of the drug in the treatment of brucellosis (AHFS Drug Information 2006).

          • Other Information:
            • Chronic brucellosis: Chronic brucellosis remains an enigma, and even the definition of this clinical syndrome is controversial (Young, 1989). Many patients who have had brucellosis describe occasional recurrences of symptoms similar to those they experienced while acutely ill; however, since the symptoms of brucellosis are nonspecific, it is often difficult to ascertain the cause (Young, 1989). Some investigators have suggested that chronic brucellosis results from cellular immune dysfunction, however there is little evidence to support this hypothesis. In fact, the contrary may be the case, since it is reported that Brucella is comparable to BCG in its ability to stimulate cellular immunity, and B. abortus strain 19 vaccine has been used to enhance resistance to neoplasms in experimental animals and humans. Nevertheless, some investigators have administered immunostimulant drugs, such as levamisole, to animals and humans to restore in vitro lymphocyte defects. Levamisole is the optical isomer of the anthelminthic drug tetramisole, which is reported to have immunostimulant properties. Several uncontrolled studies have claimed benefit from using levamisole to treat brucellosis in humans (Young, 1989).

    4. Prevention:
      1. Infection control (Godfroid et al., 2005):
        • Description: As a general rule, prevention of human brucellosis depends predominantly on the control of the disease in animals (Godfroid et al., 2005). It may also be assisted through personal hygiene, environmental sanitation, pasteurization of dairy products, and health education (Nicoletti, 1989).
      2. No authorised human Brucella vaccine (Bossi et al., 2004):
        • Description: There is currently no authorised human Brucella vaccine in the European Union or the US. There is some limited clinical data on a live, attenuated vaccine-candidate strain from the former Soviet Union and China (Bossi et al., 2004). An intradermally administered vaccine derived from B. abortus 19 strain has been used extensively in the Asian Republics of the former Soviet Union, causing a 5-11 fold reduction in the annually reported cases of human brucellosis (Pappas et al., 2006). The vaccine offers limited protection of short duration and requires booster doses (Pappas et al., 2006). An increased number of hypersensitivity reactions were reported, with 76% local reactions and 3-7% generalized adverse effects (Pappas et al., 2006). Due to the frequency of adverse events and the short duration of immunity, it seems that it is no longer used (Bossi et al., 2004). Strains of B. abortus 104M have been used in China. A phenol-insoluble peptidoglycan fraction of B. melitensis strain M15 was used in France (Pappas et al., 2006). Sub-unit vaccine candidates have also been studied but evidence of efficacy is inconclusive. The potential to develop human Brucella vaccine is limited by the small market potential, which will probably restrict development efforts to national defense agencies only (Bossi et al., 2004).

    5. Model System:
      1. Guinea Pig (Garcia-Carrillo, 1990):
        1. Model Host: Guinea Pig (Cavia porcellus)
        2. Model Pathogens:
        3. Description: Studies using guinea pigs to determine virulence of recently isolated brucella strains are almost as old as records of documenting brucella itself. Guinea pigs are probably the animals that are most susceptible to brucella infection. As few as 11 cells of some brucella strains are sufficient to cause infection (Garcia-Carrillo, 1990). All brucellae, with exceptions such as B. ovis, were pathogenic for guinea pigs. Lesions were consistently recorded in the liver, spleen, lungs, and lymph nodes a few hours after subcutaneous inoculation. The organism could be isolated from blood, lymph nodes and internal organs (Garcia-Carrillo, 1990). The guinea pig may be used as an experimental model to study the congenital or neonatal transmission of brucellosis (Garcia-Carrillo, 1990). The guinea pig model is considerably more valuable than the mouse model for the evaluation of new vaccines. Occasionally, results that were promising in mice failed to materialize in guinea pigs, whereas the results obtained using guinea pigs were usually correlated with those obtained in other animals (Garcia-Carrillo, 1990). Delayed hypersensitivity is a common manifestation of human brucellosis, and the phenomenon plays an important role in the pathogenesis of this disease. Allergic tests are used for diagnosis of brucellosis in humans and animals, mainly in goats and sheep. The allergens, methods of sensitization, and concentration of allergens are usually tested in guinea pigs (Garcia-Carrillo, 1990). The guinea pig has been used experimentally to evaluate the therapeutic effects of tetracycline and other chemotherapeutic agents (Garcia-Carrillo, 1990).
      2. Mouse (Garcia-Carrillo, 1990):
        1. Model Host: Mouse (Mus musculus)
        2. Model Pathogens:
        3. Description: Mice develop chronic infections, not only when challenged with virulent brucella strains, but also with attenuated vaccine strains (Garcia-Carrillo, 1990). Virulent brucella multiplies in the spleen and other organs of mice, and resistance is acquired. One month after inoculation, mice are very resistant to intravenous challenge with other virulent brucella strains (Garcia-Carrillo, 1990). Mice have been used to study the in vivo action of antibiotics. Trials showed that, in these animals, rifampicin was as good as or even better than tetracycline. Ampicillin and the trimethoprim-sulfametoxazole combination showed slight activity (Garcia-Carrillo, 1990).
      3. Rabbit (Garcia-Carrillo, 1990):
        1. Model Host: Rabbit (Oryctolagus cuniculus)
        2. Model Pathogens:
        3. Description: Normal rabbits are partially susceptible to brucella infection (Garcia-Carrillo, 1990). Local inflammatory reactions were produced in rabbits by intradermal injection of living brucella, and no differences were observed in the degree of dermal pathogenicity of the 3 species of brucella (Garcia-Carrillo, 1990).
      4. Rat (Garcia-Carrillo, 1990):
        1. Model Host: Rat (Rattus Norvegicus)
        2. Model Pathogens:
        3. Description: Rats are relatively resistant to Brucella infection. It was necessary to administer a high dose (several million cells) to produce generalized infection, and the spleen was usually free of Brucella a month after experimental infection (Garcia-Carrillo, 1990). Some reports show that gray rats (Rattus norvegicus) are more susceptible to B. abortus than to B. melitensis and B. suis (Garcia-Carrillo, 1990).
      5. Hamster (Garcia-Carrillo, 1990):
        1. Model Host: Hamster (Mesocricetus auratus - Syrian or Golden Hamster)
        2. Model Pathogens:
        3. Description: Based on results reported to date, the hamster does not appear to be a good animal model in which to study B. abortus infection, because great individual differences in susceptibility exist. Other experimental conditions, such as routes of inoculation, lapsed time from challenge to sacrifice, etc., need to be studied, however, before the usefulness of this species in brucellosis research can be completely evaluated (Garcia-Carrillo, 1990).
  2. Goats and Sheep:
    1. Taxonomy Information:
      1. Species:
        1. Goat (NCBI Taxonomy):
          • GenBank Taxonomy No.: 9925
          • Scientific Name: Capra hircus (NCBI Taxonomy)
          • Description: Brucella melitensis is primarily responsible for brucellosis in sheep and goats. The disease in goats closely resembles the disease produced in cattle infected with B. abortus (Enright, 1990).
        2. Sheep (NCBI Taxonomy):
          • GenBank Taxonomy No.: 9940
          • Scientific Name: Ovis aries (NCBI Taxonomy)
          • Description: The natural hosts of Brucella melitensis are adult goats and certain breeds of sheep, among which it spreads readily, causing abortion (Alton, 1990).

