Brucella spp.: Interest in human and animal pathology and food contamination - Culture; Antibodies; Molecular diagnosis (PCR).

Brucella spp.: Interest in human and animal pathology and food contamination – Culture; Antibodies; Molecular diagnosis (PCR)

 

Information 11-02-2018.

 

Brucella is a genus of gram-negative bacteria composed of small, immobile cocobacilli, lacking capsules and flagella, of the Brucellaceae family, with a wide global distribution. This microorganism infects mainly cattle, although it can infect other mammalian animals and people. Brucella infection causes a zoonosis called brucellosis, also known as Malt fever, Mediterranean fever, Rock of Gibraltar fever or gastric fever, among others. Brucellosis is considered to be one of the most important zoonoses in the world, with 500,000 reported human cases each year has become a serious economic and health problem in certain countries, although it is currently controlled in most developed countries. In addition, due to its low infectious dose and its ease of transmission as an aerosol, it is listed as a Category B bioterrorism agent in the USA.

The genus Brucella, established in 1920 by Meyer and Shaw, is currently composed of twelve species: B. melitensis, B. abortus, B. canis, B. suis, B. ovis, B. neotomae, B. pinnipedialis, B. ceti, B. microti, B. inopiata, B. papionis and B. vulpis. These species are differentiated by their tropism by the host, their pathogenicity, and their phenotypic traits. Brucella members infect a wide variety of mammals, however, each species has its preferred host. The species that can infect humans and their most frequent hosts are: B. melitensis (goat and sheep), B. abortus (cattle), B. suis (swine), B. ovis (sheep and hares), B canis (canines), and B. neotomae (desert rats).

The microorganism is transmitted by direct contact with an infected animal or its fluids (blood, urine, feces, vaginal fluids, semen, aborted fetuses, and placenta) through ingestion, skin or mucous wounds, and by inhalation of aerosol sprays. Among animals, brucellosis is transmitted mainly when a sick animal give birth. In the birth liquid of this animal there is a large quantity of bacteria, which can survive several months in the external environment, especially in cold and humid conditions, and they are still infectious for other animals, which will be infected when ingested. Veterinarians, farmers and slaughterhouse employees are exposed to infection when they handle infected animals and carcasses, their fluids, or fetuses or placentas after an abortion. People can also become infected indirectly, through the ingestion of contaminated food, such as unpasteurized milk and cheese, and undercooked meat products from infected animals. Also, it can be transmitted from contaminated drinking water, and fresh fruits and vegetables irrigated with contaminated water. Brucella survives in food at refrigeration and freezing temperatures, being able to survive years in frozen meat products. On the other hand, Brucella does not survive thermal treatments above 60ºC, so in all food subjected to these temperatures there will be no viable bacteria. Although transmission from person to person is rare, some cases have been detected in certain circumstances, such as congenital infections, through breastfeeding, bone marrow transplant, sexual contact, or blood transfusion. Finally, brucellosis is one of the most easily transmitted infections in the laboratory, when handling cultures or samples with a large number of bacteria.

Members of the genus Brucella are facultative intracellular pathogens. When it enters the organism, the microorganism is phagocytosed by polymorphonuclear neutrophils and monocytes, surviving intracellularly and evading cellular and humoral defense mechanisms. Within phagocytes, Brucella multiplies and propagates through the blood colonizing tissues rich in reticuloendothelial system such as liver, spleen, bone marrow and genito-urinary tissue, for an indefinite period of time. Brucella is also capable of inducing its own internalization in cells that are not active phagocytic such as fibroblasts and epithelial cells. Sexually mature or pregnant mammals are more susceptible to disease because Brucella has an affinity for the tissues of the reproductive organs. In pregnant animals, the final location of Brucella is the placenta.

The clinical picture and the evolution of the infection vary depending on the animal species affected. In man it has a great tendency to chronicity and is characterized by fever and localization of bacteria in different tissues. If it is not treated in time with the appropriate antibiotics, it can generate a permanent disability. Brucella spreads in phagocytes to different organs and produces when it destroys its transporter cells, the characteristic bacteremias that define the clinical picture. The signs and symptoms of brucellosis manifest between 2 and 4 weeks after infection and may include acute cyclic fever, headache, arthralgia, night sweats, diarrhea, fatigue and anorexia. This disease can produce complications such as arthritis, spondylitis, neurobrucellosis, osteomyelitis, meningitis, encephalitis, peripheral neuropathy, radiculopathy, hepatic and intracerebral abscess formation, hepatic dysfunction and endocarditis, which can be life-threatening. In addition, hematologic signs include anemia, leukopenia, and thrombocytopenia. Congenitally infected children can have low birth weight, developmental delay, jaundice, hepatomegaly, splenomegaly, respiratory distress, fever, and vomiting.

In animals, it is usually a mild illness. The affinity of Brucella for the reproductive organs, makes pathogenic Brucella species can cause abortion in females by colonization of the placental trophoblast, as well as testicular inflammation and sterility in males. In addition, Brucella can permanently infect the retromamary lymph nodes in cows, generating the release of the microorganism in the milk. Other symptoms in both sexes can include fever, arthritis, weakness and lethargy.

Recommended tests for diagnosis:

The isolation of Brucella spp. it constitutes the definitive diagnostic method. It is usually obtained by blood culture or bone marrow culture and, more rarely, by cerebrospinal fluid culture, joint fluid, purulent exudate, tissue, etc. However, the culture of the microorganism is a tedious, slow process, since it requires a minimum incubation time of 15 days, and difficult, due to the intracellular nature of the bacteria. In addition, handling samples with viable bacteria poses a risk to laboratory personnel. Secondly, the serological tests are those used in the surveillance and control programs of brucellosis. There are several tests available, the agglutination and enzyme immunoassay (ELISA) methods are the preferred ones. Serological tests have the disadvantages that they are not always sensitive or specific due to cross-reactivity with other bacterial antigens. More recently, several molecular methods (PCR) have been developed, especially useful in cases of bacteremia. In addition, they have a high sensitivity and allow the detection of Brucella spp. in body fluids and food, even with a low number of non-viable bacteria. In addition, they allow molecular typing of Brucella without having to handle living organisms. However, since PCR can detect DNA from live, damaged or dead bacteria, in the case of food it is difficult to conclude the zoonotic risk in case of positive result.

Tests carried out in IVAMI:

  • Isolation in culture of Brucella spp. in clinical samples and foods.
  • Molecular diagnosis (PCR) to detect Brucella DNA.
  • Detection of total antibodies, IgM, and IgG against Brucella spp. by agglutination or ELISA.
  • Molecular identification of Brucella spp. at the species level (sequencing).

Recommended sample:

  • Total blood extracted with EDTA for culture (5 mL), or molecular diagnosis (PCR) (2 mL).
  • Other types of animal or human samples for culture or molecular diagnosis: CSF, serum or plasma, exudates, bone marrow, tissues or suspicious fluids (similar volume).
  • Animal milk (25 mL), and cheeses (25 g).
  • Serum separated from blood for detection of antibodies (2 mL).

Conservation and shipment of the sample:

  • Refrigerated (preferred) for less than 2 days.
  • Frozen: more than 2 days.

Delivery of results:

  • Isolation in culture: 15 days.
  • Molecular detection (PCR): 24 to 48 hours.
  • Molecular identification at species level: 48 to 72 hours
  • Detection of antibodies: 48 hours.

Cost of the tests:

  • Molecular identification at the species level (sequencing): Consult ivami@ivami.com.