Laboratory diagnosis and control

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Laboratory diagnosis and control of brucellosis
Dr. Ihsan M. Ahmad
Central Veterinary Diagnostic Laboratory
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Introduction

• Brucellosis is considered as the most wide spread
  zoonosis in the world and it is considered as
  True zoonosis ( That mean it is Basically
  transmitted from animal to human).
• The importance of this contagious disease is the
  economic impact on livestock industry.
• Causes sever hazard to human health, through
  either direct contact with infected animals or
  the consumption of contaminated milk and dairy
  products.

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Causative bacteria of the disease

• Brucellosis is named after Sir David Bruce, who
  is in 1886
• isolated the causative agent from a soldier in
  Malta.
• Brucella species are recognized based on
  the natural
• animal host to the following species as shown
  in table ( 1).

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Table (1) show Brucella species
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Morphology and staining

• The bacteria are strictly parasitic and prefer
  the intracellular habit.
• The species of the genus Brucella are small non
  motile, non spore forming, Gram negative rods and
  they do not produce true capsules.
• They are somewhat resistant to decolonization by
  weak acids and thus stain red by the modified
  Ziehl - Neelsen method.

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Gram stain of Brucella (Gram ve ) Coccobacilli
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Antigenic Structure
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The designation of the antigens in cultures
composed of smooth and rough colonies are shown
in the table 2.

• The Production of monospecific antisera to A and
  M antigen can be used in the identification of
  the Brucella species.
• Br.canis and Br.ovis grow as rough colonies that
  do not possess either of the surface antigens A
  and M, but instead of that they have R antigen.

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Cultural characteristics

• Culture media
• There are two major types of media for
  cultivation of Brucella.
• A. Basal Medium
• Direct isolation and culture of Brucella are
  usually performed on solid media.
• This enables the developing colonies to be
  isolated and limit the development of
  contaminants.
• There is many Kinds of commercial media ,e.g.
  Brucella medium base, Trypticase soy agar,
  Columbia agar, Serum- dextrose agar or
  Glycerol-dextrose agar.
• The addition of 2-5 Bovine or Equine serum is
  necessary for the growth of strains such as B.
  abortus biovar 2.
• B. Selective Media
• Appropriate antibiotics are added in order to
  suppress the growth of organisms other than
  Brucella. The most widely used medium is
  Farrells medium, which is prepared by the
  addition of six antibiotics
• Polymyxin B sulphate,Bacitracin, Cycloheximide,
  Nalidixic acid, Nystatin, Vancomycin

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• 2. Colony Morphology
• Brucella colonies are visible after 3-5 days
  incubation period at 37 ºC on suitable solid
  media, and they are aerobic or microaerophilic.
• Cultures should not be discarded as negative
  until 8-10 days have elapsed.
• Brucella colonies are 1-2 mm in diameter, round,
  entire, smooth, glistening, translucent, and a
  pale honey color when plates are viewed in the
  daylight through a transparent medium.

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Brucella colonies on blood agar
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Brucella colonies on blood agar
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Epidemiology of the disease
1.Transmission of the disease

• Animal to animal Transmission
• The oral route, Contamination of the
  udder during milking and contact with aborted
  fetuses and infected newborn lambs are considered
  to be common methods of spread, also the venereal
  transmission of the disease is occur due to
  infected male or contaminated semen.
• Animal to human Transmission
• Infected tissues, and contaminated
  materials must be handled under (biosafty 3)
  conditions. Transmission could be either by
  contaminated food, invasion by intact skin,
  inhalation of aerosols containing the bacteria
  and aerosol contamination of the conjunctiva.

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One of the most important route of animal to
human transmission of Brucella
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• Brucella melitensis ( biovar 1, 2 or 3) is the
  main causative agent of caprine and ovine
  brucellosis. Sporadic cases caused by
  B.abortus have been observed. The infection is
  widespread world-wide.
• Brucella abortus is usually causes bovine
  brucellosis, less frequently by brucella
  melitensis.