    2. Infection Process:
      1. Infectious Dose: The 50% infective dose for virulent B. melitensis, administered via the conjunctiva, has been calculated as 2 x 10^4 for Swedish goats and 4 x 10^4 for Swedish sheep. In both cases the animals were adult and not pregnant. It needs to be borne in mind that the sheep may not have been representative of the more susceptible breeds (Alton, 1990).
      2. Description: The infection in female sheep and goats follows a course very similar to B. abortus infection in cattle. Briefly, B. melitensis usually enters the animal's body through the mouth, nose, or eye, though invasion through the skin, especially if abraded, has been shown to be possible (Alton, 1990). Male sheep and goats in an infected herd not infrequently become infected with B. melitensis themselves (Alton, 1990).

    3. Disease Information:
      1. Caprine/Ovine Brucellosis (CDC):
        1. Pathogenesis Mechanism: Brucellae are facultative intracellular parasites of the reticuloendothelial system. After gaining entrance to the body, the organisms encounter the cellular and humoral defenses of the body but generally succeed in arriving via the lymph channels at the nearest lymph node. The fate of the invading bacteria is mainly determined by the cellular defenses of the host, chiefly macrophages and T lymphocytes, though specific antibody undoubtedly plays a part. The outcome depends on the age, immune status of the host, whether or not it is pregnant, and the virulence of the invading bacteria and their numbers. When the bacteria prevail over the body defenses, a bacteremia is generally established (Alton, 1990). During the bacteremic phase the brucellae may be found in almost any organ or tissue, but this does not necessarily indicate that the bacteria are established in these sites. If the animal is pregnant, bacteremia often leads to invasion of the uterus where, in sheep and goats, the synthesis of the sugar erythritol in the developing membranes greatly aids the multiplication of brucellae, as it does in cattle. At the same time, infection generally becomes established in various lymph nodes and organs, often in the udder and sometimes in the spleen (Alton, 1990). The udder is the most important predilection site for B. melitensis. Infection in lactating nonpregnant goats is likely to lead to colonization of the udder with excretion of B. melitensis in the milk (Alton, 1990). Infection of the pregnant uterus often ends in abortion (Alton, 1990).

        2. Prognosis: During the initial bacteremic phase of the infection in goats, individuals may become severely ill and die suddenly, while other individuals are asymptomatic (Enright, 1990). Infection of the pregnant uterus often ends in abortion. Following the parenteral inoculation of large doses of B. melitensis, pregnant goats are almost certain to abort, after a delay of 3 to 4 weeks. However, one study showed that out of 40 pregnant goats that became massively infected in the uterus following natural exposure, 24 aborted and 16 gave birth at the normal time, i.e., only 60% aborted. Goats that have aborted at one pregnancy are less likely to do so at the next, but are likely to shed following parturition. Pregnant sheep, likewise, almost always abort 4 to 12 weeks after experimental parenteral or conjunctival infection. However, when pregnant sheep become infected naturally, they also are less likely to abort (Alton, 1990). Sheep seldom abort a second time (Alton, 1990).

        3. Diagnosis Overview: Isolation and identification of the bacterium is the only certain way of diagnosing B. melitensis infection, and this is often a practical method, especially in goats. However, there are many circumstances where culture methods are not practical or even possible, and it becomes necessary to rely on serological or allergic tests (Alton, 1990). The serological tests generally used for the diagnosis of B. melitensis infection -- Rose Bengal, agglutination and complement fixation -- employ the same particulate antigens that are used for the diagnosis of B. abortus infection in cattle. The versatility of these antigens depends on their ability to react equally with anit-b. abortus sera (with the A antigen) and anti-B. melitensis sera (Alton, 1990). The delayed-type hypersensitivity (DTH) demonstrable in brucella-infected animals has been exploited for diagnosis to a greater extent in sheep and goats than in cattle (Alton, 1990).

        4. Symptom Information :
          • Syndrome -- Caprine or Ovine Brucellosis:
            • Description: Infection of the pregnant uterus often ends in abortion (Alton, 1990). Retention of the uterus often follows involvement of the uterus, but highly satisfactory levels of fertility in infected goats have been reported (Alton, 1990). Symptoms, other than abortion, sometimes followed by retention of the fetal membranes are not usually noticed in naturally infected sheep and goats. However, when animals are artificially infected with large doses of bacteria given parenterally, symptoms including loss of condition, mastitis, and lameness have been reported in goats and in sheep. These, no doubt, occur on rare occasions in natural infection (Alton, 1990). In the acute phase there may be some swelling of lymph nodes and perhaps of the spleen and liver caused by infiltration of plasma cells and polymorphs with swelling and proliferation of the reticuloendothelial cells (Alton, 1990). Male sheep and goats in an infected herd not infrequently become infected with B. melitensis themselves (Alton, 1990). In sheep the infection may be short lived and symptomless When symptoms do develop, orchitis is normally the only manifestation, usually appearing as a large firm swelling of one testicle. Reports of hygromas and lesions in joints have been quoted by Renoux. It is likely that such lesions are very rare. In male goats orchitis, generally unilateral, is quite common. It has the outward appearance of the same condition in bulls infected by B. abortus but appears to have been little studied (Alton, 1990).

