Brucellosis is a disease caused by bacteria in the genus Brucella.

The disease infects animals such as swine, cattle,and sheep; humans can become infected indirectly through contact with infected animals or by drinking Brucella-contaminated milk. In the United States, most domestic animals are vaccinated against the bacteria, but brucellosis remains a risk with imported animal products.Brucella are rod-shaped bacteria that lack a capsule around their cell membranes. Unlike most bacteria, Brucella cause infection by actually entering host cells. As the bacteria cross the host cell membrane, they are engulfed by host cell vacuoles called phagosomes. The presence of Brucella within host cell phagosomes initiates a characteristic immune response, in which infected cells begin to stick together and form aggregations called granulomas. Bacteria of the genus Brucella cause disease primarily in domestic, feral and some wild animals and most are also pathogenic for humans. In animals, brucellae typically affect the reproductive organs, and abortion is often the only sign of the disorder. Human brucellosis is either an acute febrile disease or a persistent disease with a wide variety of symptoms. It is a true zoonosis in that virtually all human infections are acquired from animals. The disease is controlled by the routine practice of pasteurizing milk and milk products, as well as by comprehensive campaigns to eradicate the disease by destroying domestic animals which exhibit positive serologic reactions to brucellae. Vaccines providing some protection to cattle, sheep and goats are available In animals, brucellosis is a self-limiting disease, and usually no treatment is necessary for the resolution of the disease. However, for a period of time from a few days to several weeks, infected animals may continue to excrete brucella into their urine and milk. Under warm, moist conditions, the bacteria may survive for months in soil, milk, and even seawater.Because the bacteria are so hardy, humans may become infected with Brucella by direct contact with the acteria. Handling or cleaning up after infected animals may put a person in contact with the bacteria. Brucella are extremely efficient in crossing the human skin barrier through cuts or breaks in the skin.The incubation period of Brucella, the time from exposure to the bacteria to the start of symptoms, is typically about three weeks. The primary complaints are weakness and fatigue. An infected person may also experience muscle aches,fever, and chills.The course of the disease reflects the location of the Brucella bacteria within the human host. Soon after the Brucella are introduced into the bloodstream, the bacteria seek out the nearest lymph nodes and invade the lymph node cells From the initial lymph node, the Brucella spread out to other

organ targets, including the spleen, bone marrow, and liver. Inside these organs, the infected cells form granulomas.Diagnosing brucellosis involves culturing the blood,liver, or bone marrow for Brucella organisms. Apositive culture alone does not signify brucellosis, since persons who have been treated for the disease may continue to harbor Brucella bacteria for several months. Confirmation of brucellosis, therefore,includes a culture positive for Brucella bacteria as well as evidence of the characteristic symptoms and a history of possible contact with infected milk or other animal products.In humans, brucellosis caused by B. abortus is a mild disease that resolves itself without treatment. Brucellosis caused by B.melitensis and B. suis, however, can be chronic and severe. Brucellosis is treated with administration of an antibiotic that penetrates host cells to destroy the invasive bacteria. Since the invention of an animal vaccine for brucellosis in the 1970s, the disease has become somewhat rare in the United States. Yet the vaccine cannot prevent all incidence of brucellosis. The Centers for Disease Control usually reports fewer than 100 total cases per year in the United States. Most of these were reported in persons who worked in the meat processing industry. Brucellosis remains a risk for those who work in close contact with animals, including veterinarians, farmers, and dairy workers. Brucellosis also remains a risk when animal products from foreign countries are imported into the United States. Outbreaks of brucellosis have been linked to unpasteurized feta and goat cheeses from the Mediterranean region and Europe. In the 1960s, brucellosis was linked to bongo drums imported from Africa; drums made with infected animal skins can harbor Brucella bacteria, which can be transmitted to humans through cuts and scrapes in the human skin surface. In the United States, preventive measures include a rigorous vaccination program that involves all animals in the meat processing industry. On an individual level, people can avoid the disease by not eating animal products imported from countries where brucellosis is frequent, and by avoiding foods made with unpasteurized milk. See also Bacteria and bacterial infection; Food safety; Infection and resistance; Pasteurization

ETIOLOGIC AGENT Human brucellosis is caused by strains of Brucella, a bacterial genus considered on genetic grounds to comprise a single species, Brucella melitensis, with a number of biologic variants that exhibit particular host preferences. For the sake of convenience, the traditional classification into nomen species is still in general use; this scheme, which closely follows the epidemiologic patterns of the infection, recognizes B. melitensis, which is the commonest cause of symptomatic disease in humans and for which the main sources are sheep, goats, and camels; B. abortus, which is usually acquired from cattle or buffalo: B. suis, which is generally acquired from swine except for one variant enzootic in reindeer and caribou and another enzootic in rodents; and B. canis, which is most often acquired from dogs. B. ovis, which causes reproductive disease in sheep, and B. neotomae, which is specific for desert rodents, have not been clearly implicated in human disease. Other brucellae have been isolated from marine mammals and probably correspond to at least three distinctive nomen species. At least one case of laboratoryacquired human disease due to these marine types has been described, and apparent cases of natural infection have been reported. All brucellae are small, gram-negative, unencapsulated, nonsporulating rods or coccobacilli. They grow aerobically on peptone-based media incubated at 37C; the growth of some types is enhanced by supplementary CO2. In vivo, brucellae behave as facultative intracellular parasites. The organisms are sensitive to sunlight, ionizing radiation, and moderate heat; they are killed by boiling and pasteurization but are resistant to freezing and drying. Their resistance to drying renders brucellae stable in aerosol form and facilitates airborne transmission. Brucellae can survive for up to 2 months in soft cheeses made from goat or sheep milk; for at least 6 weeks in dry soil contaminated with infected urine, vaginal discharge, or placental or fetal tissues; and for at least 6 months in damp soil or liquid manure kept

