Clinical signs

Brucellosis is characterized by reproductive disorders: i) abortion, retention of placenta, metritis, subclinical mas­titis, infertility; ii) orchitis or epididymitis with frequent sterility; and iii) articular and peri-articular hygromas, seen in chronic infections.

DIAGNOSIS

Laboratory diagnosis includes indirect (antibody detec­tion) tests that can be applied to milk or blood as well as direct tests (classical bacteriology, polymerase chain reac­tion (PCR)-based methods). Isolation of Brucella spp. (or Brucella spp. DNA detection by PCR) allows certainty of diagnosis. Biotyping provides valuable epidemiological information, which allows tracing of infection sources in countries where several biotypes are co-circulating. However, when one particular biovar is the overwhelming isolated strain, classical typing techniques are of no help. In this context, new fingerprinting methods such as mul­tiple locus variable (number of tandem repeat) analysis (MLVA) and multilocus sequence analysis (MLSA), have gained wider acceptance and will, in coming years, be used as routine typing and fingerprinting methods for molecu­lar epidemiological purposes⁽⁴¹⁾.

Several techniques are available to identify Brucella spp. The Stamp staining method is still often used, and even if this technique is not specific (other abortive agents such as Chlamydophila abortus, or Coxiella burnetii are also stained), it provides valuable information for the analysis of abortive material. Bacterial isolation is nevertheless always preferable and even required for the typing of the strain.

CULTURE

For the definitive diagnosis of brucellosis, the choice of samples depends on the observed clinical signs. In the case of clinical brucellosis, valid samples include aborted fetuses (stomach, spleen and lung), fetal membranes, vaginal secre­tions, colostrum, milk, sperm or fluid collected from arthritic lesions or fluid-filled joint bursae (hygroma). At post mortem examination, in order to confirm acute or chronic brucellosis, the preferred tissues for sampling are the genital and oropharyngeal lymph nodes, the spleen, the mammary gland and associated lymph nodes. For the isola­tion of Brucella spp., the most commonly used medium is Farrell’s medium, which contains antibiotics to inhibit the growth of other bacteria present in clinical samples. Some Brucella species need CO₂ for growth. For liquid samples (milk or blood), sensitivity is increased by the use of a biphasic medium such as the Castaneda medium. Growth may appear after 2—3 days, but cultures can only be con­sidered negative after 10—15 days of incubation.

Biotyping of B rucella spp. is performed by using differ­ent tests, the most important being agglutination tests, the dependence on CO₂ for growth, production of H₂S, and growth in the presence of specific factors. These techniques need standardization and experience, and they are per­formed in reference laboratories.

Molecular identification

Several PCR-based methods have been developed, and the best validated are those that use the detection of specific sequences of Brucella spp. These techniques were mostly validated on human samples but have become more widely applied in veterinary medicine. Relative high cost and the lack of standardization have limited their application to specific problems such as wildlife studies or rapid identi­fication of incidents in uninfected areas. As a general rule, these techniques show a lower diagnostic sensitivity than culture; however, their specificity is close to 100%.

molecular typing

For the typing of Brucella spp., the AMOS PCR is often used⁽⁴²⁾. This PCR and derived PCR allow the discrimina­tion between Brucella species and between vaccine and wild-type strains but do not allow discrimination between biovars of a given Brucella species. A newly developed multiplex PCR assay (Bruce-ladder) can identify and dif­ferentiate for the first time all of the B rucella species and the vaccine strains in the same test⁽⁴³⁾. These new tech­niques are valuable tools for the identification and charac­terization of B. suis, B. ceti and B. pinnipedialis, which are now the most important Brucella species found in Euro­pean wildlife.

Brucella suis biovars

The taxonomic position of B. suis within the genus Bru­cella is subject to an ongoing debate. A number of genetic observations supported by independent studies have dem­onstrated that, with the exception of B. suis biovar 5 (prob­ably misnamed), all B. suis and B. canis strains form a consistent group. A drawback persists for the distinction of B. suis biovar 4 from B. canis, but a single nucleotide polymorphism (SNP) in the omp25 gene unique to B. canis can be included in a typing scheme⁽⁴⁴⁾.

BRUCELLA CETI AND B. PINNIPEDIALIS

A marker specific for the marine mammal strains was identified when amplification of the gene encoding the immunodominant BP26 protein revealed a larger than expected PCR product, reflecting the insertion of an /5711 element downstream of the gene⁽⁴⁵⁾. A PCR based around bp26 has become a well-used test for differentiation of Brucella spp. associated with marine mammals from clas­sical species associated with terrestrial mammals. Eventu­ally, two new Brucella species labelled (with corrected etymology) as B. ceti for isolates from cetaceans and B. pinnipedialis for isolates from pinnipeds were validly pub­lished®. Further, MLSA studies suggested that Brucella strains from marine mammals corresponded to a cluster of five sequence types distinct from all previously described Brucella species from terrestrial mammals⁽⁴⁴⁾.

Brucellosis serology is usually performed using the same antigens as in domestic ruminant serology because the Brucella immunodominant antigens are associated with the surface ‘smooth’ lipopolysaccharide (LPS) and are to a large extent shared by all the naturally occurring biovars of B. abortus, B. melitensis, B. suis, B. microti, B. ceti, B. pinnipedialis and B. inopinata. It is important to note that B. ovis and B. canis present on their surface a ‘rough’ LPS, which is not antigenically related to smooth LPS. This means that infections by these two Brucella species cannot be detected by serological tests detecting anti-smooth LPS antibodies. Tests detecting rough LPS Brucella infections will not be discussed further, as to date neither B. ovii nor B. canis have been reported in wildlife in Europe.

It is virtually impossible to determine which species of Brucella induce anti-smooth LPS antibodies in the host. Some assays, such as indirect enzyme-linked immunosorb­ent assay (ELISA), rely on species-specific reagents that are not commercially available. This limitation of the lack of polyclonal or monoclonal antibodies to many wildlife species immunoglobulins can be partly overcome by the use of either Protein A or Protein G conjugates⁽⁴⁷⁾. Other techniques such as competitive ELISA or the fluorescent polarization assay (FPA) do not rely on species- specific reagents and have been useful in marine mammals⁽⁴⁷⁾.

It is not appropriate to propose a single serological test as a reference test to assist veterinarians in the diagnosis of wildlife brucellosis. The ecology of wildlife brucellosis has become more and more important in order to understand serological results. In a first screening programme, a paral­lel interpretation of the first line tests should be chosen to improve the specificity of the detection of anti-Brucella antibodies⁽²⁷⁾. The Rose Bengal plate test and/or the newly developed ELISA and the FPA should first be used. Cross­reactive bacteria such as Yersinia enterocolitica O:9 induce serological cross-reactions in the brucellosis serological tests that are almost indistinguishable from true brucellosis serological reactions¹-⁴⁸).

The ‘gold standard’ in brucellosis remains the isolation of Brucella strains. If brucellosis is suspected in an animal or a wildlife population following positive serological results, attempts to isolate the organism are mandatory and thus should always be performed.