    4. Prevention:
      1. B. melitensis Rev. 1 vaccine (Alton, 1990):
        • Description: Rev. 1 vaccine is a living attenuated vaccine prepared from the Rev. 1 strain of B. melitensis. Rev. 1 is a nondependent reverse mutant derived from a streptomycin-dependent strain of B. melitensis, which itself originated from a virulent strain (Alton, 1990). In routine use the recommended dose of Rev. 1 is 10^9 viable organisms in a volume of 1 to 2 ml injected subcutaneously (Alton, 1990).
        • Efficacy:
          • Rate: The ability of the vaccine to produce a high level of immunity against both artificial and natural challenge has been convincingly demonstrated both for sheep and goats (Alton, 1990).
          • Duration: The duration of immunity conferred by vaccination with Rev. 1 was investigated by vaccinating Maltese goats when they were 4 to 12 months of age and challenging some at 2.5 and others at 4.5 years after vaccination. Those challenged at 4.5 years were as resistant as those challenged at shorter intervals after vaccination, and it was concluded that immunity could be considered lifelong. Similar results were observed in Iranian sheep challenged 2.5 years after vaccination (Alton, 1990).
        • Contraindicator: Because of the ability of Rev. 1 vaccine to cause abortion, pregnant animals should not be vaccinated, and at least 1 month should be allowed between vaccination and mating (Alton, 1990).
        • Complication: The Rev. 1 strain retains some virulence. The normal vaccinal dose (10^9 cells) injected subcutaneously during pregnancy will regularly produce abortion with excretion from the vagina, and sometimes in the milk of both sheep and goats (Alton, 1990). Rev.1 is a smooth organism, therefore it induces positive serology which interferes with the diagnosis (Schurig et al., 2002). Though greatly attenuated, the Rev. 1 strain is capable, under certain circumstances, of causing local hypersensitivity reactions or generalized brucellosis. Workers have contracted brucellosis from aerosols produced in the manufacturing process, and accidental self-inoculation with vaccine may occur in the field (Alton, 1990).
      2. Orally administratable B. suis strain 2 vaccine (Alton, 1990):
        • Description: This vaccine, which can be given orally, was developed in China. It is of special interest for the vaccination of sheep and goats in difficult and arid terrain where it can be given in drinking water. The vaccine consists of a smooth attenuated strain of B. suis biovar 1, apparently indistinguishable, except in its virulence, from field strains (Alton, 1990). The vaccine has been used in Inner Mongolia for almost 20 years to vaccinate many millions of sheep and goats. Here the vaccine is usually administered in drinking water. No problems have emerged, and it is claimed that vaccination has reduced the incidence of brucellosis in the region to a very low level (Alton, 1990).
        • Efficacy:
          • Rate: Good protection has been demonstrated in sheep and goats, persisting for up to 4 years (Alton, 1990).
          • Duration:
        • Complication: It causes abortion in pregnant sheep and goats if injected in full doses, but not when given orally (Alton, 1990).
      3. B. melitensis H38 adjuvant vaccine (Alton, 1990):
        • Description: This is a killed vaccine prepared from a virulent smooth strain of B. melitensis biovar 1, called H38, emulsified in an oily adjuvant. It is available commercially (Alton, 1990). The vaccine has been used in Inner Mongolia for almost 20 years to vaccinate many millions of sheep and goats. Here the vaccine is usually administered in drinking water. No problems have emerged, and it is claimed that vaccination has reduced the incidence of brucellosis in the region to a very low level (Alton, 1990). The advantage of H38 over Rev. 1 is that it can be given to animals of any age whether they are pregnant or not (Alton, 1990).
        • Efficacy:
          • Rate: Good results have been reported following the vaccination of small ruminants with H38 vaccine in the field, but there have been disappointments. Because of the disadvantages mentioned [below], H38 vaccine has not received wide acceptance in field use (Alton, 1990).
          • Duration:
        • Complication: H38 vaccine has two considerable disadvantages. First, it gives rise to a prolonged serological and allergic response. Second, it produces a local reaction at the site of inoculation that sometimes leads to suppuration and often antagonizes owners (Alton, 1990).
      4. Hygienic measures (Alton, 1990):
        • Description: Hygienic measures help to limit the spread of infection in the flock. Furthermore, the resistance provided by vaccination against B. melitensis is by no means absolute, and measures that will lessen the natural challenge presented to vaccinated sheep and goats will improve the chances of success. Briefly, there are five main areas needing attention. (1) Owners need to be educated in the basic principles and to know whether their flocks are infected or not. (2) The provision of clean accommodation at parturition that can be disinfected is very advantageous. Individual pens, where possible, are advantageous. In contrast, the repeated use of fouled, crowded pens for lambing and kidding aids the spread and the persistence of infection. (3) In an infected herd, all parturitions should be regarded as potential sources of infection and the nonliving birth products (membranes and aborted fetuses) should be incinerated where possible, otherwise buried deeply. Heavy, leak-proof, agricultural plastic sacks are useful for transporting potentially contaminated materials. Contaminated areas should be disinfected before and after sweeping or hosing down. Animals that abort should be removed, but not sold, except for immediate slaughter. (4) Unnecessary visitors, especially children, should be kept away during the lambing period. (5) Hygiene at milking should be adequate to prevent the spread of infection from udder to udder or to the milker (Alton, 1990).
      5. Eradication by test and slaughter (Alton, 1990):
        • Description: The most certain way to eradicate B. melitensis infection is to identify infected herds and to slaughter the whole herd. Few countries with a B. melitensis problem will have the necessary means to eradicate the infection on a national scale by test and slaughter of reactors and will need to rely on vaccination to control the disease. Nevertheless, where the overall individual prevalence of the disease is below 2% and where flocks are maintained under closely controlled conditions and can be protected against reentry of infection, eradication may be feasible, perhaps on a farm or regional basis, provided (1) an adequate veterinary organization for surveillance and laboratory testing is available and (2) the administrative means and will to implement the program, including the control of movements of animals into clean herds, is present (Alton, 1990).

    5. Model System:

      No model system information is currently available here.

IV. Labwork Information

A. Biosafety Information:
  1. Biosafety information for : Brucella melitensis :
    • Biosafety Level: Biosafety Level 2, Biosafety Level 3, and Animal Biosafety Level 3 (CDC)
    • Applicable: Brucellosis continues to be the most commonly reported laboratory-associated bacterial infection. B. abortus, B. canis, B. melitensis, and B. suis have all caused illness in laboratory personnel. Hypersensitivity to Brucella antigens is also a hazard to laboratory personnel. Occasional cases have been attributed to exposure to experimentally and naturally infected animals or their tissues (BMBL). Laboratory-acquired infections are occasionally reported and the inhalation of infective aerosols produced accidentally by microbiological techniques is the most frequent source of infection (Godfroid et al., 2005). The agent may be present in blood, cerebrospinal fluid, semen, and occasionally urine. Most laboratory-associated cases have occurred in research facilities and have involved exposure to Brucella organisms being grown in large quantities. Cases have also occurred in a clinical laboratory setting: direct skin contact with cultures or with infectious clinical specimens from animals (e.g., blood, uterine discharges) are commonly implicated in these cases. Aerosols generated during laboratory procedures have caused large outbreaks. Mouth pipetting, accidental parenteral inoculations, and sprays into eyes, nose and mouth have also resulted in infection (CDC).
    • Precautions:
      • Biosafety Level 2 practices are recommended for activities with clinical specimens of human or animal origin containing or potentially containing pathogenic Brucella spp. Biosafety Level 3 and Animal Biosafety Level 3 practices, containment equipment and facilities are recommended, respectively, for all manipulations of cultures of the pathogenic Brucella spp. listed in this summary [above], and for experimental animal studies. Vaccines are not available for use in humans (CDC).
    • Disposal:
      • Preserve original specimens pursuant to a potential criminal investigation and possible transfer to an appropriate Laboratory Response Network (LRN) laboratory. FBI and state public health laboratory/state public health department will coordinate the transfer of isolates/specimens to a higher-level LRN laboratory as appropriate (American Society for Microbiology)
B. Culturing Information:
  1. Brucella broth or solid medium culture (Ozkurt et al., 2002):
    1. Description: Selective media are usually required for the culture of specimens from the field, especially fetal membranes which are usually grossly contaminated (Alton, 1990). The selective medium of Kuzdas and Morse, containing the antibiotics cycloheximide, bacitracin, and polymyxin B has been used extensively for the culture of B. melitensis and is adequate in most situations (Alton, 1990). Culture in static liquid medium is not recommended and much better yields are obtained in well-aerated shaken liquid medium. Smooth strains are particularly liable to undergo dissociation to nonsmooth variants in static liquid culture (Corbel, 1989). On suitable medium growth is not usually evident in less than 48 h, especially on primary isolation. Laboratory-adapted strains tend to grow more vigorously and will often produce visible colonies after 24-h incubation (Corbel, 1989). After 48 h growth at 37C on serum dextrose agar or similar transparent solid medium, most Brucella strains will produce colonies 0.5 to 1.0 mm in diameter. These are raised, convex, and have a circular outline. Their color and texture depend upon the colonial phase of the organism. In the case of smooth strains, the colonies are transparent and pale yellow ("droplet of honey" appearance) with a shiny surface when viewed in transmitted light. In reflected light, the colonies have a bluish translucence. Nonsmooth colonies tend to be more opaque, with a granular surface and can vary in color from dead white to dark brown; off-white to buff are probably the most frequently seen colors. Unlike smooth colonies which are easily emulsified in water or saline to produce uniform suspensions, nonsmooth colonies are difficult to disperse and tend to form gritty, slimy, or lumpy suspensions depending on the degree of dissociation (Corbel, 1989). Growth of B. melitensis is not dependent on serum in the medium or an atmosphere enhanced by the addition of CO2, but is somewhat improved by the presence of either or both of these (Alton, 1990).