under cool, dark conditions. The bacteria are easily killed by a wide range of common disinfectants used under optimal conditions but are likely to be much more resistant at low temperatures or in the presence of heavy organic contamination. EPIDEMIOLOGY Brucellosis is a zoonosis whose occurrence is closely related to its prevalence in domesticated animals. The true global prevalence of human brucellosis is unknown because of the imprecision of diagnosis and the inadequacy of reporting and surveillance systems in many countries. Even in developed countries, the true incidence may be 10 to 20 times higher than the reported figures. Bovine brucellosis has been the subject of control programs in many parts of the world and has been eradicated from the cattle populations of Australia, New Zealand, Bulgaria, Canada, Cyprus, Great Britain (including the Channel Islands), Japan, Luxembourg, Romania, the Scandinavian countries, Switzerland, and the Czech and Slovak Republics. Its incidence in cattle has been reduced to a low level in the United States and most Western European countries, with a varied picture in other parts of the world. There is evidence of some resurgence of brucellosis in cattle in Eastern Europe following economic changes in recent years. Efforts to eradicate B. melitensis infection from sheep and goat populations have been much less successful. These efforts have relied heavily on vaccination programs, which have tended to fluctuate with changing economic and political conditions. In some countries, such as Israel, B. melitensis has caused serious outbreaks in cattle. Brucellosis still represents a major public health problem in Mediterranean countries; in western, central, and southern Asia; and in parts of Africa and South and Central America. Human brucellosis is usually associated with occupational or domestic exposure to infected animals or their products. Farmers, shepherds, goatherds, veterinarians, and workers in slaughterhouses and meat-processing plants in endemic areas are occupationally exposed to infection. Family members (including children) of

individuals involved in animal husbandry may also be at risk, although it is often difficult to differentiate food-borne infection from environmental contamination under these circumstances. Laboratory workers involved in handling cultures or infected samples are also at risk. Travelers and urban dwellers usually acquire the infection through consumption of contaminated foods. In countries that have eradicated the disease, new cases are most often acquired abroad. Dairy products, especially soft cheeses, unpasteurized milk, and ice cream, are the most frequently implicated sources; raw meat and bone marrow may be sources of infection under exceptional circumstances. Infection resulting from contact with cosmetic products containing infected fetal materials has been recorded. Person-to-person transmission is extremely rare, as is transfer of infection by blood or tissue donation. Although brucellosis is a chronic intracellular infection, there is no evidence for increased prevalence or severity among individuals with HIV infection or other forms of immune deficiency or immune suppression. Brucellosis may be acquired by ingestion or inhalation or through mucosal or percutaneous exposure. B. melitensis and B. suis are known to have been developed as biological weapons by several countries and could be exploited as weapons of bioterrorism ( Chap. 205). This possibility should be borne in mind in the event of sudden unexplained outbreaks

IMMUNITY AND PATHOGENESIS The mechanisms of protective immunity against human brucellosis are presumed to be similar to those documented in laboratory animals. Exposure to infection generates both humoral and cell-mediated immune responses. Although antibodies promote clearance of extracellular brucellae by bactericidal action and through the facilitation of phagocytosis by polymorphonuclear phagocytes, the antibody response cannot eradicate the infection. Organisms taken up by macrophages and other cell types can establish persistent intracellular infections. Early in the course of infection, cytokines such as interleukin (IL) 12 promote production of interferon ?, which drives TH1-type responses and stimulates macrophage activation. Activated macrophages can kill intracellular brucellae (probably mainly through the production of reactive oxygen intermediates) and can clear the infection. Tumor necrosis factor a (TNF-a) is produced early in the immune response and stimulates cytotoxic lymphocytes, which can achieve partial clearance; however, the ability of virulent Brucella cells to suppress the TNF-a response may explain its limited role

in protection. Inflammatory cytokines, including IL-6 and IL-10, downregulate the protective response. As in other types of intracellular infection, it is assumed that initial replication of brucellae takes place within cells of the lymph nodes draining the point of entry. Subsequent hematogenous spread may result in chronic localizing infection at almost any site, although the reticuloendothelial system, musculoskeletal tissues, and the genitourinary system are most frequently involved. Both acute and chronic inflammatory responses develop in brucellosis, and the local tissue response may include granuloma formation, with or without necrosis and caseation. Abscesses may also develop, especially in chronic localized infection. The determinants of pathogenicity of Brucella have not been fully characterized, and the mechanisms underlying the manifestations of brucellosis are incompletely understood. The survival strategy of the organism is centered on processes that enable it to persist within monocytic cells. The smooth Brucella lipopolysaccharide, which has an unusual O-chain composition, possesses endotoxin activity and plays a key role in pyrogenicity and in resistance to phagocytosis and serum killing in the nonimmune host. Specific exotoxins have not been isolated, but a type IV secretion system responsible for secreting proteins that regulate intracellular survival and trafficking has been identified. This system is activated by low pH, and brucellae produce acid-stable proteins that facilitate survival in phagosomes and depress activation of the oxidative burst. Macrophage apoptosis and phagosomelysosome fusion are also suppressed. Virulent brucellae are resistant to defensins and produce a Cu-Zn superoxide dismutase that enhances resistance to reactive oxygen intermediates.

