Brucellosis

History:

The isolation of Brucella melitensis as a human pathogen occured in the British Army, whilst they were stationed in Malta in the late 19th century. The discovery was made by Dr. David Bruce (Scottish pathologist and microbiologist in 1887). The "Mediterranean Fever" Commission concluded that on Malta, native goats were the reservoir of infection, and raw goat's milk was the vehicle of transmission from animal to human.

Pathophysiology:

Brucella species are facultative intracellular pathogens that are capable of surviving and replicating within phagocytic cells of the host. Shortly after gaining entry to the body, brucellae are ingested by polymorphonuclear leukocytes (PMLs), which are attracted to the site of inoculation. The principal virulence factor is cell wall lipopolysaccharide (LPS). Normal serum factors, including complement, are involved in opsonization of the organisms to allow phagocytosis, but PMLs have limited ability to kill bacteria within phagocytes. A copper-zinc superoxide dismutase, o-polysaccharide, and nucleotidelike substances are among the factors that protect brucellae from being killed by PMLs.

Brucellae that are not killed by PMLs are ingested by macrophages, where they become localized within organs of the reticuloendothelial system (ie, liver, spleen, bone marrow) and multiply in macrophages and monocytes. However, any organ system can be involved in brucellosis (ie, CNS, heart, joints, genitourinary system, pulmonary system, and skin), and localization of the process may cause focal symptoms or findings.

Shortly after infection, humoral antibodies directed against LPS and other cell wall antigens are produced. However, development of cell-mediated immunity is the principle mechanism of recovery. The host response to infection with Brucella abortus is characterized by the development of tissue granulomas indistinguishable from those of sarcoidosis. In contrast, infection with the more virulent species (Brucella melitensis, Brucella suis) more commonly results in visceral microabscesses.

Transmission and Incubation:

The disease is transmitted primarily through contaminated or untreated milk (and its derivates) or through direct contact with infected animals, which may include dogs, pigs, camels and ruminants, primarily sheep, goats, cattle, bison. This also includes contact with their carcasses. Leftovers from parturition are also extremely rich in highly virulent brucellae. Infection may also occur by injesting contaminated grass, roughage, feed, or water. Once the susceptable animal injests the organism, the bacteria progress to the regional lymph nodes where it resides during the incubation period. The incubation period, which is the time between exposure to the organism and the ability to detect the disease, may range from two weeks to two months and longer. After a subsequent brief phase when the bacteria are in the bloodstream, the organism localizes in the uterus, placenta, udder, and/or regional lymph nodes. Although the most common clinical sign of brucellosis in cattle is abortion, the brucellosis-infected cow often shows no overt clinical signs. She is often seronegative shortly after exposure due to the lag time between exposure and seroconversion or clinical disease. Brucellae, along with leptospira have the unique property of being able to penetrate through intact human skin, so infection by mere hand contact with infectious material is likely to occur.

Clinical Features:

Duration of symptoms for more than 30 days before diagnosis is the major risk factor for developing focal disease. Mortality is low (<2%) and is most frequently found in those with endocarditis due to brucellosis.

• The most common focal complications are as follows:
  • Osteoarticular complications - Especially, sacroiliitis (20-30% - but rarer in children)
  • Genitourinary tract complications - Especially, epididymoorchitis in males (2-49%)
  • Neurobrucellosis- Meningitis (1-2%) and, less commonly, papilledema, optic neuropathy, radiculopathy, stroke, and intracranial hemorrhage
  • Endocarditis (1%) - Responsible for most mortality associated with the disease
  • Hepatic abscess (1%)
• Other less common complications include the following:
  • Splenic abscess
  • Thyroid abscess
  • Epidural abscess
  • Pneumonitis
  • Pleural empyema
  • Uveitis
  • Aneurysm of the aorta
  • Aneurysm of the cerebral vessels
  • Peritonitis

Laboratory Identification:

Morphology

Brucella are small, Gram-negative coccobacilli that lack capsules,endospores or native plasmids.

The cell wall consists of an outerlayer of lipopolysaccharide (LPS) protein approximately 9 nm thick. Thin-section electron micrographs reveal an electron-dense layer3–5 nm thick consisting of a highly cross-linked muramyl–mucopeptide complex associated with lipoproteins. Matrix and porinproteins penetrate the peptidoglycan layer at irregular intervals. A low-density periplasmic space separates the peptidoglycan layer from the cell membrane.Brucellae are nonmotile and do not form spores.

Cultural Diagnosis

They grow aerobically, although some species require supplemental CO₂ for primary isolation. Brucella can be cultured on any high-quality peptone-based media. Growth is enhanced by the addition of blood or serum, but primary isolation may require prolonged (several weeks) incubation. Culture of bone marrow is reported to give a higher yield than the culture of peripheral blood.

All strains are catalase positive, but oxidase and urease activities and the production of H₂S are variable. The major species of Brucella and their biovars are differentiated on the basis of oxidative metabolism tests, growth on media containing dyes, and lysis by brucella-phages. Because the symptoms of brucellosis are nonspecific, it is important to obtain a detailed history that includes occupation, avocations, travel to enzootic areas, and ingestion of high-risk foods, such as unpasteurized dairy products. The white blood cell count is often normal or low and the erythrocyte sedimentation rate is variable.

The diagnosis is made with certainty when brucellae are recovered from blood, bone marrow, or other tissues. The rate of isolation ranges from 15% to more than 90% depending on the methods used. Most laboratories now use continuous-monitoring automated blood culture systems (e.g., BACTEC or BacT/Alert) that have improved the time to isolation and have obviated the need for biphasic media techniques. Nevertheless, the handling of brucellae is a risk to laboratory personnel, and appropriate precautions should be taken. Brucellae are isolated most often from blood and bone marrow; however, in selected cases, urine, CSF, synovial fluid, and biopsies of liver, lymph nodes, and other tissues can be successful.Rapid automated bacterial identification systems should be interpreted with caution, as not all contain the appropriate profiles, and brucellae have been misidentified as Moraxella phenylpyruvica.. Molecular techniques, such as polymerase chain reaction (PCR), employing random or selected primers have been used widely in veterinary medicine and to differentiate Brucella spp. PCR appears to be highly sensitive and specific when used on peripheral blood or other tissues; however, additional studies are needed to establish its role in brucella diagnostics.