CHAPTER 66: BRUCELLOSIS

Brucellosis is a bacterial zoonosis transmitted directly or indirectly to humans from infected animals, predominantly domesticated ruminants and swine. The disease is known colloquially as undulant fever because of its remittent character. Although brucellosis commonly presents as an acute febrile illness, its clinical manifestations vary widely, and definitive signs indicative of the diagnosis may be lacking. Thus the clinical diagnosis usually must be supported by the results of bacteriologic and/or serologic tests.

Human brucellosis is caused by strains of Brucella, a bacterial genus that was previously suggested, on genetic grounds, to comprise a single species, B. melitensis, with a number of biologic variants exhibiting particular host preferences. This view was challenged on the basis of detailed differences in chromosomal structure and host preference. The traditional classification into nomen species is now favored both because of these differences and because this classification scheme closely reflects the epidemiologic patterns of the infection. The nomen system recognizes B. melitensis, which is the most common 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 generally is acquired from swine but has one variant enzootic in reindeer and caribou and another in rodents; and B. canis, which is acquired most often 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. Two new species, B. ceti and B. pinnipedialis, have recently been identified in marine mammals, including seals and dolphins. At least one case of laboratory-acquired human disease due to one of these species has been described, and apparent cases of natural human infection have been reported. As infections in marine mammals appear to be widespread, more cases of zoonotic infection in humans may be identified. Other newly reported species include B. microti (isolated from field voles) and B. inopinata (isolated from a patient with a breast implant). Additional novel strains have been described from diverse species, including baboons, foxes, frogs, and various rodents, and the genus likely will expand further in forthcoming years. Moreover, it has become apparent that Brucella is closely related to the genus Ochrobactrum, which includes environmental bacteria sometimes associated with opportunistic infections. Genomics-based studies are beginning to elucidate the pathway of evolution from free-living soil bacteria to highly successful intracellular pathogens.

All brucellae are small, gram-negative, unencapsulated, nonsporulating, nonmotile rods or coccobacilli. They grow aerobically on peptone-based medium incubated at 37°C; the growth of some types is improved 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, facilitating airborne transmission. The organisms can survive for up to 2 months in soft cheeses made from goat’s or sheep’s 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. Brucellae 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.

Brucellosis is a zoonosis whose occurrence and control are closely related to its prevalence in domesticated animals. Its distribution is worldwide apart from the few countries where it has been eradicated from the animal reservoir. 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. Recently, there has been increased recognition of the high incidence of brucellosis in India and parts of China and of importations to countries in Oceania, such as Fiji. In Europe, the incidence of brucellosis in a country is inversely related to gross domestic product, and, in both developed and less well-resourced settings, human brucellosis is related to rural poverty and inadequate access to medical care. Failure of veterinary control programs due to conflict or for economic reasons contributes further to the emergence and re-emergence of disease, as seen currently in some eastern Mediterranean countries.

Even in well-resourced settings, the true incidence of brucellosis in domesticated animals may be 10–20 times higher than the reported figures. Bovine brucellosis has been the target 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, among other nations. Its incidence 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. 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 (e.g., Israel), B. melitensis has caused serious outbreaks in cattle. Infections with B. melitensis still pose 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 employees in slaughterhouses and meat-processing plants in endemic areas are occupationally exposed to infection. Family members of individuals involved in animal husbandry may be at risk, although it is often difficult to differentiate food-borne infection from environmental contamination under these circumstances. Laboratory workers who handle cultures or infected samples also are at risk. Travelers and urban residents usually acquire the infection through consumption of contaminated foods. In countries that have eradicated the disease, new cases are most commonly acquired abroad. Dairy products, especially soft cheeses, unpasteurized milk, and ice cream, are the most frequently implicated sources of infection; raw meat and bone marrow may be sources under exceptional circumstances. Infections acquired through cosmetic treatments using materials of fetal origin have been reported. 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 with immunodeficiency or immunosuppression of other etiologies.