Structure Brucellae are Gram-negative coccobacilli (short rods) measuring about 0.6 to 1.5 m by 0.5-0.7 m. They are non-sporing and lack capsules or flagella and, therefore, are non-motile. The outer cell membrane closely resembles that of other Gram-negative bacilli with a dominant lipopolysaccharide (LPS) component and three main groups of proteins. The guanine-plus-cytosine content of the DNA is 55-58 moles/cm. No Brucella species has been found to harbor plasmids naturally although they readily accept broad-host-range plasmids. The metabolism of the brucellae is mainly oxidative and they show little action on carbohydrates in conventional media. They are aerobes but some species require an atmosphere with added CO2 (5-10 percent). Multiplication is slow at the optimum temperature of 37 C and enriched medium is needed to support adequate growth. Brucella colonies become visible on suitable solid media in 2-3 days. The colonies of smooth strains are small, round and convex but dissociation, with loss of the O chains of the LPS, occurs readily to form rough or mucoid variants. These latter forms are natural in B canis and B ovis as the LPS of these lack O chains. Classification and Antigenic Types Distinguishing features of the six species of Brucella and their preferred hosts are shown in Table 28-1. B abortus, B melitensis and B suis are serious pathogens in humans, B canis causes mild disease and the other two species have not affected humans

A culture can be identified as belonging to the genus Brucella on the basis of colonial morphology, staining and slide agglutination with antiBrucella serum, smooth or rough. Further classification is best done in a specialized laboratory. Identification to species level may be done by the procedures shown in Table 28-1. Further differentiation to biovars may be useful and is illustrated in Table 28-2. As a further refinement, tests for the oxidative metabolism of certain aminoacids and carbohydrates have been devised. Modern DNA hybridization tests, however, show that the currently named species show a high degree of homology and suggest that the genus could be appropriately reclassified as having a single species.

The application of techniques of molecular biology have allowed the cloning and characterization of several genes coding for outer membrane proteins, the use of PCR to identify the presence of brucellar DNA at genus and species level and the demonstration of species specific patterns of restriction fragment length polymorphism. It is predictable that this work will be extended to improve diagnostic tests and even vaccine development. Two different O chains in brucellae occur in the LPS of the brucellae with smooth colonies. These are called A and M, nominally indicating abortus and melitensis antigens. ('Nominally', because some abortus biovars

carry the M antigen and some common melitensis biovars the A antigen.) Both O chains have been shown to be homopolymers of 4,6dideoxy-4-formamido-d-mannopyranose; they differ only in that in the A chain the sugar molecules are always linked 2-1 whereas the M chain has every fifth junction a 3-1 linkage. In routine serology, smooth species of brucellae cross-react almost completely with each other, but not with rough species and vice versa. Monospecific polyclonal sera reacting only to A or M antigens are prepared by cross absorption and monoclonal antibodies specific for A and M antigens are now available, indicating that there is at least one unique epitope on each type of chain

Pathogenesis Brucellae are facultative intracellular parasites, multiplying mainly in monocyte-macrophage cells. This characteristic dominates the pathology, clinical manifestations and therapy of the disease. The organisms may gain entry into the body through a variety of portals (Fig. 28-1). Because the infection is systemic it is often not possible to determine which portal was involved in a particular case. Oral entry, by ingestion of contaminated animal products (often raw milk or its derivatives) or by contact with contaminated fingers, probably represents the most common route of infection even though this portal may not be the most vulnerable one. Inhalation of aerosols containing the bacteria, or aerosol contamination of the conjunctivae, is another route. Inhalation probably underlies some industrial outbreaks. Percutaneous infection through skin abrasions or by accidental inoculation has frequently been demonstrated.

Figure 28-1 Portals of entry for Brucella species Brucella species differ markedly in their capacity to cause invasive human disease. Brucella melitensis is the most pathogenic; B abortus is associated with less frequent infection and a greater proportion of subclinical cases. The virulence of B suis strains for humans varies but is generally intermediate.

Animal studies suggest that invading brucellae are rapidly phagocytosed by polymorphonuclear leukocytes. Brucellae are frequently able to survive and multiply in these cells because they inhibit the bactericidal myeloperoxidase- peroxide-halide system by releasing 5'-guanosine and adenine. Early in infection, macrophages are also relatively ineffectual in killing the intracellular brucellae (Fig. 28-2). In systemic spread, it is not clear whether the bacteria are transported within neutrophils and macrophages or in the blood stream outside cells but organisms may disseminate widely from regional lymphoid tissue appropriate to the portal of entry and may localize in certain target organs such as lymph nodes, spleen, liver, bone marrow, and (especially in animals) the reproductive organs. The presence of meso-erythritol in the testicles and seminal vesicles of bulls, rams, goats, and boars and in the products of conception in pregnant ruminants and pigs stimulates enormous multiplication of brucellae. Erythritol represents a potent localizing factor in the relevant species, but is absent in humans

Figure 28-2 Spread of Brucella in the body In humans, the tissue lesions produced by Brucella species consist of minute granulomas that are composed of epithelioid cells, polymorphonuclear leukocytes, lymphocytes and some giant cells. In cases of infection with B melitensis these granulomas are particularly small although the toxemia associated with this organism is great.