    2. Medium:
      1. For most purposes, serum dextrose agar is a suitable solid medium. Serum dextrose broth is the equivalent liquid medium (Corbel, 1989). Brucella broth: tryptone 10 g, peptamine 10 g, dextrose 1 g, yeast extract 2 g, sodium chloride 5 g, sodium bisulfite 0.1 g, supplement [cycloheximide, bacitracin, circulin and polymyxin B], and 0.25 mL of 4% sodium citrate as anticoagulant (Ozkurt et al., 2002).
    3. Optimal Temperature: 37C (Corbel, 1989)
  2. Blood culture (Diaz and Moriyon, 1989):
    1. Description: The blood broth culture in 10% CO2 is the simplest and most often used bacteriologic procedure. However, the relative proportion of successful blood cultures reported varies between 85.4 and 17% (Diaz and Moriyon, 1989). Conventional blood cultures are seldom positive by the 4th day of incubation; the majority are positive between the 7th and 21st days and 2% are positive after the 27th day. For this reason, it is recommended that cultures be maintained for at least 45 days before declaring them negative (Diaz and Moriyon, 1989). Unless Castaneda's method is used, a culture suspected of Brucella has to be subcultured on solid media for identification (Diaz and Moriyon, 1989).

  3. Bone marrow culture (Pappas et al., 2006):
    1. Description: Bone marrow cultures are considered the gold standard for the diagnosis of brucellosis, since the relatively high concentration of brucella in the reticuloendothelial system makes it easier to detect the organism. Furthermore, bacterial elimination from the bone marrow is equivalent to microbial eradication. However, harvesting bone marrow for culture remains an invasive, painful technique, and results have not been universally reproducible (Pappas et al., 2006).

C. Diagnostic Tests :
  1. Organism Detection Tests:
    1. Light Microscopy, General Culture Characteristics, and Biochemical Tests (Corbel, 1989):
      1. Time to Perform: 1-to-2-days
      2. Description: MAJOR CHARACTERISTICS OF BRUCELLA: (a) Small, gram-negative coccobacilli. (b) Grows only in aerobic blood culture bottles after 2-4 days. (c) Grows as typical colonies on BAP (Sheep blood agar) and CHOC (chocolate agar) within 48 h. Isolates typically do not grow on MAC (MacConkey) or EMB, although pinpoint colonies have been infrequently observed on these media after extended incubation times (7 days). Colony morphology on BAP: Brucella will appear as punctate colonies after 48 h. Colonies are non-pigmented and non-hemolytic. (d) Positive for oxidase, catalase, and urea (American Society for Microbiology). PRESUMPTIVE IDENTIFICATION OF BRUCELLA SPECIES: (a) Brucella species will grow on subculture after 48 h of incubation in 5 to 10% CO2 on CHOC and BAP. (b) The organism does not show typical gram negative rod colony morphology on MAC within 48 hours, which will allow it to be separated from some other gram-negative coccobacilli. (c) The colonies typically show "dust-like" growth after overnight incubation, and a minimum of 48 h is necessary to get sufficient growth for further identification. (d) Colonies are smooth, convex, and raised with an entire edge (i.e., they have no distinguishing features) (American Society for Microbiology).
      3. Picture(s):
        • Brucella spp. are gram-negative in their staining morphology (Gram staining):

          Description: Brucella spp. are poorly staining, small gram-negative coccobacilli (0.5-0.7 x 0.6-1.5 um), and are seen mostly as single cells and appearing like "fine sand" (CDC PHIL).
        • Brucella melitensis colonies (Colonies):

          Description: Brucella spp. Colony Characteristics: - A. Fastidious, usually not visible at 24h. - B. Grows slowly on most standard laboratory media (e.g. sheep blood, chocolate and trypticase soy agars). Pinpoint, smooth, entire translucent, non-hemolytic at 48h (CDC PHIL).

