BW-ch09.pdf

Brucellosis

Chapter 9

BRUCELLOSIS

BRET K. PURCELL, P h D, MD * ; DAVID L. HOOVER, MD † ; and ARTHUR M. FRIEDLANDER, MD ‡

INTRODUCTION

INFECTIOUS AGENT

DISEASE

Epidemiology

Pathogenesis

Clinical Manifestations

Diagnosis

Treatment

PROPHYLAXIS

SUMMARY

* LieutenantColonel,MedicalCorps,USArmy;Chief,BacterialTherapeutics,DivisionofBacteriology,USArmyMedicalResearchInstituteofInfectious

Diseases,1425PorterStreet,FortDetrick,Maryland21702

† Colonel,MedicalCorps,USArmy(Ret);MedicalDirector,DynportVaccineCompanyLLC,ACSCCompany,64ThomasJohnsonDrive,Frederick,

Maryland21702;formerly,ScientificCoordinator,BrucellaProgram,DepartmentofBacterialDiseases,WalterReedArmyInstituteofResearch,Silver

Spring,Maryland

‡ Colonel,MedicalCorps,USArmy(Ret);SeniorScientist,DivisionofBacteriology,USArmyMedicalResearchInstituteofInfectiousDiseases,1425

PorterStreet,FortDetrick,Maryland21702;andAdjunctProfessorofMedicine,UniformedServicesUniversityoftheHealthSciences,4301Jones

BridgeRoad,Bethesda,Maryland20814

185

MedicalAspectsofBiologicalWarfare

INTRODUCTION

Brucellosis is a zoonotic infection of domesticated

and wild animals caused by organisms of the genus

Brucella . Humans become infected by ingesting animal

food products, directly contacting infected animals, or

inhaling infectious aerosols either by accident or as a

result of bioterrorism.

Military medicine has played a major role in study-

ing and describing brucellosis in humans. 1 In 1751 G

Cleghorn, a British army surgeon stationed on the

Mediterranean island of Minorca, described cases of

chronic, relapsing febrile illness and cited Hippocrates’

description of a similar disease more than 2,000 years

earlier. 2 Three additional British army surgeons work-

ing on the island of Malta during the 1800s were re-

sponsible for important observations of the disease. JA

Marston described clinical characteristics of his own

infection in 1861. 3 In 1887 David Bruce, for whom the

genus Brucella is named, isolated the causative organ-

ism from the spleens of five patients who died from

the disease and placed the microorganism within the

genus Micrococcus . 4 Ten years later, ML Hughes, who

coined the name “undulant fever,” published a mono-

graph that detailed clinical and pathological findings

in 844 patients. 5

That same year, Danish investigator B Bang iden-

tified an organism, which he called the “bacillus of

abortion,” in the placentas and fetuses of cattle suf-

fering from contagious abortion. 6 In 1917 AC Evans

recognized that Bang’s organism was identical to that

described by Bruce as the causative agent of human

brucellosis. The organism infects mainly cattle, sheep,

goats, and other ruminants, in which it causes abor-

tion, fetal death, and genital infections. 7,8 Humans,

who are usually infected incidentally by contact with

infected animals or ingestion of dairy foods, may de-

velop numerous symptoms in addition to the usual

ones of fever, malaise, and muscle pain. Because of the

worldwide distribution of brucellosis, international

travel and military deployments increase the risk of

exposure. 9 The disease frequently becomes chronic and

may relapse, even with treatment . Laboratory-acquired

infections have been documented as awareness of this

disease has increased. 10-13 Laboratory accidents may

become more frequent and significant as biodefense

research expands in the academic and biotechnology

industries. Strict adherence to proper engineering con-

trols, good laboratory and microbiology techniques,

and personal protective equipment, in addition to

vaccination (when possible), significantly reduce the

incidence of laboratory-acquired infections. 14,15 How-

ever, no human brucellosis vaccine is available for

laboratory workers.

