Brucella

HISTORY:

It’s D. Bruce, a British physician, who isolated in Malta in 1887 the bacterium responsible for the “Mediterranean fever” also called Malta fever, undulant fever or undulant fever. The role of the goat in its transmission was quickly established.

I – GENERAL CHARACTERISTICS:

Bacteria of the genus Brucella are small coccobacilli (0.5 to 1.5 | along Jm) Gram-negative, immobile, do not form spores, poorly growing on ordinary media. Strictly aerobic, growth is often enhanced by CO2. Facultative bacteria intracellular multiplication, they can infect animals and humans causing a disease, brucellosis, acute first, then chronic.

II – CLASSIFICATION:

The genus Brucella contains six species.

– Three main species can infect humans.

They are:

– B. melitensis: found in goats and sheep;

– B. abortus: cattle abortion agent;

– B. am: found in pigs and the hare.

– Three other species, much more rare, are: B. ovis, B. canis and B. neotomae.

III – HABITAT AND EPIDEMIOLOGY:

Brucella are responsible for anthropozoonosis affecting rural and spread around the world. Brucellosis is especially common in the Mediterranean basin.

A – The reservoir of germs:

It consists of farm animals: goats and sheep typically for B. melitensis, cattle for B. B. abortus and pigs for am. In fact, it is only because the preferred host species specificity is not strict.

Animal disease is often unapparent. In pregnant female, it is manifested by abortions. The presence of erythritol in fetal and placental tissues of animals stimulates the multiplication of Brucella and explains what viscerotropism.Vaginal secretions of sick animals spread the bacteria in their environment (litter, manure). The achievement of the mammary gland results in excretion of Brucella in milk.

Better control of animal brucellosis causes a decrease in the number of human cases.

B – Human contamination:

Brucella melitensis remains dominant in human brucellosis in France, but the proportion of B. abortus increased significantly.

1. It is direct in the majority of cases (70%):

Humans are contaminated through the skin. Brucella enter the body through a skin excoriation however small. They can even pass through healthy skin. Conjunctival contamination is possible.

Brucellosis is an occupational disease that affects mainly rural (veterinarians, shepherds, cattle dealers etc …). The frequency of laboratory personnel contamination is noteworthy. In the latter the aerosol contamination has been described. Two patients out of three men of working age.

2. Indirect contamination is rare digestive:

It is the origin of the disease among vacationers or urban. It is through ingestion of raw milk (goat or sheep) or cheese consumption craftsmanship.

There is no human transmission.

The number of cases reported annually has decreased since 1978. In 1989, 164 cases of brucellosis were reported to health authorities.

 

IV – PATHOPHYSIOLOGY:

Brucellosis is a lymphatic sepsis. From the door skin or digestive entrance, Brucella win by the first lymph node relays and multiply.

Then they swarm lymphatic or blood stream to colonize organs with significant reticuloendothelial frame (nodes, bone marrow, liver, spleen). Repeating bacterial discharges translates undulant fever.

Osteoarticular locations, glandular, hepato-splenic or neuromeningeal may arise and continue to evolve during the subacute phase.

Brucella intracellular persist for years in the body, which causes an immune reaction of delayed-type hypersensitivity.

V – PATHOGENICITY HUMANS:

After incubation for 2 to 4 weeks, septicemic acute brucellosis is characterized by Algonkian-sudoro undulant fever. Well supported, the fever may go unnoticed.

Forms focused (osteoarticular, neuro-meningeal etc …) can appear at the waning of septicemic forms or be immediately isolated.

Minor unapparent, characterized by the absence of location, resulting in disabling chronic brucellosis or “patraquerie” are common.

In rural areas, should be considered as a possible brucellosis any unexplained febrile syndrome.

VI – ISOLATION AND IDENTIFICATION OF BRUCELLA:

It is essential that the precise clinical laboratory that requires Brucella for the appropriate culture conditions are in onp had n v e r p vr.

A – Blood culture:

During brucellosis is:

consistently positive during the acute phase frequently positive during the subacute phase, exceptionally positive during the chronic phase.

It is recommended to sow the blood in vials Castaneda biphasic bottle (containing agar and broth) with an atmosphere of 10% Cu 2.

The bottles are conventionally stored 6 weeks at 37 ° C for Brucella colonies are sometimes very slow to develop in primary culture. However, the bacteria growth occurs most commonly in some days. “

B – Other pathological Products:

Brucella can also be done from lymph nodes, bone marrow, joint aspiration fluid, cerebrospinal fluid, pus diverse.