Brucellosis may be acquired by ingestion, inhalation, or mucosal or percutaneous exposure. Accidental injection of the live vaccine strains of B. abortus (S19 and RB51) and B. melitensis (Rev 1) can cause disease. B. melitensis and B. suis have historically been developed as biological weapons by several countries and could be exploited for bioterrorism (Chap. 10). This possibility should be borne in mind in the event of sudden unexplained outbreaks.

Exposure to brucellosis elicits both humoral and cell-mediated immune responses. The mechanisms of protective immunity against human brucellosis are presumed to be similar to those documented in laboratory animals, but such generalizations must be interpreted with caution. The response to infection and its outcome are influenced by the virulence, phase, and species of the infecting strain. Differences have been reported between B. abortus and B. suis in modes of cellular entry and subsequent compartmentalization and processing. Antibodies promote clearance of extracellular brucellae by bactericidal action and by facilitation of phagocytosis by polymorphonuclear and mononuclear phagocytes; however, antibodies alone cannot eradicate infection. Organisms taken up by macrophages and other cells can establish persistent intracellular infections. The key target cell is the macrophage, and bacterial mechanisms for suppressing intracellular killing and apoptosis result in very large intracellular populations. Opsonized bacteria are actively phagocytosed by neutrophilic granulocytes and by monocytes. In these and other cells, initial attachment takes place via specific receptors, including Fc, C3, fibronectin, and mannose-binding proteins. Opsonized—but not unopsonized—bacteria trigger an oxidative burst inside phagocytes. Unopsonized bacteria are internalized via similar receptors but at much lower efficiency. Smooth strains enter host cells via lipid rafts. Smooth lipopolysaccharide (LPS), β-cyclic glucan, and possibly an invasion-attachment protein (IalB) are involved in this process. Tumor necrosis factor α (TNF-α) produced early in the course of infection stimulates cytotoxic lymphocytes and activates macrophages, which can kill intracellular brucellae (probably mainly through production of reactive oxygen and nitrogen intermediates) and may clear infection. However, virulent Brucella cells can suppress the TNF-α response, and control of infection in this situation depends on macrophage activation and interferon γ (IFN-γ) responses. Cytokines such as interleukin (IL) 12 promote production of IFN-γ, which drives T_H1-type responses and stimulates macrophage activation. Inflammatory cytokines, including IL-4, 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 genitourinary system are most frequently targeted. 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 organism is a “stealth” pathogen whose survival strategy is centered on several processes that avoid triggering innate immune responses and that permit survival within monocytic cells. The smooth Brucella LPS, which has an unusual O-chain and core-lipid composition, has relatively low endotoxin activity and plays a key role in pyrogenicity and in resistance to phagocytosis and serum killing in the nonimmune host. In addition, LPS is believed to play a role in suppressing phagosome–lysosome fusion and diverting the internalized bacteria into vacuoles located in endoplasmic reticulum, where intracellular replication takes place. Specific exotoxins have not been isolated, but a type IV secretion system (VirB) that regulates intracellular survival and trafficking has been identified. In B. abortus this system can be activated extracellularly, but in B. suis it is activated (by low pH) only during intracellular growth. Brucellae then produce acid-stable proteins that facilitate the organisms’ survival in phagosomes and may enhance their resistance to reactive oxygen intermediates. A type III secretion system based on modified flagellar structures also has been inferred, although not yet confirmed. Virulent brucellae are resistant to defensins and produce a Cu-Zn superoxide dismutase that increases their resistance to reactive oxygen intermediates. A hemolysin-like protein may trigger the release of brucellae from infected cells.

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 recognized in the nineteenth century 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. (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. B. canis infections are reported to present frequently with acute gastrointestinal symptoms.