Necrosis is not common, and abscesses do not form, except in B suis infection. The fact that humans rapidly develop hypersensitivity to brucellar antigens suggests that many of the symptoms of human brucellosis result from the reaction of the host defenses Host Defenses The specific host defenses against brucellae resemble those against other intracellular bacteria and are both humoral (antibody-mediated) and cell-mediated. Passively administered monoclonal antibody directed against LPS has been shown to reduce the numbers of brucellae surviving in the spleens and livers of experimental mice, indicating a role for antibody in protection. However, the principal component in defense against brucellae is cell-mediated. Macrophages have been shown to process brucellar antigen and present this to T lymphocytes which produce lymphokines. These agents, of which interferon is the most active in this context, activate the formerly ineffective macrophages to greater bactericidal potency. Depletion of gamma interferon makes experimental animals vulnerable to infection. T cell-derived lymphokines are also involved in attracting cells to the foci of infection. This leads to granuloma

formation. While this contributes to the pathology, it also delivers the activated macrophages to the site where they are needed. This inflammatory response is enhanced by cytokines, such as the colonystimulating factors, tumor necrosis factor and interleukin-1, produced by a number of cell types. Mice which have survived brucellosis are protected against further challenge, and there is clinical evidence that complete recovery from a natural infection is associated with at least a degree of residual resistance in humans. HOST RESPONSE (animal) The alimentary tract is the major route in the transmission of B. abortus in cattle. Licking aborted fetuses and placental membranes or ingesting contaminated milk by calves introduces brucellae to the oral mucosa, tonsils, and gastrointestinal mucosa. Passage of B. abortus through epithelial barriers results in acute regional lymphadenitis and bacteremia. Epithelium covering domes of ileal Peyers patches, an important site of entry for several bovine pathogens that traverse the intestinal mucosa, is important in uptake of brucellae. Studies of infected ligated ileal loops in calves have shown that transepithelial migration of B. abortus occurs chiefly by dome lymphoepithelial cell endocytosis and transport,and that bacteria are degraded by macrophages and neutrophils of the gut-associated lymphoid tissue (Ackermann et al. 1988).The interaction of bacteria with serum components (i.e., antibody or complement), neutrophils, mononuclear macrophages, fibroblasts, and epithelial cells results in the production of a variety of biologically active substances that activate macrophages, expand clones of antigen-recognizing T lymphocytes, stimulate lymphocytes to secrete cytokines, stimulate hematopoiesis, and induce inflammation. Several fractions have been isolated from B.abortus that generate chemotactic factors derived from serum (Bertram et al. 1986). A carbohydraterich,aqueous methanol fraction of B. abortus inhibited chemotactic activity at high concentration;however, a nondialyzable component of this fraction contained a potent stimulator of chemotaxis.Preheating the serum at 56C for 30 minutes prevented generation of chemotactic activity by different fractions. Protein-rich fractions of B. abortus strain 2308 or B. abortus strain 19 failed to stimulate chemotaxis.Neutrophils are considered an important line of defense against infection with the Brucella spp.(Riley and Robertson 1984). A component of B.abortus is capable of inhibiting release of myeloperoxidase by dose-dependent preferential inhibition of

primary granule release from bovine neutrophilic leukocytes (Bertram et al. 1986). Failure of polymorphonuclear leukocytes (PMNs) to kill the organism at the primary site of infection may be responsible in part for the dissemination of Brucella spp. to other tissues of the body. Lysates from granules of guinea pig, human, or bovine PMNs were highly toxic to smooth B. abortus strain 45/0 or to rough B. abortus 45/20 (Riley and Robertson 1984).However, an oxygen-dependent killing system appeared to be lethal to both strains. Iodine was more active than chlorine in the presence of H2O2 and granule lysate in killing the organism. Ingestion of either strain by PMNs failed to stimulate the hexose monophosphate shut; therefore, Brucella spp. survive possibly because certain surface properties fail to generate a suitable stimulus to activate killing mechanisms during interaction with the plasma membrane. Long-term protective immunity against intracellular pathogens such as Brucella is associated with development of strong cell-mediated responses with production of cytokines such as IL-2, IL-12, IL-18, and IFN-_ from CD4+ T cells that are associated with Th1-type responses (Manetti et al. 1993; Ismail et al. 2002). Production of IFN-_ and TNF-_ from activated CD8+ cells contribute to macrophage activation and killing of brucellae (Ismail et al. 2002;Murphy et al. 2001). Although ntibody responses may be beneficial, they do not appear to be associated with long-term protection. Cell-mediated responses have been evaluated in cattle infected with virulent B. abortus and in cattle vaccinated with B. abortus strain 19. Lymphocyte-stimulation responses were significantly greater in cattle infected with virulent B. abortus as compared to responses observed in cattle vaccinated with attenuated B.abortus strain 19. However, no correlation was found between lymphocyte responses to specific antigen and humoral antibody responses in B. abortusinfected cattle.Differences in subpopulations of lymphocytes and mononuclear macrophages may explain in part how an organism stimulates bactericidal activity of macrophages in one instance and suppresses this activity at other times. The chronic persistence of B.abortus infections may be due to intracellular localization of brucellae in macrophages whose bactericidal mechanisms are resistant or refractory to activation. Overcoming bactericidal incompetence in these cells may be necessary for elimination of brucellae. Studies on the nonspecific and specific immunity to brucellae in rodents have provided valuable information on host cell-cell interactions and on host cell-brucellae interactions, which cannot be adequately studied in domestic animals due to lack of genetically identical individuals (Cheers 1984).