Liquids are seeded as the blood, Castaneda bottle.

The tissues were ground and plated on appropriate solid media (Brucella agar agar Albimi, tryptic soy agar) incubated at 37 ° C in an atmosphere of 10% CO2.

C – Characterization of the genus Brucella:

1. The isolation of slow growth is typical:

II always more than 48 hours and sometimes several weeks for

colonies from a pathological product. The colonies are small (0.5 mm

diameter), smooth, translucent, with smooth edges. They sometimes have a honey color

and consist of small coccobaciiïes Gram negative.

2. The following characters are positive s:

– Strict aerobic

– Catalase

– Oxidase

– Nitrate reductase

– Urease (immediate for B. suis, negative for B. ovis)

3. The other metabolic traits are negative:

D – Characterization of the species (see Table I)

– CO2 Requirement

In France, 96% of strains of B. abortus require an atmosphere of 10% CO 2 for growth. B. melitensis and B. am never demanding. This is a good guiding criterion.

– Production of H 2 S.

B. melitensis does not occur while the strains of B. abortus and B. am occur within 24 hours (method from paper to lead sub-acetate).

– Action bacteriostatic dyes.

Basic fuchsin and thionin at certain concentrations have a bacteriostatic action. Thionine inhibit B. abortus and fuchsin inhibit B. am.

The text above can differentiate the main species of Brucella. However there is atypical strains that do not fit this identification scheme. Using a specialized laboratory is required.

E – Determination of biotypes:

Made specialized laboratory, it uses three types of techniques

1. Agglutination:

by monospecific sera, anti-Abortus (A), anti-melitensis (M).

Surface antigens of Brucella:

– Bacteria phase S (smooth)

All of the following species have two surface antigens A and M are agglutinogens: B. melitensis, B. abortus, B. suisand B. . neotomae The amount of these antigens differs depending on the species: M is predominant in B.melitensis, A is predominant among three other species.

A comprehensive anti-brucella serum agglutinates 4 species. Saturation, it is possible to obtain monospecific anti-A or anti M.

– Phase Bacteria R (rough)

Specificities A and M are replaced by a common R antigen to all Brucella, including B. ovis and B. canis who have neither A nor Mr.

2. Phage:

phage Tbilisi (Tb) and Weybridge (We).

3. Study of metabolism oxydatifdes sugars:

It is through a manometric method.

It is recognized biotypes 3 for B. melitensis biotypes 9 for B. abortus biotypes and 4 for B. am.

VII – IMMUNOLOGICAL DIAGNOSIS:

Symptoms of brucellosis are uncharacteristic, so the diagnosis of the disease is often referred to as a stage where blood cultures are negative. In this situation, immunological methods are very important because they allow indirect diagnosis.

A – serological reactions:

They can give conflicting results because they do not always detect the same classes of antibodies. Also it may be desirable to simultaneously perform two or three of these reactions.

1. Sérodiagnostic Wright or slow agglutination reaction:

a / Principle:

This is the classic serological reactions bmcellose. It is to seek the agglutination of Brucella (B. abortus strain) in the presence of serum dilutions studied. This reaction shows the IgM and IgG. It is the most positive early in the disease (10 to 15 days after the start).

This is a good method for diagnosis of acute brucellosis, but negative quickly. It is sometimes negative in subacute brucellosis and almost always negative in chronic brucellosis.

b / Interpretation

Agglutination 1/80 is positive. But agglutination at a lower title should be considered suspect of brucellosis in early to decline and make practice reconsider one or two weeks later.

EXPRESSION OF WRIGHT SERODIAGNOSIS in International Units order to properly compare the results obtained in various regions, the need has emerged to prepare agglutinating brucella antigen identical and therefore have a standard agglutinating serum.

To express the Wright serology in International Units, there must be a standard 1 000 units, which is manipulated in parallel with the sera studied. We note, first, the title obtained with the standard and, secondly, with a serum, and we practice a rule of three:

Title = UI

1000 x inverse of the serum under study

Unlike the standard serum obtained from DTRE

Example:

Titles Achieved standard serum. . . . . . 1/1280

test serum 1/640 ……

Title in the IU studied serum:

1000 × 640/1280 = 500 units

Unitary notation, we consider that the serology is positive for S as 100 IU.

c / Error Causes:

– False Positives

False agglutination can be due to the antigenic relationship between Brucella and other bacteria, Yersinia enterocolitica serotype 09, Vibrio cholerae (vaccination), Francisella tularensis and rarely Escherichia coli 0157.

