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Brucella spp. infections

Brucella spp. infections

J. Godfroid -, Faculty of Biosciences, Fisheries and Economics, UiT - The Arctic University of Norway, Hansine Hansens veg 18, Tromsø 9019, Norway and Faculty of Veterinary Science, University of Pretoria, Private, Bag X04, Onderstepoort, Gauteng, South Africa, 0081

Brucellosis is caused by facultative intracellular α-Proteobacteria of the genus Brucella, which is related phylogenetically to other bacterial genera such as Agrobacterium, Phyllobacterium, Rhizobium and Ochrobactrum.

Bacteria of the genus Brucella are Gram-negative cocci, coccobacilli or short rods with straight or slightly convex sides and rounded ends that do not ferment carbohydrates in conventional media.4 They are transmissible to a wide range of animal species. Early bacteraemia is followed by localization of the infection in the genital organs and cells of the monocyte-macrophage series. Brucellosis is an important disease of humans and domestic and wild animals worldwide. In humans the disease caused by Brucella spp. (mainly Brucella melitensis, Brucella abortus and Brucella suis) is also known as Malta fever, Mediterranean fever or Bang’s disease, or as undulant fever.4, 8, 14 Among domestic animals, cattle, sheep, goats and pigs are mainly affected (Table 1). A number of wildlife species, such as African buffalo, antelope, bison, camels, elk, reindeer, caribou and other wild ruminants and carnivores, are also susceptible to infection.80 (see Brucella infections in terrestrial wildlife).

The genus Brucella contains 12 validly published species that include B. abortus, B. melitensis, B. suis, B. ovis, B. canis and B. neotomae. Seven are recognized for B. abortus, three for B. melitensis and five for B. suis. However, the degree of genetic relatedness, as shown by DNA hybridization studies and whole genome sequencing techniques, is consistent with the existence of a single species within the genus Brucella.14, 73

Brucella abortus, B. melitensis, B. suis and B. neotomae generally occur in the smooth form, while B. ovis and B. canis are invariably rough species. Differentiation of the biovars of the species of the genus Brucella is shown in Table 2.

Recently  new Brucella species have been described besides the six classical species: Brucella ceti and Brucella pinnipedialis in cetaceans and pinnipeds, respectively; Brucella microti in vole and soil; Brucella vulpis in red foxes; Brucella inopinata in frogs and from a woman’s breast implant, whose origin has not been determined; and Brucella papionis in baboon.14

In animals, the most important clinical manifestations are abortion, the birth of weak offspring, orchitis and epididymitis. Signs of arthritis may be evident in chronic infections. The primary sources of contamination of the environment are foetal membranes and fluids and vaginal discharges expelled by infected females when they abort or at parturition. Brucellae are also commonly shed in milk and semen.

The rare occurrence of brucellosis in domestic animals in many developed countries is directly linked to the success of brucellosis control/eradication programmes.14

Table 1Brucella species and preferential livestock species

B. abortus Abortion and orchitis Cattle
B. ovis Epididymitis/orchitis Sheep
B. melitensis Abortion and orchitis Sheep and goats
B. suis Abortion, stillbirth, sterility in sows, and orchitis Pigs


In cattle, biovars of B. abortus are usually the cause of brucellosis but in some countries, particularly in southern Europe, central Asia and in the Middle East, B. melitensis has also been implicated as a cause of abortion in cattle where they are kept in close association with infected sheep or goats. Occasionally Brucella suis may infect the mammary gland of cattle but it has not been reported to cause abortion in this species.14

Caprine and ovine brucellosis (excluding Brucella ovis infections) are most commonly caused by B. melitensis (see Brucella melitensis infection) but sporadic cases are caused by B. abortus. In sheep and goats the disease caused by B. melitensis and its epidemiology are very similar to those of B. abortus infection in cattle. Importantly, in West Africa there is no report of the isolation of B. melitensis in sheep and goats, and infection in small ruminants seem to be due to B. abortus.14

Brucellosis in pigs (see Brucella suis infection) is caused by B. suis and is characterized by an initial bacteraemia followed by the production of chronic lesions in the bones and reproductive organs of both sexes. Brucella suis consists of five biovars. Pigs are infected by B. suis biovars 1, 2 or 3: disease caused by biovars 1 and 3 is similar, while that caused by biovar 2 differs in its pathology, host range, and geographical distribution (mainly in Europe). Brucella suis biovar 2 is rarely pathogenic to humans, whereas biovars 1 and 3 are highly pathogenic and cause severe disease in humans.14

Brucella ovis is one of the most common causes of epididymitis in rams but is a rare cause of abortion in ewes and neonatal mortality in lambs (see Brucella ovis infection). Low reproductive rates may occur in affected flocks. It is not a zoonosis.

