Introduction

Brucellosis is an infectious bacterial disease of humans and ungulates caused by Brucella species. It is a worldwide zoonosis that has significant economic and human health impacts. The disease has been afflicting humans and animals since ancient times as anthropological evidence suggests the presence of the organism in carbonized cheese and the typical bone lesions in human remains from areas affected by the eruption of Mount Vesuvius in ad 79.¹ Human infection is generally due to contact with animals or animal products contaminated with the bacteria. Animal disease in the United States has been controlled through vaccination and an eradication program, although the bacteria still persists in certain wildlife populations. Brucellosis remains a problem in countries where animal disease control programs have not reduced the infection among animals. Management and control of animal brucellosis is the key to prevention of human disease.

The genus Brucella consists of six classical species which are generally associated with their host of preference: Brucella abortus (cattle); B. melitensis (goats and sheep); B. suis (pigs); B. canis (dogs); B. ovis (sheep); and B. neotomae (desert wood rat). Relatively new species have been isolated from marine mammals: B. pinnipedialis (seals, sea lions and walruses) and B. ceti (whales, porpoises and dolphins) are of unknown significance and have the potential to infect humans.^2,3 Brucella microti, found in the common vole, red foxes, and the soil in some areas of Europe, is of unknown pathogenicity for livestock and humans.⁴ Because of the advent of molecular technologies, several new atypical species have been proposed due to their genetic similarities with classical brucellae.^5–8 Although they have a distinct preference for their chosen host, the brucellae are capable of infecting other vertebrates also. Bovine brucellosis is primarily caused by B. abortus and results in abortion and infertility, but B. melitensis and B. suis can infect cattle and cause a range of symptoms.

Brucella spp. are facultative intracellular pathogens that can be grown in the laboratory environment in nutrient-rich media selected for fastidious organisms in an aerobic atmosphere at 37 °C. Laboratory strains usually produce individual colonies in 2–5 days, but animal isolates from infected tissues may take up to 4 weeks to grow on selective media. The organisms are small Gram-negative coccobacilli that are catalase-positive, nonmotile, and have variable oxidase and urease activity. Species and biovars identification is achieved through the use of dye sensitivities, phage typing, and molecular analysis. The species are classified as “smooth” or “rough” based on the composition of their lipopolysaccharide (LPS) outer membrane. Smooth species (B. abortus, B. melitensis, B. suis) have a complete O-polysaccharide group (OPS) while rough species (B. canis and B. ovis) lack this polysaccharide chain or possess only a portion of it.⁹ This affects colonial morphology and may be used both microbiologically and serologically in species and strain determination.

Animal brucellosis is classically a reproductive disease, resulting in abortion, stillbirth, and weak offspring. It may affect fertility and milk production. Chronically infected animals which exhibit no clinical signs pose a threat to naive animals and their handlers. The presence of the disease in a region or country can result in international regulations that restrict animal movement and trade, adversely affecting exports and the economy.

Brucella melitensis

Brucella melitensis, the first species in the genus Brucella to be described, causes abortions in female goats and sheep, unilateral orchitis in males, and Malta fever in humans.¹⁰ Sir David Bruce, a British army surgeon, discovered the organism in 1887 as the causative agent of Mediterranean or Malta fever.¹¹ The preferred hosts may be goats and sheep, but this organism is the least animal species-specific of the brucellae,¹⁰ and in countries with a high prevalence of this infection in small ruminants it is not uncommon to isolate it from cattle.¹²

Although sheep and goats and their products are the main source of human infection, B. melitensis in cattle has emerged as an important problem in some southern European countries, Israel, Kuwait, and Saudi Arabia. The disease in goats closely resembles the disease observed in B. abortus-infected cattle.¹³ Brucella melitensis infection is particularly problematic because B. abortus vaccines do not effectively protect against this species. Thus bovine B. melitensis infection is emerging as an increasingly serious public health problem in some countries. The United States was thought to be free of this pathogen; however, in October 1999, four cows and some goats and sheep in South Texas were found to be infected with B. melitensis.¹⁴ The likely source of the outbreak was goats from northern Mexico that may have crossed the border and mixed with this herd.