The ease of transmission by aerosol underscores

the concern that Brucella might be used as a biological

warfare agent. The United States began developing

Brucellasuis as a biological weapon in 1942. The agent

was formulated to maintain long-term viability, placed

into bombs, and tested in field trials in 1944 and 1945

with animal targets. By 1969 the United States termi-

nated its offensive Brucella program and destroyed all

its biological weapon munitions. Although the muni-

tions developed were never used in combat, studies

conducted under the offensive program reinforced

the concern that Brucella organisms might be used

against US troops as a biological warfare agent. 16 Even

before the 2001 anthrax attacks, civilian populations

were recognized as potential high-yield targets. A

1997 model of aerosol attack with Brucella on an urban

population included an estimated economic impact of

$477.7 million per 100,000 persons exposed. 17 Brucella

represents one of many biological agents of zoonotic

disease that could pose a threat as a terrorist weapon

against human or agricultural targets. 18 An excellent

review of brucellosis was published in 2005. 19

INFECTIOUS AGENT

Brucellae are small, nonmotile, nonsporulating,

nontoxigenic, nonfermenting, facultative, intracellular,

gram-negative coccobacilli parasites that may, based

on DNA homology, represent a single species. 20,21

Taxonomically, brucellae are classified as a- Proteobac-

teria and subdivided into six species, each comprising

several biovars. 22 Each species has a characteristic,

but not absolute, predilection to infect certain animal

species (Table 9-1). Brucellamelitensis , Bsuis , Babortus ,

and B canis are the classic causative agents of disease

in humans. Human infection with recently discovered

marine strains (see Table 9-1) has also been noted. 23

Human infections with Brucella ovis and Brucella

neotomae have not been described. Brucellae grow

best on trypticase soy-based media or other enriched

media with a typical doubling time of 2 hours in

liquid culture. Although Bmelitensis bacteremia can

be detected within 1 week by using automated cul-

ture systems, 24 cultures should be maintained for at

least 4 weeks with weekly subculture for diagnostic

purposes. Most biovars of Babortus require incuba-

tion in an atmosphere of 5% to 10% carbon dioxide

186

Brucellosis

TABLE 9-1

of variable number tandem repeats have rapidly

expanded information on virulence determinants,

identification of pathogenicity islands, and evolution-

ary relatedness among the Brucella . 25-30

The LPS component of the outer cell membranes

of brucellae is different—both structurally and func-

tionally—from that of other gram-negative organ-

isms. 31,32 The lipid A portion of a Brucella organism

LPS contains fatty acids that are 16-carbons long, and

it lacks the 14-carbon myristic acid typical of lipid A

of Enterobacteriaceae . This unique structural feature

may underlie the remarkably reduced pyrogenicity

of Brucella LPS, compared with the pyrogenicity of

Escherichiacoli LPS (less than 1/100th). 33 In addition,

the O-polysaccharide portion of LPS from smooth

organisms contains an unusual sugar, 4,6-dideoxy-

4-formamido-alpha- d -mannopyranoside, which is

expressed either as a homopolymer of alpha-1,2-linked

sugars (A type), or as a repetitive series of 3-alpha-1,2

and 2-alpha-1,3-linked sugars (M type). These varia-

tions in O-polysaccharide linkages lead to specific,

taxonomically useful differences in immunoreactivity

between A and M sugar types. 34 A unique feature of this

organism, unlike most pathogenic bacteria, is the lack

of many classical virulence factors, such as exotoxins;

capsule; flagella; fimbriae; plasmids; lysogenic phage;

antigenic variation; cytolysins; pathogenic islands; or

type I, II, or III secretion systems; making characteriza-

tion of pathogenic mechanisms in this organism highly

challenging. Recently, however, a type IV secretion

system 35 has been identified as an important contribu-

tor to virulence.