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 experiencing fever and sweats, patients become increasingly apathetic and fatigued; lose appetite and weight; and have nonspecific myalgia, headache, and chills. Overall, the presentation of brucellosis often fits one of three patterns: febrile illness that resembles typhoid but is less severe; fever and acute monoarthritis, typically of the hip or knee, in a young child; and long-lasting fever, misery, and low-back 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 proven 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, accidental inoculation with veterinary Brucella vaccines, 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 are musculoskeletal pain and physical findings in the peripheral and axial skeleton (~40% of cases). Osteomyelitis more commonly involves the lumbar and low 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; the pattern may be one of monoarthritis or polyarthritis. Osteomyelitis may also accompany septic arthritis.

In addition to the usual causes of vertebral osteomyelitis or septic arthritis, the most important disease in the differential diagnosis is tuberculosis. This point influences the therapeutic approach as well as 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 66-1).

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. Sputum or pleural effusion cultures are rarely positive in such cases, which respond well to standard brucellosis treatment. One-quarter of patients have hepatosplenomegaly, and 10–20% have significant lymphadenopathy; the differential diagnosis includes glandular fever–like illness such as that caused by Epstein-Barr virus, Toxoplasma, cytomegalovirus, HIV, or Mycobacterium tuberculosis. Up to 10% of men have acute epididymoorchitis, which must be distinguished from mumps and from 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 toward 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, atypical leptospirosis, or noninfectious conditions and that may be complicated by intracerebral abscess, a variety of cranial nerve deficits, or 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 develop.

Because the clinical picture of brucellosis is not distinctive, the diagnosis must be based on a history of potential exposure, a presentation consistent with the disease, and supporting laboratory findings. Results of routine biochemical assays are usually within normal limits, although serum levels of hepatic enzymes and bilirubin may be elevated. Peripheral leukocyte counts are usually normal or low, with relative lymphocytosis. Mild anemia may be documented. Thrombocytopenia and disseminated intravascular coagulation with raised levels of fibrinogen degradation products can develop. The erythrocyte sedimentation rate and C-reactive protein levels are often normal but may be raised.

In body fluids such as cerebrospinal fluid (CSF) or joint fluid, lymphocytosis and low glucose levels are the norm. Elevated CSF levels of adenosine deaminase cannot be used to distinguish tubercular meningitis, as they may also be found in brucellosis. Biopsied samples of tissues such as lymph node or liver may show noncaseating granulomas (Fig. 66-1) without acid/alcohol-fast bacilli. The radiologic features of bony disease develop late and are much more subtle than those of tuberculosis or septic arthritis of other etiologies, with less bone and joint destruction. Isotope scanning is more sensitive than plain x-ray and continues to give positive results long after successful treatment.

Isolation of brucellae from blood, CSF, bone marrow, or joint fluid or from a tissue aspirate or biopsy sample is definitive, and attempts at isolation are usually successful in 50–70% of cases. Duplicate cultures should be incubated for up to 6 weeks (in air and 10% CO₂, respectively). Concentration and lysis of buffy coat cells before culture may increase the isolation rate. Cultures in modern nonradiometric or similar signaling systems (e.g., Bactec) usually become positive within 7–10 days but should be maintained for at least 3 weeks before the results are declared negative. All cultures should be handled under containment conditions appropriate for dangerous pathogens. Brucella species may be misidentified as Agrobacterium, Ochrobactrum, or Psychrobacter (Moraxella) phenylpyruvicus by the gallery identification strips commonly used in the diagnostic laboratory. In recent years, matrix-assisted laser desorption ionization time-of-flight spectrometry (MALDI-TOF MS) has emerged as a powerful tool in bacterial identification. The relative homogeneity of classical Brucella species makes identification beyond the genus level by routine approaches challenging, although further improvements may facilitate discrimination at the species level. The place of this technique in routine diagnostic practice will depend on such refinements. Meanwhile, the authors are aware of cases in which blood culture isolates have been identified incorrectly using MALDI-TOF MS.