Brucella suis

Brucellosis caused by B. suis was first described by J. Traum in 1914 in swine herds in Indiana. Although originally considered a pathogenic B. abortus, it was later named B. suis by I.F. Huddleson.¹⁵ Domestic and feral swine are natural hosts of B. suis.^16–18 In sows, abortion is the primary indicator of disease, which may occur at any stage of the pregnancy. An infected sow may deliver healthy live piglets as well as dead or weak ones. In boars, there may be brucellae present in the semen without any visual indications of disease. Boars may exhibit unilateral swelling and atrophy of the epididymides and testes usually resulting in infertility. Reports of lameness; swollen joints, bursae, and tendons; and paralysis because of abscess formation near the spine have also been documented. Brucellosis caused by B. suis is considered to be a venereal disease with the infected boar passing the disease on to uninfected sows.¹⁵

Since pigs do not produce dairy products, contraction of the human disease is primarily limited to feral pig hunters and as an occupational hazard of farmers, veterinarians, and abattoir workers. However, B. suis can infect cattle that have had direct contact with feral swine, leading to the eventual consumption of unpasteurized dairy products and the perpetuation of this zoonotic pathogen.^19,20

Brucella abortus

Brucella abortus, initially isolated as Bacillus abortus by Bang in 1897 and eventually renamed in 1920, is the etiological agent of bovine brucellosis, an infection that leads to spontaneous abortion, premature calving, and infertility in cattle. At parturition, the fetus, placenta, uterine fluid, and milk contain large quantities of infectious bacteria, which are shed into the environment, potentially infecting naive animals or human handlers.^13,21

In cattle, brucellosis is primarily a disease of the female. Bulls can be infected, but they do not readily spread the disease. The organism localizes in the testicles of the bull resulting in orchitis. In the female, the organism localizes in the udder, uterus, and lymph nodes adjacent to the uterus. The infected cows exhibit symptoms that may include abortion during the last trimester of pregnancy, retained afterbirth, and weak calves at birth.¹³ Infected cows typically abort only once. Subsequent calves may be born weak or healthy and normal. Some infected cows will not exhibit any clinical symptoms of the disease and give birth to normal calves. Millions of organisms are shed in the afterbirth and fluids associated with calving and abortion. The disease is spread when cattle ingest contaminated feed or lick calves or aborted fetuses from infected cattle. Once lactation begins, the organism migrates to the lymphatics associated with the mammary gland, thus making milk a potential source of human infection.

This species is able to cross the species barrier affecting both livestock and humans.²² In livestock, it causes billions of dollars in losses due to the reproductive consequences in cattle. It is also listed as a civilian, military, and agricultural bioterrorism agent.

Pathogenicity

Pathogenic Brucella spp. are intracellular pathogens that are capable of survival and replication inside host phagocytic and nonphagocytic mammalian cells, which is essential for virulence.²³ Following penetration of the mucosal epithelium, the bacteria are transported to the regional lymph nodes. The spread and multiplication of Brucella in lymph nodes, spleen, liver, bone marrow, mammary glands, and sex organs occurs via macrophages. The increase of brucellae in the host is mainly due to the organism’s ability to avoid the killing mechanisms of the macrophages. Virulent Brucella species not only resist killing by neutrophils following phagocytosis,^24–26 but they also replicate inside macrophages²⁷ and nonprofessional phagocytes.²⁸ Brucellae are capable of establishing themselves in replicative phagosomes inside host macrophages for extended intracellular survival. They also appear to be capable of withstanding exposure to reactive oxygen intermediates, acidic pH, and nutrient deprivation during their time inside a variety of host macrophages, including in mice, humans, and cattle.^23,29–33 There is evidence that smooth LPS probably plays a vital role in intracellular survival since smooth organisms tend to survive much more effectively than rough ones.^34,35 Macrophages containing the bacteria journey to the draining lymph node via the lymphatics.^13,36 Within 2–3 weeks, the lymph node becomes hemorrhagic due to destruction of the vasculature within the node. Some macrophages within the lymph node are lysed, and the brucellae are released to enter the bloodstream which results in a subsequent bacteremia.^13,36,37 The organisms migrate throughout the host, localizing in the reticuloendothelial system.