TYPICAL HOST SPECIFICITY OF BRUCELLA

SPECIES

Brucella Species Animal Host Human Pathogenicity

Bsuis

Swine

High

Bmelitensis

Sheep, goats High

Babortus

Cattle, bison Intermediate

Bcanis

Dogs

Intermediate

Marine species

Marine

Rare

mammals

Bovis

Sheep

None

Bneotomae

Rodents

None

for growth. Brucellae may produce urease and may

oxidize nitrite to nitrate; they are oxidase- and cata-

lase-positive. Species and biovars are differentiated

by their carbon dioxide requirements; ability to use

glutamic acid, ornithine, lysine, and ribose; produc-

tion of hydrogen sulfide; growth in the presence

of thionine or basic fuchsin dyes; agglutination by

antisera directed against certain lipopolysaccharide

(LPS) epitopes; and susceptibility to lysis by bacte-

riophage. Brucella can grow on blood agar plates and

does not require X or V factors for growth. Analysis

of fragment lengths of DNA cut by various restriction

enzymes has also been used to differentiate brucellae

groupings. 21 Recent studies using proteomics, com-

plete genomic sequencing, and multilocus analysis

DISEASE

Epidemiology

In Kuwait, for example, disease with a relatively high

proportion of respiratory complaints has occurred in

individuals who have camped in the desert during the

spring lambing season. 36 In Australia an outbreak of Bsuis

infection was noted in hunters of infected feral pigs. 37 B

canis , a naturally rough strain that typically causes genital

infection in dogs, can rarely infect humans. 38

Brucellae are highly infectious in laboratory set-

tings; numerous laboratory workers who culture the

organism have become infected. However, fewer than

200 total cases per year (0.04 cases per 100,000 popula-

tion) are reported in the United States. The incidence

is much higher in other regions such as the Middle

East; countries bordering the Mediterranean Sea; and

China, India, Mexico, and Peru. Jordan, for example,

had 33 cases per 100,000 persons in 1987; Kuwait had

88 cases per 100,000 persons in 1985; and Iran had 469

cases from 1997 to 2002. 39-41

Animals may transmit Brucella organisms during

septic abortion, during slaughter, and through their

milk. Brucellosis is rarely, if ever, transmitted from

person to person. The incidence of human disease is

thus closely tied to the prevalence of infection in sheep,

goats, and cattle, and to practices that allow exposure

of humans to potentially infected animals or their

products. In the United States, where most states are

free of infected animals and where dairy products are

routinely pasteurized, illness occurs primarily in in-

dividuals who have occupational exposure to infected

animals, such as veterinarians, shepherds, cattlemen,

and slaughterhouse workers. In many other countries,

humans more commonly acquire infection by ingesting

unpasteurized dairy products, especially cheese.

Less obvious exposures can also lead to infection.

187

MedicalAspectsofBiologicalWarfare

Pathogenesis

with a long O-polysaccharide side chain); Bmelitensis

may be less susceptible than Babortus to complement-

mediated killing. 48,49 Administration of antibody to

mice before challenge with rough or smooth strains

of brucellae reduces the number of organisms that ap-

pear in the liver and spleen. This effect is attributable

mainly to antibodies directed against LPS, with little

or no contribution of antibody directed against other

cellular components. 50

Reduction in intensity of infection in mice can be

transferred from immune to nonimmune animals by

both cluster of differentiation 4 + (CD4 + ) and CD8 + T

cells 51 or by the immunoglobulin (IgG) fractions of

serum. In particular, the T-cell response to Brucella

appears to play a key role in the development of im-

munity and protection against chronic disease. 52,53

Neutralization of B abortus -induced host interferon

gamma (IFN–g) during infection in pregnant mice

prevents abortion. 54 Moreover, macrophages treated

with IFN-g in vitro inhibit intracellular bacterial repli-

cation. 55 Studies in humans support a role for IFN-g in

protection; homozygosity for the IFN-g + 874A allele is

associated with about a 2-fold increase in the incidence

of brucellosis. 56 In ruminants, vaccination with killed

bacteria provides some protection against challenge,

but live vaccines are more effective. 57-59 The most effica-

cious live vaccines express surface O-polysaccharide;

at a minimum, a complete LPS core is required for

rough mutant vaccine efficacy against Babortus and B

ovis infections in the mouse model. 60

These observations suggest that brucellae, like other

facultative or obligate intramacrophage pathogens,

are primarily controlled by macrophages activated

to enhanced microbicidal activity by IFN-g and other

cytokines produced by immune T lymphocytes. It is

likely that antibody, complement, and macrophage-

activating cytokines produced by natural killer cells

play supportive roles in early infection or in controlling

growth of extracellular bacteria.