The protective effects of submucosal immune responses and inflammatory reactions against invading organisms may substantially alter the ability of Brucella spp. to colonize the local lymph nodes. Bacterial and host factors that allow brucellae to penetrate intact mucosa should be examined.Submucosal inflammatory responses composed of macrophages, lymphocytes, plasma cells, and large numbers of eosinophils and neutrophils have been observed as early as 2 and 4 days after conjunctival inoculation of cattle with B. abortus. These reactions are present in the submucosa of the conjunctiva,lachrymal duct, and in tonsils. Alteration of submucosal inflammatory reactions by presensitization and their effectiveness in destroying brucellae are unknown.Some pregnancy-associated or pregnancyspecific factors that suppress immune responses may alter the effectiveness of vaccines. The responses of apregnant host to Brucella spp. may represent a useful model to elucidate various mechanisms of immune suppression during pregnancy.

CLINICAL FEATURES: Brucellosis almost invariably causes fever, which may be associated with profuse sweats, especially at night. In endemic areas, brucellosis may be difficult to distinguish from the many other causes of fever. However, two features were recognized in the nineteenth century to distinguish brucellosis from other tropical fevers, such as typhoid and malaria: (1) Left untreated, the fever of brucellosis shows an undulating pattern that persists for weeks before the commencement of an afebrile period that may be followed by relapse, and (2) the fever of brucellosis is associated with musculoskeletal symptoms and signs in about one-half of all patients. The clinical syndromes caused by the different nomen species are similar, although B. melitensis tends to be associated with a more acute and aggressive presentation and B. suis with focal abscess induction. B. abortus infections may be more insidious in onset and more likely to become chronic.

The incubation period varies from 1 week to several months, and the onset of fever and other symptoms may be abrupt or insidious. In addition to fever and sweats, patients become increasingly apathetic and fatigued; lose appetite and weight; and have nonspecific myalgia, headache, and chills. Overall, the presentations of brucellosis often fit into one of three patterns: febrile illness that resembles typhoid but is less severe; fever and acute monarthritis, typically of hip or knee, in a young child; or long-lasting fever, misery, and low-back pain or hip pain in an older man. In an endemic area (e.g., much of the Middle East), a patient with fever and difficulty walking into the clinic would be regarded as having brucellosis until it was proved otherwise. Diagnostic clues in the patient's history include travel to an endemic area, employment in a diagnostic microbiology laboratory, consumption of unpasteurized milk products (including soft cheeses), contact with animals, and in an endemic setting a history of similar illness in the family (documented in almost 50% of cases). Focal features are present in the majority of patients. The most common is musculoskeletal pain and physical findings in the peripheral and axial skeleton (~40% of cases). Osteomyelitis more commonly involves the lumbar and lower thoracic vertebrae than the cervical and high thoracic spine. Individual joints that are most commonly affected by septic arthritis are the knee, hip, sacroiliac, shoulder, and sternoclavicular joints, and the pattern may be one of either monarthritis or polyarthritis. Osteomyelitis may also accompany septic arthritis. In addition to the usual causes of vertebral osteomyelitis or septic arthritis, the most important differential diagnosis is tuberculosis. This point has an impact on the therapeutic approach as well as on the prognosis, given that several antimicrobial agents used to treat brucellosis are also used to treat tuberculosis. Septic arthritis in brucellosis progresses slowly, starting with small pericapsular erosions. In the vertebrae, anterior erosions of the superior end plate are typically the first features to become evident, with eventual involvement and sclerosis of the whole vertebra. Anterior osteophytes eventually develop, but vertebral destruction or impingement on the spinal cord is rare and usually suggests tuberculosis ( Table 141-1).

Other systems may be involved in a manner that resembles typhoid. About one-quarter of patients have a dry cough, usually with few changes visible on the chest x-ray, although pneumonia, empyema, intrathoracic adenopathy, or lung abscess can occur. One-quarter of patients have hepatosplenomegaly, and 10 to 20% have significant lymphadenopathy; the differential diagnosis includes glandular fever-like illness such as that caused by Epstein-Barr virus, Toxoplasma, and cytomegalovirus; HIV1 infection; or tuberculosis. Up to 10% of men have acute epididymoorchitis, which must be distinguished from that due to mumps or surgical problems such as torsion. Prostatitis, inflammation of the seminal vesicles, salpingitis, and pyelonephritis all occur. There is an increased incidence of fetal loss among infected pregnant women, although teratogenicity has not been described and the tendency to cause abortions is much less pronounced in humans than in farm animals. Neurologic involvement is common, with depression and lethargy whose severity may not be fully appreciated by either the patient or the physician until after treatment. A small proportion of patients develop lymphocytic meningoencephalitis that mimics neurotuberculosis or noninfectious conditions and that may be complicated by intracerebral abscess, a variety of cranial nerve deficits, and ruptured mycotic aneurysms. Endocarditis occurs in ~1% of cases, most often affecting the aortic valve (natural or prosthetic). Any site in the body may be involved in metastatic abscess formation or inflammation; the female breast and the thyroid gland are affected particularly often. Nonspecific maculopapular rashes and other skin manifestations are uncommon and are rarely noticed by the patient even if they are present BRUCELLA ABORTUS Brucella abortus infection in pregnant cattle involves the placenta, fetus, and mammary gland.One investigation indicated initial lesions occurred within the placenta as early as 4 weeks after challenge; at 5 weeks, a more extensive endometritis with erosion of endometrial epithelium was evident (Payne 1959; Anderson and Cheville 1986).