Brucella canis causes epididymitis and orchitis in male dogs and metritis in bitches, and is a rare infection in humans. It does not infect other animal species.

Brucella neotomae has been isolated from a rodent species in western USA.14

The presence of Brucella spp. in abortion material or vaginal discharge can be demonstrated microscopically in tissues or smears using modified acid-fast staining (Stamp’s staining), and is considered presumptive. Polymerase chain reaction (PCR) methods are nowadays used routinely in diagnostic laboratories to identify Brucella DNA in  clinical samples. The first species-specific multiplex PCR assay for the differentiation of Brucella, named AMOS-PCR (on the basis of the initials of the species that it identifies) was described in 1994.31 This assay was later updated to identify B. abortus vaccine strains S19 and RB51.30 A real-time PCR was developed in a multiplex format in 2004, allowing the rapid identification of Brucella spp., B. abortus, and B. melitensis in a single test.163 A multiplex PCR assay (Bruce-ladder) for rapid and simple one-step identification of Brucella has been developed and validated in 2008. It can identify and differentiate in a single step most Brucella species as well as the vaccine B. abortus strains S19 and RB51 and the vaccine B. melitensis strain Rev.1.116 In 2011, another multiplex PCR assay (Suis-ladder), has been developed for fast and accurate identification of B. suis strains at the biovar level.117

Brucella spp. should be isolated by culturing samples on selective media (Farrell) allowing the further isolation of DNA to perform molecular typing by methods such as Multiple Locus Variable-Number-Tandem-Repeat Analysis (MLVA)110, 196 and Multilocus sequence typing (MLST),200 as well as Whole Genome Sequencing (WGS).73 On-line resources on MLVA and MLST can be found on the MicrobesGenotyping website The Brucella database is regularly updated and contains data for more than 5000 strains. Presently, whole-genome data from many strains of Brucella spp. have been published and are available at the NCBI (National Center for Biotechnology Information) On line resources like PATRIC (Pathosystems Resource Integration Center), provide integrated data and analysis tools to support biomedical research on Brucella spp.

Species and biovars identification by classical biotyping methods remains the reference method.14 However, PCR-based typing methods as well as WGS become important tools for studies related to the phylogeny, taxonomy, epidemiology and evolution of Brucella spp.73, 196, 200

Most of the serological tests for the diagnosis of smooth Brucella spp. infections (i.e. B. melitensis, B. abortus and B. suis) have been developed to detect antibodies directed against antigens (mainly A and/or M epitopes) associated with the smooth lipopolysaccharide (S-LPS) and are shared by all the naturally occurring biovars of B. abortus, B. melitensis and B. suis.14, 80 Diagnostic antigens must be prepared from smooth strains of B. abortus (strain 1119-3 or strain 99) or B. melitensis (strain 16M) and comply with minimum standards for purity, sensitivity and specificity. Such antigens are suitable for the detection of antibodies directed against B. melitensis, B. abortus and B. suis in different animal species infected with smooth brucellae. Because the S-LPS is absent in the naturally rough B. ovis and B. canis, specific B. ovis and B. canis antigens (mainly R epitopes) associated with the rough lipopolysaccharide (R-LPS) have to be used for the diagnosis of infections caused by these organisms.14

Table 2Biovar differentiation of the six classical species of the genus Brucella

        Thionin* Fuchsin* Safranin O** A M R Tb°
B. abortus 1 (+) + + + + -   L
  2 (+) + +   L
  3 (+) + + + + +   L
  4 (+) + +*** + +   L
  5 + + + +   L
  6 (–) + + + +   L
  9 + + + + +   L
B. suis 1 + + –**** +   NL
  2 + +   NL
  3 + –**** +   NL
  4 + (–) + +   NL
  5 + +   NL
B. melitensis 1 + + + +   NL
  2 + + + +   NL
  3 + + + + +   NL
B. ovis   + + (+)   NL
B. canis   +   NL
B. neotomae   + +     NL/PL

* Concentration = 1/50000 p/v

** Concentration = 1/10000 p/v

*** Certain strains are Fuchsin sensitive

**** Certain strains are Fuchsin and Safranin O resistant

Tb °= Tbilisi phage; L = confluent lysis; PL = partial lysis; NL = no lysis

A = monospecific antiserum; M = monospecific antiserum; R = monospecific antiserum

(+) = most of the strains are positive; (–) = most of the strains are negative

CO2 = CO2 requirement

H2 S=H2 S production

The brucellin allergic skin test can be used as a screening or complementary test in unvaccinated animals, provided that a purified lipolysaccharide (LPS)-free, standardized antigen preparation (consisting mainly of brucellae cytoplasmic proteins) is used.14