Among former mélitococciques, the agglutination rate can rise to a transient manner following an ordinary infection or heterologous vaccination.

Injection of melittin did not appear to modify the rate of Brucella agglutinins in the serum of a patient. However, it is prudent to take the sample for serology before the skin test.

– False negatives

Search blocking antibodies. Blocking or incomplete antibodies appear in the serum of some patients, especially chronic brucellosis phase. These are IgA or IgG blocking the antigenic sites on the surface of the bacteria used for the serodiagnosis of Wright, without causing agglutination. Wright’s HIV status appears to be negative in these patients.

A Wright serodiagnosis negative must search the blocking antibodies or by the indirect Coombs’ method or by a “blocking test”. This process consists in adding the Wright serology tubes remained negative, a drop of positive serum. If it does not produce agglutination is that it is inhibited by blocking antibodies attached to the Brucella.

2. Testing the buffered antigen (EA.T.) or reaction to the antigen or Rose Bengal Test Card.

It’s a quick slide agglutination reaction. It uses a buffered in acid suspension of Brucella inactivated and stained with rose bengal.

Not showing that IgG, this reaction is positive a little later than the Wright serology, but it is very sensitive and remains longer positive. Its good specificity and simplicity make it a very useful reaction for epidemiological investigations.

3. Complement fixation reaction:

Sensitive implementation, the complement fixation test highlights IgG. It is therefore also, later positive and remains longer positive.

For negative sera agglutination and having equal or higher rates than 1/10 in complement fixation, brucellosis seems to be criminalized. However, this reaction can be falsely positive in the same circumstances as the Wright serology.

4. Indirect immunofluorescence:

This reaction is positive a little later that the serum agglutination Wright. It is very useful in chronic brucellosis, because it still detects the presence of antibodies while other reactions became negative.

The relative complexity of handling make it preferable for epidemiological surveys the test, buffered antigen, much simpler.

5. Counter immunoelectrophoresis Counter immunoelectrophoresis:

Contrary to the above techniques using lipopolysaccharide antigen, this technique uses an extractable protein antigen Brucella which is very specific.

B – skin test:

The identification of Brucella allergy skin test may be the only biological sign of chronic brucellosis.

1. melittin Burnet:

Melittin of Burnet is a broth culture filtrate of Brucella melitensis. Intradermal injection (0.1 ml) is at the front of the forearm. To avoid the error cause due to hypersensitivity to protein broth that was used for the preparation of melittin is practiced in other forearm a broth control injection, which should remain negative.

The reading is made 24 to 48 hours after injection. A positive reaction is characterized by erythematous area and local edema can be assessed in touch. We must not allow for an early and transient response.

The appearance of an allergy to melittin is later than that of blood agglutinins. A positive intradermal melittin persists long after the cure of the disease, and often a lifetime. It does not necessarily mean active disease.

2. The phenol-soluble fraction:

This fraction is extracted from a strain of B. Delipidated abortus. It is used as antigen intradermally to detect the allergic state of a subject with respect to brucellosis. Used for the diagnosis of the disease, skin test is recommended for individuals exposed candidates for vaccination.

VIII – TREATMENT:

A – Forms and acute forms focused:

Cyclins are very active due to their good penetration into cells.

Resistant Brucella strains are rare.

Other assets are antibiotics streptomycin, rifampicin, chloramphenicol, and sulfonamides. The usual treatment, which lasts six weeks, combines doxycycline rifampicin. He proved more efficient than the old tetracycline and streptomycin.

B – Chronic forms:

The antigénothérapie is the only effective in these forms. Desensitization graded is made using:

– A suspension of B. melitensis killed by heating, injected subcutaneously;

– Either the phenol-insoluble fraction antigen injected intradermally.

IX – PREVENTION:

The incidence of human brucellosis is based on the importance of the disease in the herd and prophylaxis of animal disease. Preventive measures are:

screening of contaminated animals (serology and Control dairy products); slaughter of sick animals; vaccination of young females.

in humans, vaccination of exposed professional is advisable. Killed or live vaccines are now replaced by the use of better tolerated antigen fractions and causing good immunity. The antigenic extract, called PI fraction (phenol-insoluble), extracted from the wall B. melitensis and consists of glycoproteins, confers immunity 18 months.Vaccination should be carried out after the exclusion of brucella achieved by screening for allergic state vis-à-vis brucellosis subject.