Following experimental infection of pregnant cattle via the conjunctival route, bacteria were first detected in the uterus 45 weeks postinoculation.Chorionic epithelium becomes infected and infection extends into the placenta. Considerable variation occurs in distribution and severity of placental lesions in natural and experimental B. abortus infections. Seldom are all placentomes involved,and frequently only portions of individual placentomes develop lesions. Moreover, fetuses that become infected during late gestation may be aborted,with an absence of grossly recognizable placental lesions. In such cases, microscopic examination of placental tissues often reveals mild focal inflammation (Enright et al. 1984). Other investigators report that extensive fetal inflammatory disease involving multiple organs leads to fetal stress, which results in premature deliveries. Bovine fetuses of a known gestation age were inoculated per os or intramuscularly with low doses of B. abortus strain 2308 and examined after abortion or delivery by cesarean section at intervals of 616 days postinoculation (Enright et al. 1984). Infected fetuses developed lymphoreticular hyperplasia of lymph nodes as early as 6 days postinoculation. Progression of lymph node changes to primary-follicle formation and germinal center formation was noted at 9 and 12 days postinoculation, respectively.Morphologic alterations of lymph nodes correlated with elevations of IgM and IgG immunoglobulin levels by day 9 postinoculation. Granulomatous responses, composed of acrophages, epithelioid cells, and giant cells, were present in lymphatic tissues, liver, and occasionally within the kidney interstitium as early as 6 and 9 days postinoculation.Inflammatory lesions were extensive in fetal lungs characterized by thickened interlobular septae and focal to diffuse, exudative interstitial pneumonia.Pulmonary lesions were composed primarily of macrophages and a few neutrophils. Measurement of fetal cortisol levels may serve as an indicator of fetal stress. Also, as fetal corticosteroids can induce the physiological process of fetal expulsion/parturition (Gaverick and Smith 1993), they may also serve as an early indicator of a physiologic process that could lead to abortion. Fetal cortisol levels were elevated substantially above normal values and were present as early as 6 days postinoculation (Enright et al. 1984). Experimentally infected bovine fetuses were aborted between 7 and 19 days postinoculation. This relatively short and consistent interval between infection and abortion suggests that the bovine fetus is a sensitive indicator of in utero infection with B. abortus. The character and early development of granulomatous inflammatory reaction and a rapid development of the fetal immune response demonstrate that the fetus is capable of

reaction to B. abortus infection similar to that of mature animals. Mammary gland infection is often clinically inapparent, and gross lesions may not be present.Histologically, there is a lobular and periductal,lymphoplasmacytic, and histiocytic interstitial mastitis with leukocytes in alveoli and ducts. Brucellae localize and replicate primarily in macrophages in mammary secretions or in phagocytes in the inter- stitium. Lesions are not seen in all infected ammals(Meador et al. 1989a). Supramammary lymph nodes are typically enlarged and are characterized by edema, lymphofollicular hyperplasia, medullary plasmacytosis, and sinus histiocytosis. Localization of B. abortus in the mammary gland is markedly influenced by nursing, that is, milk retention in the mammary gland correlates with the degree of infection.Studies in goats have shown that failure to nurse or release milk enhances localization and replication of bacteria in mammary glands after parturition (Meador et al. 1989b). In turn, mammary infection may result in increased systemic spread and persistence of brucellae in the host. BRUCELLA MELITENSIS Due to its high pathogenicity in humans, B. melitensisis one of the most serious zoonoses in the world with distribution in the Mediterranean, Arabian peninsula, and Asia, and parts of Africa, Latin America, and South America (Alton 1990).Brucella melitensis is the principal cause of brucellosis in sheep and goats, is endemic in camels in some areas (Abbas and Agab 2002), and is becoming problematic as a cause of cattle brucellosis in some countries (Corbel 1997). It is the least species specific of the brucellae, and transmits to many other species (Alton 1990). With the exception of small ruminants, camels, and cattle, most species are considered to be end hosts for B. melitensis (Alton 1990). The pathogenesis of B. melitensis infection in goats and early localization within the mammary gland and pregnant uterus of sheep is similar to that of B. abortus in cattle. In sheep and goats, excretion in milk is a significant route of B. melitensis transmission to offspring (Grill et al. 1997). Following intravenous injection in sheep, B. melitensis induced necrosis and edema of placentomes and glandular endometritis (Mollelo et al. 1963). In general, B.melitensis is considered to cause more necrosis in placental tissue and less exudation than B. abortus.