In ruminants, Brucella organisms bypass the most

effective host defenses by targeting embryonic and tro-

phoblastic tissue. In cells of these tissues, the bacteria

grow not only in the phagosome but also in the cyto-

plasm and the rough endoplasmic reticulum. 61 In the

absence of effective intracellular microbicidal mecha-

nisms, these tissues permit exuberant bacterial growth,

which leads to fetal death and abortion. In ruminants,

the presence in the placenta of erythritol may further

enhance growth of brucellae. Products of conception

at the time of abortion may contain up to 10 10 bacteria

per gram of tissue. 62 When septic abortion occurs, the

intense concentration of bacteria and aerosolization of

infected body fluids during parturition often result in

infection of other animals and humans.

Brucellae can enter mammalian hosts through skin

abrasions or cuts, the conjunctiva, the respiratory tract,

and the gastrointestinal tract. 42 In the gastrointestinal

tract, the organisms are phagocytosed by lymphoepi-

thelial cells of gut-associated lymphoid tissue, from

which they gain access to the submucosa. 43 Organisms

are rapidly ingested by polymorphonuclear leuko-

cytes, which generally fail to kill them, 44,45 and are also

phagocytosed by macrophages (Figure 9-1). Bacteria

transported in macrophages, which travel to lymphoid

tissue draining the infection site, may eventually local-

ize in lymph nodes, liver, spleen, mammary glands,

joints, kidneys, and bone marrow.

In macrophages, brucellae inhibit fusion of phago-

somes and lysosomes, 46 and replicate within compart-

ments that contain components of endoplasmic reticu-

lum 47 via a process facilitated by the type IV secretion

system. 35 If unchecked by macrophage microbicidal

mechanisms, the bacteria destroy their host cells and

infect additional cells. Brucellae can also replicate

extracellularly in host tissues. Histopathologically,

the host cellular response may range from abscess

formation to lymphocytic infiltration to granuloma

formation with caseous necrosis.

Studies in experimental models have provided

important insights into host defenses that eventu-

ally control infection with Brucella organisms. Serum

complement effectively lyses some rough strains (ie,

those that lack O-polysaccharide side chains on their

LPS), but has little effect on smooth strains (ie, bacteria

Fig. 9-1. Impression tissue smear from a bovine aborted fetus

infected with Brucellaabortus . The bacteria appear as lightly

stained, gram-negative cells.

Photograph: Courtesy of John Ezzell, PhD, US Army Medi-

cal Research Institute of Infectious Diseases, Fort Detrick,

Maryland.

188

Brucellosis

Clinical Manifestations

the same frequency as sacroiliitis. In contrast to septic

arthritis caused by pyogenic organisms, joint inflam-

mation seen in patients with Bmelitensis is mild, and

erythema of overlying skin is uncommon. Synovial

fluid is exudative, but cell counts are in the low thou-

sands with predominantly mononuclear cells. In both

sacroiliitis and peripheral joint infections, destruction

of bone is unusual. Organisms can be cultured from

fluid in about 20% of cases; culture of the synovium

may increase the yield. Spondylitis, another important

osteoarticular manifestation of brucellosis, tends to af-

fect middle-aged or elderly patients, causing back (usu-

ally lumbar) pain, local tenderness, and occasionally

radicular symptoms. 72 Radiographic findings, similar

to those of tuberculous infection, typically include

disk space narrowing and epiphysitis, particularly

of the antero-superior quadrant of the vertebrae, and

presence of bridging syndesmophytes as repair occurs.