  2. Immunoassay Tests:
    1. Serum Agglutination Test (SAT) (Diaz and Moriyon, 1989):
      1. Time to Perform: 1-to-2-days
      2. Description: Because of its simplicity, the SAT is the most widely used of all serologic tests for brucellosis. Wright and Smith were the first to describe the presence of agglutinins in sera of both animals and humans infected with Brucella (Diaz and Moriyon, 1989). This test may be done as a tube or a plate test (Alton, 1990). The SAT is normally conducted by making doubling dilutions of the serum in phenol saline in round-bottomed tubes and adding an equal volume of the standard antigen. After mixing, the tubes are incubated overnight at 37C, and the degree of agglutination is then read by comparing the opacity against standards representing various degrees of agglutination. The resulting titer should then be converted to international units. Antigens may be prepared from suspensions grown in liquid medium by batch or continuous culture and are equally satisfactory, provided the satisfactory precautions are taken to prevent dissociation (MacMillan, 1990). Titers above 1:160 are considered diagnostic in conjunction with a compatible clinical presentation. However, in areas of endemic disease, using a titer of 1:320 as diagnostic may be more specific (Pappas et al., 2006). Serum agglutination tests have a major drawback in that they are not suitable for patient follow-up, since titers can remain high for a prolonged period (Pappas et al., 2006). The agglutination test is very sensitive to antibody resulting from vaccination (Alton, 1990).
      3. False Positive: Specificity: 94% and 99.5% when interpreted at the 30- and 60-IU levels, respectively (Dohoo et al., 1986).
      4. False Negative: Sensitivity: 98.3% and 93.1% for each of these levels (Dohoo et al., 1986).
    2. Rose Bengal (RB) Test (Diaz and Moriyon, 1989):
      1. Time to Perform: minutes-to-1-hour
      2. Description: The RB test is a modification of the acid-plate-agglutination test introduced by Pietz for the screening of bovine brucellosis, and uses a suspension of B. abortus smooth cells stained with Rose Bengal dye buffered at pH 3.65. The potential usefulness of the RB test for diagnosing human brucellosis was suggested by the Food and Agriculture Organization/World Health Organization Committee and this method has replaced the rapid slide test of Huddleson which was prone to false-negative results (Diaz and Moriyon, 1989). The test is usually conducted on ruled enamel strips, on a glass or ceramic tile, or in a WHO hemagglutination plate. Thirty microliters of the serum and antigen are applied, each having been allowed sufficient time to attain room temperature. The reaction mixture is rocked or oscillated for 4 min, and then the tests are read by examining for agglutination in good light (MacMillan, 1990). The RBT is very sensitive to vaccinal antibody, seriously limiting its use in vaccinated animals (Alton, 1990).
      3. False Positive: The RB test can give false-positive reactions with sera from patients infected with Yersinia enterocolitica O:9, or healthy individuals who have been exposed to smooth Brucella. However, no false-positive reactions were found with patients infected with tularemia or those vaccinated against Vibrio cholerae (Diaz and Moriyon, 1989).
      4. False Negative: In our laboratory, of 173 sera from patients with acute brucellosis, only one was RB-negative (Diaz and Moriyon, 1989).
    3. Complement Fixation (CF) Test (Diaz and Moriyon, 1989):
      1. Time to Perform: 1-hour-to-1-day
      2. Description: As early as 1938, complement fixation was considered by Russian workers as the most specific and valuable serologic test for brucellosis (Diaz and Moriyon, 1989). Despite its complexity and the diversity of techniques employed in different laboratories there is practically unanimous agreement that the complement fixation test is the most effective of those commonly used for the diagnosis of brucellosis in individual sheep and goats (Alton, 1990). The basis for the complement fixation test (CFT) is that dilutions of serum from cattle, sheep or goats (swine sera may be used as well but require addition of normal porcine serum to supplement as a modifying factor), antigen (usually whole cells) and a pretitrated amount of complement (guinea pig serum is used as a source of complement while indigenous complement in the test serum is inactivated by heating for 30-60 min at 56 C) are added together. If antibody is present in the serum, it will bind to the antigen and providing the antibody is IgG1 isotype, complement will be activated. An indicator system is then added. It consists of sheep erythrocytes sensitized with rabbit antibody. If the test serum contained antibody, complement is not available and lysis of the erythrocytes will not take place. Alternately, if no antibody was present, the available complement activated by interaction with receptors on the Fc portion of the rabbit antibody will lyse the erythrocytes, releasing hemoglobin, which is assessed visually, or using a spectrophotometer (Nielsen et al., 2002). The CFT is outstanding among the available diagnostic tests in its relative lack of sensitivity to antibody resulting from vaccination, particularly vaccination with Rev. 1 (Alton, 1990).
      3. False Positive: Nonspecific reactions are rare, although recently a phenomenon was recognized in Great Britain in which 0.5% of all samples tested in the county of Lancashire failed the CFT (MacMillan, 1990).
      4. False Negative: To cite a few reports from the literature, Alton et al. found that all culturally positive animals reacted to the CFT. Nicoletti reported that the CFT correctly identified 98% of culturally positive animals (MacMillan, 1990).
    4. Anti-globulin (Coombs') Test (MacMillan, 1990):
      1. Time to Perform: 1-to-2-days
      2. Description: The antiglobulin test was developed to detect antibodies which, although they combine with the antigen, do not give rise to agglutination. The presence of these so-called "incomplete agglutinins" can be detected using an antibody directed against the IgG fraction of the species of animal being tested. These reagents can be obtained commercially. The test is performed in two phases. First, the conventional SAT is carried out, and after reading, those tubes that do not show any agglutination are centrifuged and the deposited cells washed thoroughly. The presence, if any, of the nonagglutinating Brucella antibody on the Brucella antigen is detected by adding the antiglobulin reagent at its working dilution and either reincubating the tubes and reading after 24-h of incubation or conducting the test on a slide (MacMillan, 1990). Depending on the specificity of the antiglobulin reagent, all the major immunoglobulins can take part in the test and it is undoubtedly more sensitive than the ordinary tube agglutination test. It is reported to be ov value in the early stages of the disease as well as in clearing nonspecific agglutination reactions (MacMillan, 1990). Diagnosis of a relapse is particularly difficult and is most often based on the presence of high titers in the Coombs test. However, this is a long and technically difficult test, requiring skilled personnel, and so it is not routinely performed in many clinical laboratories (Orduna et al., 2000).
      3. False Positive: Specificity: 99.8% (> 1/160 diagnostic threshold titer) (Orduna et al., 2000).
      4. False Negative: Sensitivity: 91.5% (> 1/160 diagnostic threshold titer) (Orduna et al., 2000).
    5. Delayed-type hypersensitivity (DTH) allergic tests (Blasco et al., 1994):
      1. Time to Perform: 2-to-7-days
      2. Description: The delayed-type hypersensitivity (DTH) demonstrable in brucella-infected animals has been exploited for diagnosis to a greater extent in sheep and goats than in cattle (Alton, 1990). A total of 291 unvaccinated sheep from Brucella melitensis-infected flocks were examined for delayed-type hypersensitivity (DTH) responses with Brucellergene commercial allergen and with cold saline extract and with cytosol from rough B. melitensis 115 (Blasco et al., 1994). The DTH response was maximal after 72 h, no matter the allergen, dose, or inoculation site, and no significant differences in the intensity of the reaction were observed among inoculation sites, doses tested (for CSA and cytosol), or allergens (Blasco et al., 1994). Unfortunately, a long-lasting allergic state (for 2 years or more) is provoked by vaccination with either Rev. 1 or H38 vaccines (Alton, 1990).
      3. False Positive: It is generally acknowledge that DTH tests show no positive reactions (100% specificity) for Brucella-free flocks, and this is supported by the results obtained in our work with the 100 Brucella-free sheep (Blasco et al., 1994).
      4. False Negative: Sensitivity: 97.1% (Blasco et al., 1994).
    6. Dipstick assay (Casao et al., 2003):
      1. Time to Perform: minutes-to-1-hour
      2. Description: The dipstick assay is a rapid test that detects Brucella-specific IgM antibodies. The technique provides a quick result, is easy to perform, and does not require trained personnel (Casao et al., 2003). The detection of Brucella-specific immunoglobulin M (IgM) antibodies allows the diagnosis of patients with brucellosis at an early stage or acute disease and also may help to discriminate between patients in the early phase of brucellosis and those with chronic brucellosis (Smits et al., 1999). The dipstick assay consists of a strip of nitrocellulose-containing specific antigen applied in a distinct line, and a non-enzymatic detection reagent (Casao et al., 2003). The detection reagent consists of a monoclonal antihuman IgM antibody conjugated to Palanyl red (Casao et al., 2003). Specific IgM antibodies that are detected in the dipstick assay are present in the serum of patients during the early stages of the disease. Patients with a long period of evolution will probably have a negative dipstick test, but can be diagnosed with the aid of the Coombs test and classical clinical findings (Casao et al., 2003).
      3. False Positive: Only 4 of 297 samples from the noncase patients gave a positive result in the dipstick assay, giving a specificity of 98.6% (Smits et al., 1999).
      4. False Negative: The sensitivity of the dipstick assay was 89.0% for the samples collected within 2 months after the onset of the disease and 83.1% for the samples collected 2 to 4 months after the onset of the disease. The sensitivity dropped to 32.6 and 29.8% for the two groups of samples collected after 4 and 6 months of treatment, respectively (Smits et al., 1999).
    7. Brucellacapt (Orduna et al., 2000):
      1. Time to Perform: 1-hour-to-1-day
      2. Description: Brucellacapt is an inmmunocapture-agglutination technique, which detects all antibodies against Brucella (Casao et al., 2004). The use of Brucellacapt in the diagnosis of human brucellosis could help to detect the disease in patients with long evolution times, which cannot be detected with SAT. Brucellacapt is also easier to perform than Coombs test (Casao et al., 2004). Although Brucellacapt can have advantages respect other classical tests, it can hardly replace a rapid screening tests as RB or dipstick in a first diagnostic, because Brucellacapt is more complex, expensive and slow. However, Brucellacapt could be a second level test such as the Coombs test which offers similar sensitivity and specificity (Casao et al., 2004). The Brucellacapt test (Vircell SL) was performed as specified by the manufacturer. Briefly, 50 ul samples of serum dilutions were added to wells of a U-bottom microtiter plate coated with anti-total human immunoglobulin. Then 50 ul of an antigen suspension was added to all the wells. The plates were sealed with adhesive tape and incubated at 37 C for 24 h in a dark humid chamber. Positive reactions show agglutination over the bottom of the well. Negative reactions are indicated by a pellet at the center of the bottom of the well (Casao et al., 2004).
      3. False Positive: Specificity of the Brucellacapt - 97.5% (Casao et al., 2004).
      4. False Negative: Ninety-six percent of the patients were Brucellacapt positive at the onset of the disease. The only negative patient became positive 15 days after. This sensitivity was similar to Coombs (100%) and higher than SAT (73%) (Casao et al., 2004).
    8. I-ELISA to differentiate between infected and Rev.1-vaccinated sheep (Cloeckaert et al., 2001):
      1. Time to Perform: 1-hour-to-1-day
      2. Description: Previously a Brucella protein named CP28, BP26, or Omp28 has been identified independently by three research groups as an immunodominant antigen in infected cattle, sheep, goats, and humans (Cloeckaert et al., 2001). The BP26 protein appeared particularly useful for the differentiation of serological responses of infected and Rev.1-vaccinated sheep, since in the latter no detectable antibody responses against BP26 were observed either by immunoblotting, I-ELISA using the partially purified native protein, or competitive ELISA (C-ELISA) using MAbs against BP26 (Cloeckaert et al., 2001).
      3. False Positive: The cutoff of the recombinant BP26 I-ELISA was determined with sera from 106 healthy sheep (group C) at an absorbance value of 0.6. Under these conditions, the specificity of the recombinant BP26 I-ELISA was 93% (Cloeckaert et al., 2001)
      4. False Negative: The sensitivity of the I-ELISA assessed with sera from naturally infected and suspected sheep found positive in the current conventional diagnostic tests was as follows: 100% for bacteriologically and serologically positive sheep (n = 50), 88% for bacteriologically negative but serologically and delayed-type hypersensitivity-positive sheep (n = 50), and 84% for bacteriologically and serologically negative but delayed-type hypersensitivity-positive sheep (n = 19) (Cloeckaert et al., 2001).
    9. Fluorescence polarization assay (Lucero et al., 2003):
      1. Time to Perform: minutes-to-1-hour
      2. Description: The fluorescence polarization assay (FPA) was developed as a test that could be performed outside the diagnostic laboratory, allowing for rapid and accurate diagnosis. The basis of the test is that a molecule in solution rotates randomly at a rate inversely proportional to its size. If the molecule is labeled with a fluorescent marker and is examined by plane polarized light, a small molecule will rotate through a given angle faster than a larger molecule. The time of rotation may be measured using horizontal and vertical measurements. For diagnosis of brucellosis, a fluorescence polarization analyzer is used to obtain a background measurement of fluorescence of diluted serum. Antigen consisting of an OPS fragment, approximately 22 kDa in size, labeled with fluorescein isothiocyanate is added and incubated for 2 min, followed by a final reading in the analyzer which automatically subtracts the background reading. The net result is presented in millipolarization units. The FPA can be performed almost anywhere using a portable analyzer which receives power from a laptop computer, using serum, milk or EDTA anticoagulated blood. The test is rugged, relatively inexpensive, simple and very rapid. The FPA has been validated for a large number of species, including cattle, swine, bison and a number of cervids and is an alternate OIE test for bovine and swine brucellosis diagnosis (Nielsen et al., 2002).
      3. False Positive: Based on 340 sera from asymptomatic blood donors with no evidence of brucellosis, the specificity of the FPA was 97.9 % using a cut-off value of 72 mP (Lucero et al., 2003).
      4. False Negative: Sera from Brucella-infected patients (11 Brucella melitensis, 32 Brucella abortus, 32 Brucella suis and one Brucella sp.) yielded a sensitivity estimate of 96.1 % (Lucero et al., 2003).