BRUCELLA OVIS B. ovis is an important cause of epididymitis in rams, but appears to be the least pathogenic of the Brucella that affects animals. Venereal exposure is probably the most frequent route of transmission (Buddle

1955). Lesions in rams are most often located in the tail of the epididymis. Initial localization in the epididymis is accompanied by perivascular edema and infiltration of peritubular tissue by lymphocytes and monocytes; subsequently, neutrophils infiltrate the exudate. Previously inflamedtubular epithelium develops papillary hyperplasia and local hydropic degeneration, with subsequent formation of inflammatory reaction leading to an extravasation of spermatozoa. Host responses to extravasated spermatozoa lead to the formation of large spermatic granulomas, which may result in complete blockage of the epididymis; testicular degeneration and fibrosis are secondary to this blockage. B. ovis can be cultured from spleens of infected ewes in which no lesions are observed.Placental pathology experimentally induced by B.ovis tends to localize in the intercotyledonary placenta and is often less severe than the placentitis caused by B. melitensis or B. abortus.Pregnant ewes inoculated with B. ovis in the conjunctiva on days 30 to 90 of gestation usually develop uterine infections and abort or deliver infected lambs. In contrast to the limited period of susceptibility of fetuses in the intraconjunctivally infected ewes, fetuses exposed to B. ovis in utero are susceptible to infection throughout pregnancy. Intervals between in utero infection with B. ovis and abortion range from 23 to 80 days postinoculation; by comparison,bovine fetuses infected in utero by B. abortus usually abort by 12 days postinoculation. This difference may be attributed to the low pathogenicity of B. ovis. The immunologic and granulomatous inflammatory responses of B. ovis-infected bovine fetuses are similar to those observed in B. abortusinfected bovine fetuses. BRUCELLA SUIS Brucellosis in swine is usually caused by B. suis,which may be transmitted by ingestion, although venereal transmission occurs (McMillan 1992).Boars are as susceptible to infection as sows, and many infected boars develop lesions in the testicles and accessory reproductive organs and shed B. suisorganisms in semen for extended periods. There is atendency for focal granulomatous inflammation to develop in the endometrium and extend to the entire nongravid uterus. B. suis also tends to secondarily localize in a greater variety of tissues than does B.abortus. The organism demonstrates a predilection for localization in bone and joints, spleen, liver, kidney,and brain. Prolonged bacteremia often occurs with B. suis infection and may contribute to localization in a wide variety of tissues.The early stages in pathogenesis of B. suis infection in swine are comparable to the early

stages of Brucella 313 B. abortus infection in cattle. However, the character of the response of swine to B. suis differs slightly from the response of cattle to B. abortus. B. suis multiplies in mononuclear phagocytes and produces granulomatous lesions composed of macrophages and epitheliod cells. The granulomas tend to undergo caseous necrosis and become encapsulated by fibrous connective tissue. BRUCELLA CANIS Brucellosis in dogs is characterized by abortions in females, and epididymitis, testicular atrophy, and infertility in males (Carmichael 1990). Brucella canis has a limited host range with canids most susceptible and with cats, laboratory animals, and humans being less susceptible to infection. The disease is rapidly transmitted via vaginal discharges,semen, urine, milk, and other body fluids. The principle clinical sign is abortion, however, many dogs do not demonstrate prominent clinical signs.Diagnosis is based on serologic tests. There is no acceptable vaccine for canine brucellosis. Diagnosis The diagnosis of brucellosis is primarily dependent on clinical suspicion allied with the taking of an adequate history of possible exposure including during travel. Presentation can, however, be highly atypical and focal lesions may present decades after exposure. Unequivocal diagnosis requires isolation of the organism. Blood culture is the method of choice but specimens need to be obtained early in the disease and cultures may need to be incubated for up to four weeks. Even so, failure to grow the organism is common, especially in cases of B abortus infection, and isolation rates of only 20-50% are reported even from experienced laboratories. Modern commercial systems are hampered by the small amount of CO2 produced during growth. Culture from bone marrow and from presenting foci may be successful. Presumptive identification of cultures from morphology and slide agglutination with specific antiserum should be followed by further work in a reference facility. Molecular techniques for typing are being developed

Figure 28-4 Diagnosis of brucellosis. Serology remains the mainstay of laboratory diagnosis, but the interpretation of results is fraught with difficulties. The large number of techniques in use is evidence of the problems. The standard serum agglutination test (SAT) has been augmented by the modified Coombs' (antiglobulin) technique and the use of 2-mercaptoethanol to separate the actions of specific IgG and IgM. These classical methods may, in time, be supplanted by EIA (enzyme immunoassay) tests, designed to differentiate between specific IgM and IgG antibodies. While the SAT titers commonly decline after recovery from infection and antiglobulin test levels are maintained much longer, the IgM antibody that is commonly measured by the SAT does not fall away as regularly as in some infections. Nevertheless, persisting levels of antibody may indicate a remaining focus of infection and specific IgG levels rise again with a true relapse. Further, because cases often are investigated late in their course, rising titers are frequently missed; the variability of individual responses and the frequency of subclinical infections make the interpretation of single high titers subject to error. All serologic tests have to be interpreted with caution in the light of clinical data and in the context of the local