The peripheral blood–based polymerase chain reaction has enormous potential to detect bacteremia, to predict relapse, and to exclude “chronic brucellosis.” This method is probably more sensitive and is certainly quicker than blood culture, and it does not carry the attendant biohazard risk posed by culture. Nucleic acid amplification techniques are now quite widely used, although no single standardized procedure has been adopted. Primers for the spacer region between the genes encoding the 16S and 23S ribosomal RNAs (rrs-rrl), various outer-membrane protein–encoding genes, the insertion sequence IS711, and the protein BCSP31 are sensitive and specific. Blood and other tissues are the most suitable samples for analysis.

Serologic examination often provides the only positive laboratory findings in brucellosis. In acute infection, IgM antibodies appear early and are followed by IgG and IgA. All these antibodies are active in agglutination tests, whether performed by tube, plate, or microagglutination methods. The majority of patients have detectable agglutinins at this stage. As the disease progresses, IgM levels decline, and the avidity and subclass distribution of IgG and IgA change. The result is reduced or undetectable agglutinin titers. However, the antibodies are detectable by alternative tests, including the complement fixation test, Coomb’s antiglobulin test, and enzyme-linked immunosorbent assay. There is no clear cutoff value for a diagnostic titer. Rather, serology results must be interpreted in the context of exposure history and clinical presentation. In endemic areas or in settings of potential occupational exposure, agglutinin titers of 1:320–1:640 or higher are considered diagnostic; in nonendemic areas, a titer of ≥1:160 is considered significant. Repetition of tests after 2–4 weeks may demonstrate a rising titer.

In most centers, the standard agglutination test is still the mainstay of serologic diagnosis, although some investigators rely on the rose bengal test, which has not been fully validated for human diagnostic use. Dipstick assays for anti-Brucella IgM are useful for the diagnosis of acute infection but are less sensitive for infection with symptoms of several months’ duration. In an endemic setting, more than 90% of patients with acute bacteremia have standard agglutination titers of at least 1:320. Other screening tests are used in some centers.

Antibody to the Brucella LPS O chain—the dominant antigen—is detected by all the conventional tests that employ smooth B. abortus cells as antigen. Since B. abortus cross-reacts with B. melitensis and B. suis, there is no advantage in replicating the tests with these antigens. Cross-reactions also occur with the O chains of some other gram-negative bacteria, including Yersinia enterocolitica O:9, Escherichia coli O157, Francisella tularensis, Salmonella enterica group N, Stenotrophomonas maltophilia, and Vibrio cholerae. Cross-reactions do not occur with the cell-surface antigens of rough Brucella strains such as B. canis or B. ovis; serologic tests for these nomen species must employ an antigen prepared from either one. The live B. abortus vaccine strain RB51 does not elicit antibody responses in serologic tests that use smooth antigens, and this fact must be taken into account if serologic tests are employed in attempts to identify or follow the course of infections in persons accidentally exposed to the vaccine.

Relapse occurs in up to 30% of poorly compliant patients. Thus patients should ideally be followed clinically for up to 2 years to detect relapse, which responds to a prolonged course of the same therapy used originally. The general well-being and the body weight of the patient are more useful guides than serology to lack of relapse. IgG antibody levels detected by the standard agglutination test and its variants 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 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 can cause prolonged inactivity, with domestic and economic consequences.

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.

Vaccines based on live attenuated Brucella strains, such as B. abortus strain 19BA or 104M, have been used in some countries to protect high-risk populations but have displayed only short-term efficacy and high reactogenicity. Subunit vaccines have been developed but are of uncertain value and cannot be recommended at present. Research in this area has been stimulated by interest in biodefense (Chap. 10) and may eventually yield new products. The mainstay of veterinary prevention is a national commitment to testing and slaughter of infected herds/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 non-occupational animal-to-human transmission. All cases of brucellosis in animals and humans should be reported to the appropriate public health authorities.

Acknowledgment

The authors thank Dr. Adrian M. Whatmore for his review of the manuscript.