It is recommended by the World Organisation for Animal Health (OIE) that sheep and goats should be immunized with B. melitensis strain Rev.1 live vaccine against both B. melitensis and B. ovis infections, and that B. abortus strain 19 live vaccine and B. abortus strain RB51 live vaccine (produced from a laboratory-derived rough mutant of smooth B. abortus strain 2308) should be used in cattle. No vaccine is recommended by the OIE for use in pigs. Vaccines for the prevention of brucellosis have to fulfil minimal requirements and their production should be based on a seed-lot system: seed cultures to be used for vaccines should originate from reference centres and they must conform to minimum standards for viability, smoothness, residual infectivity and immunogenicity.14

The World Health Organization (WHO) laboratory biosafety manual classifies Brucella in Risk group III. Brucellosis in humans is an acute influenza-like clinical disease characterized by a febrile illness — undulant fever — that may progress to a chronic form with serious complications in which the musculo-skeletal, cardiovascular and central nervous systems are affected. Infection is acquired mainly via the oral, respiratory or conjunctival routes. Ingestion of infected dairy products is the main public health risk but veterinarians, farmers, and abattoir and laboratory workers are also at risk of becoming infected by handling infected animals, carcasses and aborted material.8

Brucellosis is one of the most easily acquired laboratory infections and strict safety precautions should always be observed when handling cultures and infected or potentially infected samples such as aborted material. The examination of infected material should be carried out in class III safety cabinets by trained staff. Protective clothing should be worn and decontamination of laboratory equipment and sterilization of cultures and hazardous material should be implemented. Specific safety recommendations have been made and must be followed when dealing with Brucella infected material. The handling of serum samples represents a minimal hazard if good laboratory practices are implemented.5, 8


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  7. FOSTER, J. T., BECKSTROM-STERNBERG, S. M., PEARSON, T., BECKSTROM-STERNBERG, J. S., CHAIN, P. S., ROBERTO, F. F., HNATH, J., BRETTIN, T. & KEIM, P., 2009. Whole-genome-based phylogeny and divergence of the genus Brucella. Journal of Bacteriology, 191, 2864-2870.
  8. GODFROID, J., GARIN-BASTUJI, B., SAEGERMAN, C. & BLASCO, J. M., 2013. Brucellosis in terrestrial wildlife. Revue Scientifique et Technique (Office International des Epizooties), 32, 27-42.
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  10. LÓPEZ-GOÑI, I., GARCÍA-YOLDI, D., MARÍN, C. M., DE MIGUEL, M. J., MUÑOZ, P. M., BLASCO, J. M., JACQUES, I., GRAYON, M., CLOECKAERT, A., FERREIRA, A. C., CARDOSO, R., CORRÊA DE SÁ, M. I., WALRAVENS, K., ALBERT, D. & GARIN-BASTUJI, B., 2008. Evaluation of a multiplex PCR assay (Bruce-ladder) for molecular typing of all Brucella species, including the vaccine strains. Journal of Clinical Microbiology, 46, 3484-3487
  11. LÓPEZ–GOÑI, I., GARCÍA–YOLDI, D., MARÍN, C. M., DE MIGUEL, M. J., BARQUERO-CALVO, E., GUZMÁN-VERRI, C., ALBERT, D. & GARIN-BASTUJI, B., 2011. New Bruce-ladder multiplex PCR assay for the biovar typing of Brucella suis and the discrimination of Brucella suis and Brucella canis. Veterinary Microbiology, 154, 152-155.
  12. PROBERT, W. S., SCHRADER, K. N., KHUONG, N. Y., BYSTROM, S. L. & GRAVES, M. H., 2004. Real-time multiplex PCR assay for detection of Brucella spp., B. abortus, and B. melitensis. Journal of Clinical Microbiology, 42, 1290-1293.
  13. VERGNAUD, G., HAUCK, Y., CHRISTIANY, D., DAOUD, B., POURCEL, C., JACQUES, I., CLOECKAERT, A. & ZYGMUNT, M. S., 2018. Genotypic Expansion Within the Population Structure of Classical Brucella Species Revealed by MLVA16 Typing of 1404 Brucella Isolates From Different Animal and Geographic Origins, 1974-2006. Frontiers in Microbiology, 9, 1545.
  14. WHATMORE, A. M. & GOPAUL, K. K., 2011. Recent advances in molecular approaches to Brucella diagnostics and epidemiology. In: Brucella: Molecular Microbiology and Genomics, LÓPEZ-GOÑI, I. & O’CALLAGHAN, D., (eds.). Caister Academic Press, Norfolk, UK, 57-88.
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