prevalence of brucellosis. Moreover, serum from persons with tularemia may show cross-reactions with Brucella antigen. The diagnosis of the chronic brucellosis syndrome, without specific localization, is often very unsatisfactory. When cultures are negative and the results of serologic tests are equivocal a confident diagnosis is often impossible TREATMENT The broad aims of antimicrobial therapy for brucellosis are to treat current infection and relieve its symptoms and to prevent relapse. Focal disease presentations may require surgical intervention (e.g., cardiac valve replacement, abscess drainage, joint replacement) in addition to more prolonged and tailored antibiotic therapy. In addition, tuberculosis must always be excluded, or to prevent the emergence of resistance the regimen must be tailored to specifically exclude monotherapy with agents active against tuberculosis (e.g., rifampin) or to include a full antituberculous regimen. Early experience with streptomycin monotherapy for brucellosis showed that relapse was common; thus dual therapy with streptomycin and tetracyclines became the norm. This is still the most effective combination, but alternatives may be used, with the options depending on local or national policy about the use of rifampin for the treatment of nonmycobacterial infection. Antimicrobial efficacy can usually be predicted by in vitro testing. The efficacy of fluoroquinolone monotherapy has been disappointing, with a high relapse rate, despite the good in vitro activity and white-cell penetration of most agents of this class. For adults with acute nonfocal brucellosis (duration, <1 month), a 6-week course of therapy incorporating at least two antimicrobial agents is required. Complex or focal disease necessitates =3 months of therapy. Adherence to the therapeutic regimen is very important, and poor compliance underlies almost all cases of apparent treatment failure; such failure is rarely due to the emergence of drug resistance, although increasing resistance to trimethoprimsulfamethoxazole (TMP-SMX) has been reported at one center. There is good retrospective evidence that a 3-week course of two agents is as good as a 6-week course for treatment and prevention of relapse in children, but this point has not yet been proved in prospective studies.

The "gold standard" for the treatment of brucellosis in adults is intramuscular streptomycin (750 mg to 1 g daily for 14 to 21 days) together with doxycycline (100 mg twice daily for 6 weeks). In both clinical trials and observational studies, relapse follows such treatment in 5 to 10% of patients. The usual alternative regimen (and the current World Health Organization recommendation) is rifampin (600 to 900 mg/d) plus doxycycline (100 mg twice daily) for 6 weeks. In trial conditions, the relapse/failure rate is ~10%, but this rate rises to >20% in many nontrial situations, possibly because doxycycline levels are reduced and clearance rates increased by concomitant rifampin administration. Patients who cannot tolerate or receive tetracyclines (children, pregnant women) can be given high-dose TMP-SMX3 instead (2 or 3 standard-strength tablets twice daily for adults, depending on weight). Evidence is beginning to accumulate that other aminoglycosides can be substituted for streptomycin e.g., netilmicin or gentamicin given at a dosage of 5 to 6 mg/kg per day for at least 2 weeks. (Shorter courses have been associated with high failure rates in adults.) A 5- to 7-day course of therapy with gentamicin (and a 3-week course of TMP-SMX3) is probably adequate for children with uncomplicated disease. Early experience with fluoroquinolone monotherapy was disappointing, but high-dose ofloxacin (400 mg twice daily) or ciprofloxacin (500 mg twice daily), given for 6 weeks with rifampin, may become accepted as an alternative to the other 6-week regimens for adults. Significant neurologic disease due to Brucella requires prolonged treatment (i.e., for 6 to 12 months), usually with ceftriaxone supplementation of a standard regimen. Brucella endocarditis is treated with at least three drugs (an aminoglycoside, a tetracycline, and rifampin), and many experts add ceftriaxone and/or a fluoroquinolone to reduce the need for valve replacement. Treatment is usually given for at least 6 months, and clinical end points for its discontinuation are often difficult to define. Surgery is still required for the majority of cases of infection of prosthetic heart valves and prosthetic joints.

There is no evidence base to guide prophylaxis after exposure to brucellae in the laboratory, inadvertent immunization with live vaccine intended for use in animals, or exposure to deliberately released brucellae. Most authorities recommend the administration of rifampin plus doxycycline for 3 weeks after a low-risk exposure (e.g., a nonspecific laboratory accident) and for 6 weeks after a major exposure to aerosol or injected material. PROGNOSIS AND FOLLOW-UP Relapse occurs in up to 30% of poorly compliant patients. Thus patients ideally should be followed clinically for up to 2 years to detect relapse, which responds to a prolonged course of the same therapy that was originally used. The general well-being and body weight of the patient are more useful guides than serology to lack of relapse. IgG antibody levels detected by SAT4 and variants of this test can remain in the diagnostic range for >2 years after successful treatment. Complement fixation titers usually fall to normal within 1 year of cure. Immunity is not solid; patients can be reinfected after repeated exposures. Fewer than 1% of patients die of brucellosis. When the outcome of this infection is fatal, death is usually a consequence of cardiac involvement; more rarely, it results from severe neurologic disease. Despite the low mortality rate, recovery from brucellosis is slow, and the illness in humans can cause prolonged inactivity, with consequent domestic difficulties and economic losses. The existence of a prolonged chronic brucellosis state after successful treatment remains controversial. Evaluation of patients in whom this state is considered (often those with work-related exposure to brucellae) includes careful exclusion of malingering, nonspecific chronic fatigue syndromes and other causes of excessive sweating, such as alcohol abuse and obesity. In the future, the availability of more sensitive assays to detect Brucella antigen or DNA may help to identify patients with ongoing infection. PREVENTION Vaccines based on live attenuated Brucella strains, such as B. abortus strain 19BA or 104M, have been used in some countries to protect highrisk populations but

have displayed only short-term efficacy and a high incidence of local and systemic side effects (local inflammation, pain, lymphadenopathy, fever, malaise, nausea). Subunit vaccines have been developed but are of uncertain value and cannot be recommended at present. Interest in biodefense has stimulated research in this area (Chap. 205). The mainstay of veterinary prevention is a national commitment to testing and slaughter of infected herds and flocks (with compensation for owners), control of animal movement, and active immunization of animals. These measures are usually sufficient to control human disease as well. In their absence, pasteurization of all milk products before consumption is sufficient to prevent animal-to-human transmission. All cases of Brucella infection in animals and humans should be reported to the appropriate public health authorities.