Bone scan of spondylitic areas is often negative or only

weakly positive. Paravertebral abscess rarely occurs. In

contrast with frequent infection of the axial skeleton,

osteomyelitis of long bones is rare. 73

Infection of the genitourinary tract (an important

target in ruminant animals) may lead to pyelonephritis,

cystitis, Bartholin’s gland abscess and, in males, epi-

didymoorchitis. Both pyelonephritis and cystitis may

mimic their tuberculous counterparts, with “sterile”

pyuria on routine bacteriologic culture. 74-76 With blad-

der and kidney infection, Brucella organisms can be

cultured from the urine. Brucellosis in pregnancy can

lead to placental and fetal infection. 77 Whether abortion

is more common in brucellosis than in other severe

bacterial infections, however, is unknown.

Lung infections have also been described, par-

ticularly before the advent of effective antibiotics.

Although up to one quarter of patients may complain

of respiratory symptoms, including mostly cough, dys-

pnea, or pleuritic pain, chest radiograph examinations

are usually normal. 78 Diffuse or focal infiltrates, pleural

effusion, abscess, and granulomas may be seen.

Hepatitis and, rarely, liver abscess also occur. Mild

elevations of serum lactate dehydrogenase and alkaline

phosphatase are common. Serum transaminases are

frequently elevated. 79 Biopsy may show well-formed

granulomas or nonspecific hepatitis with collections of

mononuclear cells. 63 Spontaneous bacterial peritonitis

has been reported. 80,81

Other sites of infection include the heart, central

nervous system, and skin. Although rare, Brucella en-

docarditis is the most feared complication and accounts

for 80% of deaths from brucellosis. 82,83 Central nervous

system infection usually manifests itself as chronic

meningoencephalitis, but subarachnoid hemorrhage

and myelitis also occur. Guillain-Barre syndrome has

Clinical manifestations of brucellosis are diverse,

and the course of the disease is variable. 63 Patients

with brucellosis may present with an acute, systemic

febrile illness; an insidious chronic infection; or a lo-

calized inflammatory process. Disease may be abrupt

or insidious in onset, with an incubation period of 3

days to several weeks. Patients usually complain of

nonspecific symptoms such as fever, sweats, fatigue,

anorexia, and muscle or joint aches (Table 9-2). Neuro-

psychiatric symptoms, notably depression, headache,

and irritability, occur frequently. In addition, focal

infection of bone, joints, or genitourinary tract may

cause local pain. Cough, pleuritic chest pain, and

dyspepsia may occur. Symptoms of patients infected

by aerosol are indistinguishable from those of patients

infected by other routes. Chronically infected patients

frequently lose weight. Symptoms often last for 3 to 6

months and occasionally for a year or more. Physical

examination is usually normal, although hepatomega-

ly, splenomegaly, or lymphadenopathy may be found.

Brucellosis does not usually cause leukocytosis. Some

patients may be moderately neutropenic 64 ; however,

cases of pancytopenia have been noted. 65 In addition,

bone marrow hypoplasia, immune thrombocytopenic

purpura, and erythema nodosum may occur during

brucellosis infections. 66-68 Disease manifestations can-

not be strictly related to the infecting species.

Infection with B melitensis leads to bone or joint

disease in about 30% of patients; sacroiliitis devel-

ops in 6% to 15% of patients, particularly in young

adults. 69-71 Arthritis of large joints occurs with about

TABLE 9-2

SYMPTOMS AND SIGNS OF BRUCELLOSIS

Symptom or Sign

Patients Affected (%)

Fever

90–95

Malaise

80–95

Body aches

40–70

Sweats

40–90

Arthralgia

20–40

Splenomegaly

10–30

Hepatomegaly

10–70

Data sources: (1) Mousa AR, Elhag KM, Khogali M, Marafie AA.

The nature of human brucellosis in Kuwait: study of 379 cases. Rev

Infect Dis . 1988;10:211–217. (2) Buchanan TM, Faber LC, Feldman

RA. Brucellosis in the United States, 1960–1972: an abattoir-associ-

ated disease, I: clinical features and therapy. Medicine (Baltimore).

1974;53:403–413. (3) Gotuzzo E, Alarcon GS, Bocanegra TS, et al.

Articular involvement in human brucellosis: a retrospective analysis

of 304 cases. SeminArthritisRheum. 1982;12:245–255.

189

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