During pregnancy, Brucella spp. replicate to high numbers inside the trophoblastic cells of the ungulate placenta.^13,36,38,39 The mechanism of invasion of nonphagocytic cells, such as placental trophoblasts, is not clearly established. Within nonphagocytic cells, brucellae tend to localize in the rough endoplasmic reticulum. Placental trophoblasts are a part of the epithelial layers of the placenta of the natural host. They serve as an important interface between the maternal and fetal circulation. During late gestation, Brucella are known to replicate within the placental trophoblasts of their natural ruminant host, causing the degradation of placental integrity, infection of the fetus, and possibly abortion or the birth of weak or infected animals.⁴⁰ Erythritol, which may serve as a growth stimulant for brucellae, is produced in large amounts by ruminant placental trophoblasts.¹³ Further experiments utilizing microscopic analysis of placental tissues from B. abortus-infected cows and goats also revealed that brucellae replicate in intracellular compartments associated with the rough endoplasmic reticulum of trophoblasts, suggesting a similar intracellular environment to that inside host macrophages.^38,41,42 The exact mechanism by which fetal infection causes abortions is unclear. The interference with fetal circulation due to placentitis has been suggested as a cause of fetal abortion.⁴³

Diagnostics

To diagnose brucellosis in cattle one could use three methodologies: (i) serology, the detection of antibodies to the bacteria; (ii) bacteriology, the isolation of the organism from animal tissues; and (iii) molecular biology, using molecular techniques to isolate and identify bacterial DNA. Serology is commonly used to test animals because results can be obtained in a short period of time. All the serologic tests are based on bovine antibodies binding to Brucella antigens. Typically a country would follow the suggested OIE or USDA serological methods.^44,45 Conventional serologic tests include the Rose Bengal test (RBT), the milk ring test, the Card test, the Rivanol test, the standard tube test (STT), buffered acidified plate antigen test, and the complement fixation test. Primary binding tests include particle concentration fluorescence immunoassays (PCFIA), indirect and competitive enzyme-linked immunosorbent assays (ELISA), and fluorescence polarization assay (FPA). Current US surveillance involves the milk ring test for dairies and serologic testing of market cattle. Ideally, one would use serology in conjunction with either Brucella culture or molecular typing for a definitive diagnosis.^46,47

Identification of a serologic positive animal will trigger an accepted regulatory process that may involve test and slaughter, quarantine, vaccination, and herd management. Human disease detection initiates the appropriate antibiotic therapy once the bacterial genus is elucidated. Epidemiologic surveillance generally requires speciation so all three methodologies may be utilized.

Immune response

The host immune response to a typical virulent strain of B. abortus includes both cell-mediated and humoral reactions. The humoral facet involves the production of antibodies that target the surface components of the cell’s outer membrane. Complement has been shown to be ineffective in combating B. abortus directly but may aid in phagocytosis.⁴⁸ The primary immunogenic element of the smooth outer membrane is the OPS portion of the LPS. The host’s antibody response to the OPS is insufficient in providing protection against a challenge infection. The efficacy of the rough RB51 vaccine strain, which lacks the OPS, also demonstrates that the humoral response is not of primary importance. The IgG₁ antibodies predominantly produced in response to B. abortus do not correlate with elimination of the pathogen.⁴⁹ Antibodies can promote intracellular killing through opsonization and slow the reproduction rate of intracellular B. abortus, but antibodies alone cannot protect the host from the bacteria.^48–50 In terms of bovine vaccination, the cell-mediated immune response must be evoked for protective immunity.

The host’s cell-mediated immune reaction to B. abortus is primarily accomplished by activation of the T-cells of the adaptive immune response. The function of cell-mediated immunity is to seek out and destroy host cells containing intracellular pathogens, such as viruses and facultative or obligate intracellular bacteria.⁵¹ The macrophage is primarily responsible for ingestion and clearance of brucellae from the extracellular environment. Antigen presentation to both CD4 and CD8 T-cells is a crucial step in the host’s protective immunity. Macrophages that are not activated prior to ingestion of B. abortus differ in their response compared with previously activated macrophages, which are considerably more effective in killing the brucellae.^52,53 Brucella abortus ingested by inactivated macrophages can inhibit phagolysosome development or neutralize the acidic environment, allowing them to avoid killing and replicate within the cell relatively undeterred.⁵² In these nonactivated phagocytes, the brucellae cause the macrophages to suppress their reaction to a strong CD4⁺ Th1 response.⁵⁴ Activated macrophages degrade B. abortus on ingestion and phagolysosomal fusion, where the bacteria can be recognized and presented to CD4⁺ Th1 cells by the major histocompatibility complex II receptor. An effector CD4⁺ Th1 cell secretes interferon (IFN)-γ which further activates macrophages, causing an increase in antimicrobial activities against the pathogen. Brucella abortus triggers a strong CD8⁺ T-cell response that mediates cytotoxic activity to lyse infected cells. If a macrophage containing replicating B. abortus is lysed, the bacteria are released into the extracellular environment where they can either infect other cells or be exposed to the immune system and ingested and destroyed by activated macrophages.^{27,53,55–57}