  3. Nucleic Acid Detection Tests: :
    1. Genus-specific identification of Brucella based on 16S rRNA gene (Herman et al., 1992):
      1. Time to Perform: 1-hour-to-1-day
      2. Description: The application of two synthetic oligonucleotides as probes and as primers in the polymerase chain reaction is presented for a specific, sensitive, and quick identification of Brucella spp. The specific oligonucleotide sequences were chosen on the basis of a 16S rRNA sequence alignment between Brucella abortus and Agrobacterium tumefaciens (Herman et al., 1992). Successful amplification of a predicted 800-bp amplicon from B. abortus and other species of Brucella demonstrated that the sequences were highly conserved and that the test could be extended to the entire genus (Bricker et al., 2002).
      3. Primers:
      4. False Positive: To assess specificity, the assay was applied to a panel of 17 other bacteria. No products were amplified from any non-Brucella species except Ochrobactrum anthropi, the closest known relative to Brucella (Bricker et al., 2002).
    2. Species-specific identification of Brucella spp. with the AMOS-PCR assay (Ewalt et al., 2000):
      1. Time to Perform: 1-hour-to-1-day
      2. Description: PCR assays have been developed to differentiate among Brucella species and/or biovars. These assays are directed toward genetic loci that are variable among the species/biovars. Such targets are uncommon in Brucella since the genus is remarkably homogeneous and has been proposed to be a single species (Bricker et al., 2002). We describe a PCR assay that comprises five oligonucleotide primers which can identify selected biovars of four species of Brucella. Individual biovars within a species are not differentiated. The assay can identify three biovars (1, 2, and 4) of B. abortus, all three biovars of B. melitensis, biovar 1 of B. suis, and all B. ovis biovars. These biovars include all of the Brucella species typically isolated from cattle in the United States, a goal of the present research. The assay exploits the polymorphism arising from species-specific localization of the genetic element IS711 in the Brucella chromosome (Bricker et al., 1994). The multiplex design consists of one common primer anchored in the IS element and a species-specific primer that binds to the unique sequence flanking that insertion site. The assay primers were chosen so that species discrimination was determined by the size of the amplicon (Bricker et al., 2002). The assay was named the AMOS-PCR assay for the Brucella species (Bricker et al., 2002).
      3. Primers:
      4. False Positive: Six closely related bacteria (Agrobacterium radiobacter, Agrobacterium rhizogenes, Ochrobactrum anthropi, Rhizobium leguminosarum, Rhizobium meliloti, and Rhzodospirillum rubrum) and two control bacteria (Bordeteila bronchiseptica and Escherichia coli) tested negative by the assay (Bricker et al., 1994).
      5. False Negative: The performance of the assay with U.S. field isolates was highly effective. When 107 field isolates were screened by the described method, there was 100% agreement with the identifications made by conventional methods (Bricker et al., 1994).
    3. PCR-EIA for diagnosis of acute human brucellosis (Vrioni et al., 2004):
      1. Time to Perform: 1-hour-to-1-day
      2. Description: A PCR assay with primers B4 and B5 was used to detect Brucella DNA in whole blood and serum. These primers specifically amplify a 223-base pair fragment from the conserved region of the gene, which encodes an immunogenic membrane protein of 31 kDa of Brucella abortus, specific to the Brucella genus and present in all its biovars (Vrioni et al., 2004). Amplified products were detected after hybridization in Hybridowell microtiter plates (Argene Biosoft, France), according to the manufacturer's instructions. Briefly, 2 ul of amplified product was coated directly onto a microtiter plate, which was incubated for 2 h at 37 C. After hybridization with a hybridization solution containing 50 ng/ml of the biotinylated oligonucleotidic probe BMpr (TCA GAC GTT GCC TAT TGG GCC) for 30 min at 37 C, hybrids were detected by a streptavidine-peroxidase conjugate. Tetramethylbenzidine was used as the chromogen, and a microplate reader was used to monitor the signal (Vrioni et al., 2004). The PCR-EIA assay can assist the accurate rapid diagnosis of acute human brucellosis efficiently; the speed, simplicity, and handling safety of the kit make it suitable for the workflow of a routine clinical microbiology laboratory (Vrioni et al., 2004).
      3. Primers:
      4. False Positive: Two hundred forty-one of the 243 patients tested had detectable Brucella DNA in either whole blood or serum specimens: 149 (61.3%) patients were positive in both whole blood and serum specimens, 43 (17.7%) were positive in serum specimens only, and 49 (20.2%) were positive in whole blood specimens only. The diagnostic specificity of the PCR-EIA assay for both specimen categories was 100% (Vrioni et al., 2004).
      5. False Negative: The sensitivity was 81.5% for whole blood specimens, 79% for serum specimens, and 99.2% for whole blood and serum specimens combined (Vrioni et al., 2004).