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Brucellosis Edema and swelling of scrotum
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2.The economic losses of brucellosis

• Losses due to abortion in the affected animal
  population.
• Diminished milk production, mastitis and
  contamination of milk.
• Cull and condemnation of infected animals due to
  breeding failure.
• Human brucellosis causing reduced work capacity
  of the affected people.
• Government costs on research and eradication
  schemes.
• Losses of financial investments.

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Collection of the samples

• For serological examinations
• Serum samples are collected for serological
  diagnosis.
• Collect 5-10 ml of blood in plain tubes (with out
  EDTA).
• Avoid shaking of the tubes (which contain blood)
  at transporting to prevent distraction of the
  RBCs and hemolysis.
• Try to separate the serum from clotted blood as
  possible as you can.
• The tubes are placed vertically at room
  temperature for 1 hour then refrigerated at
  (4 - 8 C) for 1- 2 hour. Dont refrigerate the
  whole blood immediately after collection.
• Dont freeze the whole blood.
• After the serum was separated distribute it
  equally in to two plastic tubes for serum and
  keep it in a refrigerator for short periods or in
  freeze for longer periods.
• Avoid repeated freezing thawing as this may
  affect the protein structure of the serum.

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• The most valuable samples from live animals are
  semen, vaginal swabs, and milk.
• After necropsy, the preferred organs are
  epididymas, seminal vesicles inguinal lymph
  nodes in rams, and the uterus, iliac and supra
  mammary lymph nodes in ewes.
• In aborted and stillborn lambs the preferred
  culture sites are abomasal content and lung.
• Samples for culture should be
  transported to the laboratory on ice as soon as
  possible after collection.

B. For bacterial isolation
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Classes of Immunoglobulin

• There are five major classes of antibodies
  which have diverse functions.
• IgM (Immunoglobulin M)
• The class of serum antibody first produced
  during an infection. It is a large pentameric
  molecule that is active in agglutination
  pathogens and activating complement.
• 2. IgG (Immunoglobulin G)
• The predominant immunoglobulin class in
  serum. Has function such as neutralizing toxins,
  opsonizing bacteria, activating complement and
  crossing the placenta to protect the fetus and
  neonate.

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• IgA (Immunoglobulin A)
• The class of immunoglobulin that is
  present in dimeric form in many body secretions
  (e.g., saliva, tears, bronchial and intestinal
  secretions) and protects body surfaces.
• 4. IgE (Immunoglobulin E)
• The class of immunoglobulin that binds
  to mast cell and basophils, and is responsible
  for type I or anaphylactic hypersensitivity. It
  is also involved in resistance to helminth
  parasites.
• 5. IgD (Immunoglobulin D)
• The class of immunoglobulin found on
  the surface of many B lymphocytes thought to
  serve as antigen receptor in the stimulation of
  antibodies synthesis.

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Types of antibody response

• Primary antibody response.
• It is stimulated during immunization
  procedures (and also in naturally acquired
  immunity) the type of immunoglobulin is (IgM) and
  it is characterized by low antibodies titer.
• B. Secondary antibody response.
• It is characterized by producing the
  (IgG) type of immunoglobulin with high level and
  affinity for the antigen. It depends on the
  formation of the memory B cells.

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Diagram show the primary and secondary antibody
response and the types of immunoglobulin
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The most common serological test

• Rose Bengal Test (RBT).
• Complement Fixation Test (CFT).
• Enzyme Linked Immuno Sorbent Assay (ELISA).
• Milk Ring Test (MRT).
• Standard Agglutination Test (SAT).

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Rose Bengal Test (RBT)

• A. Principle
• The buffered acid antigen stained with
  Rose Bengal is used for the early detection of
  Brucella specific agglutinins.
• B. Reagents
• Rose Bengal Antigen.
• Positive and Negative controls.
• Important notes
• Shake before use.
• Do not freeze.
• Store at 4 C in a dark place.