    4. PCR-RFLP (Al Dahouk et al., 2005):
      1. Time to Perform: 1-hour-to-1-day
      2. Description: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) relies on PCR producing considerable amounts of an amplicon of known nucleic acid sequence and subsequent restriction enzyme digestion of the amplified DNA products. The generated fragments are separated by gel electrophoresis according to their respective size (Al Dahouk et al., 2005). Although Brucella spp. represent a highly homogenous group of bacteria, RFLPs of selected genes display sufficient polymorphism to distinguish Brucella species and biovars. PCR-RFLP analysis shows excellent typeability, reproducibility, stability, and epidemiological concordance. Consequently, PCR-RFLP assays of specific gene loci can serve as tools for diagnostic, epidemiological, taxonomic, and evolutionary studies (Al Dahouk et al., 2005). PCR-RFLP analysis was applied to various Brucella genes, i.e. omp22, omp25, omp31, omp2, rpsL, dnaK, and ery. Among all these loci, the omp genes exhibited the most pronounced polymorphism (Al Dahouk et al., 2005).

  4. Other Types of Diagnostic Tests:

    No other tests available here.

V. References

A. Journal References:
Al Dahouk et al., 2005: Al Dahouk S, Tomaso H, Prenger-Berninghoff E, Splettstoesser WD, Scholz HC, Neubauer H. Identification of brucella species and biotypes using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Crit Rev Microbiol. 2005; 31(4): 191 - 196. [PubMed: 16417200].
Blasco et al., 1994: Blasco JM, Marin C, Jimenez de Bagues M, Barberan M, Hernandez A, Molina L, Velasco J, Diaz R, Moriyon I. Evaluation of allergic and serological tests for diagnosing Brucella melitensis infection in sheep. J Clin Microbiol. 1994; 32(8): 1835 - 1840. [PubMed: 7989528].
Bossi et al., 2004: Bossi P, Tegnell A, Baka A, Van Loock F, Hendriks J, Werner A, Maidhof H, Gouvras G; Task Force on Biological and Chemical Agent Threats, Public Health Directorate, European Commission, Luxembourg. Bichat guidelines for the clinical management of brucellosis and bioterrorism-related brucellosis. Euro Surveill. 2004; 9(12): E15 - E16. [PubMed: 15677842].
Bricker et al., 1994: Bricker BJ, Halling SM. Differentiation of Brucella abortus bv. 1, 2, and 4, Brucella melitensis, Brucella ovis, and Brucella suis bv. 1 by PCR. J Clin Microbiol. 1994; 32(11): 2660 - 2666. [PubMed: 7852552].
Bricker et al., 1995: Bricker BJ, Halling SM Enhancement of the Brucella AMOS PCR assay for differentiation of Brucella abortus vaccine strains S19 and RB51. J Clin Microbiol. 1995; 33(6): 1640 - 1642. [PubMed: 7650203].
Bricker et al., 2002: Bricker BJ PCR as a diagnostic tool for brucellosis.. Vet Microbiol. 2002; 90(1-4): 435 - 446. [PubMed: 12414163].
Casao et al., 2003: Casao MA, Smits HL, Navarro E, Solera J. Clinical utility of a dipstick assay in patients with brucellosis: correlation with the period of evolution of the disease. Clin Microbiol Infect. 2003; 9(4): 301 - 305. [PubMed: 12667240].
Casao et al., 2004: Casao MA, Navarro E, Solera J. Evaluation of Brucellacapt for the diagnosis of human brucellosis. J Infect. 2004; 49(2): 102 - 108. [PubMed: 15236916].
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Cloeckaert et al., 2001: Cloeckaert A, Baucheron S, Vizcaino N, Zygmunt MS. Use of recombinant BP26 protein in serological diagnosis of Brucella melitensis infection in sheep. Clin Diagn Lab Immunol. 2001; 8(4): 772 - 775. [PubMed: 11427425].
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Ewalt et al., 2000: Ewalt DR, Bricker BJ. Validation of the abbreviated Brucella AMOS PCR as a rapid screening method for differentiation of Brucella abortus field strain isolates and the vaccine strains, 19 and RB51. J Clin Microbiol. 2000; 38(8): 3085 - 3086. [PubMed: 10921983].
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Herman et al., 1992: Herman L, De Ridder H. Identification of Brucella spp. by using the polymerase chain reaction. Appl Environ Microbiol. 1992; 58(6): 2099 - 2101. [PubMed: 1377903].
Ko et al., 2003: Ko J, Splitter GA. Molecular host-pathogen interaction in brucellosis: current understanding and future approaches to vaccine development for mice and humans. Clin Microbiol Rev. 2003; 16(1): 65 - 78. [PubMed: 12525425 ].
Lucero et al., 1999: Lucero NE, Foglia L, Ayala SM, Gall D, Nielsen K. Competitive enzyme immunoassay for diagnosis of human brucellosis. J Clin Microbiol. 1999; 37(10): 3245 - 3248. [PubMed: 10488186 ].
Lucero et al., 2003: Lucero NE, Escobar GI, Ayala SM, Silva Paulo P, Nielsen K. Fluorescence polarization assay for diagnosis of human brucellosis. J Med Microbiol. 2003; 52(Pt 10): 883 - 887. [PubMed: 12972582].
Mendez et al., 2003: Mendez Martinez C, Paez Jimenez A, Cortes-Blanco M, Salmoral Chamizo E, Mohedano Mohedano E, Plata C, Varo Baena A, Martiinez Navarro F. Brucellosis outbreak due to unpasteurized raw goat cheese in Andalucia (Spain), January - March 2002. Euro Surveill. 2003; 8(7): 164 - 168. [PubMed: 12941982].
Moreno et al., 2002: Moreno E, Cloeckaert A, Moriyon I. Brucella evolution and taxonomy. Vet Microbiol. 2002; 90(1-4): 209 - 227. [PubMed: 12414145].
Nielsen et al., 2002: Nielsen K Diagnosis of brucellosis by serology.. Vet Microbiol. 2002; 90(1-4): 447 - 459. [PubMed: 12414164].
Olle-Goig et al., 1987: Olle-Goig JE, Canela-Soler J. An outbreak of Brucella melitensis infection by airborne transmission among laboratory workers. Am J Public Health. 1987; 77(3): 335 - 338. [PubMed: 3812841].
Orduna et al., 2000: Orduna A, Almaraz A, Prado A, Gutierrez MP, Garcia-Pascual A, Duenas A, Cuervo M, Abad R, Hernandez B, Lorenzo B, Bratos MA, Torres AR. Evaluation of an immunocapture-agglutination test (Brucellacapt) for serodiagnosis of human brucellosis. J Clin Microbiol. 2000; 38(11): 4000 - 4005. [PubMed: 11060059].
Ozkurt et al., 2002: Ozkurt Z, Erol S, Tasyaran MA, Kaya A. Detection of Brucella melitensis by the BacT/Alert automated system and Brucella broth culture. Clin Microbiol Infect. 2002; 8(11): 749 - 752. [PubMed: 12445015].
Pappas et al., 2006: Pappas G, Panagopoulou P, Christou L, Akritidis N. Brucella as a biological weapon.. Cell Mol Life Sci. 2006; 63(19-20): 2229 - 2236. [PubMed: 16964579].
Pappas et al., 2006: Pappas G, Papadimitriou P, Akritidis N, Christou L, Tsianos EV. The new global map of human brucellosis. Lancet Infect Dis. 2006; 6(2): 91 - 9. [PubMed: 16439329 ].
Rajashekara et al., 2006: Rajashekara G, Eskra L, Mathison A, Petersen E, Yu Q, Harms J, Splitter G. Brucella: functional genomics and host-pathogen interactions. Anim Health Res Rev. 2006; 7(1-2): 1 - 11. [PubMed: 17389050].
Schurig et al., 2002: Schurig GC, Sriranganathan N, Corbel MJ. Brucellosis vaccines: past, present and future. Vet Microbiol. 2002; 90(1-4): 479 - 496. [PubMed: 12414166].
Smits et al., 1999: Smits HL, Basahi MA, Diaz R, Marrodan T, Douglas JT, Rocha A, Veerman J, Zheludkov MM, Witte OW, de Jong J, Gussenhoven GC, Goris MG, van Der Hoorn MA. TEXT. Development and evaluation of a rapid dipstick assay for serodiagnosis of acute human brucellosis. 1999; 37(12): 4179 - 4182. [PubMed: 10565959].
Staszkiewicz et al., 1991: Staszkiewicz J, Lewis CM, Colville J, Zervos M, Band J. Outbreak of Brucella melitensis among microbiology laboratory workers in a community hospital. J Clin Microbiol. 1991; 29(2): 287 - 290. [PubMed: 2007637].
Stothard et al., 2005: Stothard P, Van Domselaar G, Shrivastava S, Guo A, O'Neill B, Cruz J, Ellison M, Wishart DS. BacMap: an interactive picture atlas of annotated bacterial genomes.. Nucleic Acids Res. 2005; 33(Database issue): D317 - D320. [PubMed: 15608206].
Thapar et al., 1986: Thapar MK, Young EJ. Urban outbreak of goat cheese brucellosis. Pediatr Infect Dis. 1986; 5(6): 640 - 643. [PubMed: 3797296].
Vrioni et al., 2004: Vrioni G, Gartzonika C, Kostoula A, Boboyianni C, Papadopoulou C, Levidiotou S. Application of a polymerase chain reaction enzyme immunoassay in peripheral whole blood and serum specimens for diagnosis of acute human brucellosis. Eur J Clin Microbiol Infect Dis. 2004; 23(3): 194 - 199. [PubMed: 14986157].
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Yagupsky et al., 2000: Yagupsky P, Peled N, Riesenberg K, Banai M. Exposure of hospital personnel to Brucella melitensis and occurrence of laboratory-acquired disease in an endemic area. Scand J Infect Dis. 2000; 31(1): 31 - 35. [PubMed: 10716074].
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B. Book References:
AHFS Drug Information 2006: McEvoy Gerald K. 1 - 3776. In: AHFS Drug Information 2006.2006. American Society of Health-System Pharmacists, Inc, Bethesda, MD, United States.
Alton, 1990: Alton Godfrey G. Brucella melitensis, 1887 to 1987. 379 - 382. In: Nielson Klaus, Duncan J Robert Animal Brucellosis1990. CRC Press., Boca Raton, Florida.
Alton, 1990: Alton Godfrey G. Brucella melitensis. 383 - 409. In: Nielson Klaus, Duncan J Robert Animal Brucellosis1990. CRC Press., Boca Raton, Florida.
Corbel, 1989: Corbel Michael J. Microbiology of the Genus Brucella. 53 - 72. In: Young Edward J, Corbel Michael J Brucellosis: Clincial and Laboratory Aspects1989. CRC Press, Inc., Boca Raton, Florida.
Corbel, 1989: Corbel Michael J. Brucellosis: Epidemiology and Prevalence Worldwide. 25 - 40. In: Young Edward J, Corbel Michael J Brucellosis: Clincial and Laboratory Aspects1989. CRC Press, Inc., Boca Raton, Florida.
Crawford et al., 1990: Crawford Richard P., Huber Jan D, Adams Bruce S Epidemiology and Surveillance. 131 - 151. In: Nielson Klaus, Duncan J Robert Animal Brucellosis1990. CRC Press., Boca Raton, Florida.
Davis, 1990: Davis Donald S. Brucellosis in Wildlife. 321 - 334. In: Nielson Klaus, Duncan J Robert Animal Brucellosis1990. CRC Press., Boca Raton, Florida.
Diaz and Moriyon, 1989: Diaz Ramon, Moriyon Ignacio Laboratory Techniques in the Diagnosis of Human Brucellosis. 73 - 83. In: Young Edward J, Corbel Michael J Brucellosis: Clincial and Laboratory Aspects1989. CRC Press, Inc., Boca Raton, Florida.
Enright, 1990: Enright Fred M. The Pathogenesis and Pathobiology of Brucella Infection in Domestic Animals. 301 - 334. In: Nielson Klaus, Duncan J Robert Animal Brucellosis1990. CRC Press., Boca Raton, Florida.
Garcia-Carrillo, 1990: Garcia-Carrillo Casimiro. Laboratory Animal Models for Brucellosis Studies. 423 - 442. In: Nielson Klaus, Duncan J Robert Animal Brucellosis1990. CRC Press., Boca Raton, Florida.
MacMillan, 1990: MacMillan Alastair. Conventional Serological Tests. 153 - 197. In: Nielson Klaus, Duncan J Robert Animal Brucellosis1990. CRC Press., Boca Raton, Florida.
Nicoletti, 1989: Nicoletti Paul L. Relationship between Animal and Human Disease. 41 - 52. In: Young Edward J, Corbel Michael J Brucellosis: Clincial and Laboratory Aspects1989. CRC Press, Inc., Boca Raton, Florida.
Nicoletti, 1990: Nicoletti Paul. Vaccination. 283 - 299. In: Nielson Klaus, Duncan J Robert Animal Brucellosis1990. CRC Press., Boca Raton, Florida.
Young, 1989: Young Edward J. Clinical Manifestations of Human Brucellosis. 97 - 126. In: Young Edward J, Corbel Michael J Brucellosis: Clincial and Laboratory Aspects1989. CRC Press, Inc., Boca Raton, Florida.
Young, 1989: Young Edward J. Treatment of Brucellosis in Humans. 97 - 126. In: Young Edward J, Corbel Michael J Brucellosis: Clincial and Laboratory Aspects1989. CRC Press, Inc., Boca Raton, Florida.
C. Website References:
NCBI_Taxonomy: Homo sapiens [ ].
CDC: Brucellosis [ ].
NIAID: Laboratory of Intracellular Parasites [ ].
CDC: AGENT: Brucella [ ].
BMBL: Section VII - A Agent Summary Statements [ ].
Colonies: Brucella melitensis colonies [ ].
American Society for Microbiology: Sentinel Laboratory Guidelines for Suspected Agents of Bioterrorism: Brucella species [ ].
American Society for Microbiology: Sentinel Laboratory Guidelines for Suspected Agents of Bioterrorism: Brucella species [ ].
Agricultural Research Council: Serology [ ].
Brucella melitensis chromosome I, complete sequence: Brucella melitensis chromosome 1 [ ].
Brucella melitensis chromosome II, complete sequence: Brucella melitensis chromosome 2 [ ].
CDC PHIL: 1901 [ ].
CDC PHIL: 1902 [ ].
Cellular traffic: Model of intracellular trafficking and survival of Brucella inside macrophages [ ].
Dennis Kunkel Microscopy, Inc: 96552F [ ].
Gram staining: Brucella spp. are gram-negative in their staining morphology [ ].
NCBI Taxonomy: Brucella melitensis [ ].
NCBI Taxonomy: Brucella melitensis 16M [ ].
NCBI Taxonomy: Brucella melitensis biovar Melitensis [ ].
NCBI Entrez Genome: Brucella melitensis 16M chromosome I, complete sequence [ ].
NCBI Entrez Genome: Brucella melitensis 16M chromosome II, complete sequence [ ].
NCBI Taxonomy: Capra hircus [ ].
NCBI Taxonomy: Ovis aries [ ].
Rose Bengal Test (RBT): Rose Bengal Test (RBT) [ ].
Serum Agglutination Test (SAT): Serum Agglutination Test (SAT) [ ].
D. Thesis References:

No thesis or dissertation references used.

VI. Curation Information