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• C. Equipments
• Precision pipettes calibrated to 30 µl.
• Enamel plate or disposable agglutination cards.
• Disposable stirring sticks.

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• D. Procedure
• Allow reagents and serum samples to reach room
  temperature (18-25 C).
• Gently shake the reagents.
• Check the reagent against positive and negative
  controls (as follows).
• Place a drop (30 µl) of undiluted serum onto a
  circle of the slide.
• Add a drop of the reagent (Rose Bengal Brucella
  antigen) next to the drop of the serum.
• Mix both drops by the disposable stirring stick,
  spreading them over the full surface of the
  circle.
• Rotate the plate (card) manually or with
  mechanical rotator at (80- 100) R.P.M (Round per
  minute) for 4 minutes.

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• E. Reading
• The reading must be carried out exactly 4 minutes
  from the beginning of shacking.
• Beyond this time nonspecific reaction may occurs
  (False Positive).
• No agglutination (- Ve) Negative result.
• Any visible agglutination (even slight) (Ve)
  Positive results (Presence of specific
  antibodies).

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Positive and negative results for RBT (Rose
Bengal test)
Positive
Negative
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• Important Notes
• The test is very sensitive especially in
  vaccinated animals.
• Positive samples should be retested by
  confirmatory test such as CFT or ELISA.
• False Negative reaction may occur and can be
  detected by retesting animals at intervals over a
  period of at least 3 months.

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Complement Fixation Test (CFT)

• The complement fixation test (CFT) is widely used
  for the diagnosis of brucellosis in cattle, sheep
  and goat.
• This test is relatively insensitive to antibody
  produced in response to vaccination with the
  living attenuated vaccine (for Br. abortus, Br.
  melitensis).
• whilst being highly sensitive and specific in
  animals naturally infected with brucellosis.
• The test is some what complex and in mass testing
  a screen test such as Rose Bengal test, is often
  used to reduce the number of samples that need to
  be tested by (CFT).

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• Principle
• In the complement fixation reactin an
  antigen-antibody reaction is demonstrated by
  binding of complement.
• The reaction can be visualized with the aid of an
  indicator reactin (hemolytic system).
• The hemolytic system consist of sheep
  erythrocytes and antiserum to sheep erythrocytes
  (amboceptor).
• If no antigen-antibody reaction develops with
  the consumption of complement, the erythrocyte
  will be lysis completely and the hemoglobin will
  be released.

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Complement fixation test
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• B. Reagents

C. Results The test is read by the eye
Sedimentation of the sensitized RBC Positive
result. Complete lysis of the sensitized RBC
Negative result.
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Milk Ring Test (MRT)

• The Ring test method is used for the detection
  of brucella antibody in milk samples. This
  technique is very easy to perform it especially
  in dairy herds.
• A. Principle
• It is uses the principle of agglutination
  between antibodies contained in milk and dyed
  colored bacterial antigen of brucella to form
  antigen-antibody complexes that are progressively
  carried by the fat towards the surface of the
  milk and formed a blue violet ring
• B. Reagents
• Inactivated bacterial culture of Brucella abortus
  S 99 inactivated by heat and phenol and stained
  with hematoxyline.
• Positive control for milk ring test.

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• C. Procedure
• Keep the antigen for 1 hour at room temperature,
  and shake it gently before the beginning of the
  test.
• Carefully mix the tested milk, and put 1 ml in a
  test tube.
• Add 50 µl of the antigen and mix carefully.
• Place in incubator for 1 hour at 37 OC, after
  that for (18-20 hours) at 4 OC, and then read the
  result.
• D. Results
• Ring of cream equal or more colored than the
  underlying milk
• Positive result.
• Ring of cream less colored than the underlying
  milk
• Negative result.

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Types of Brucella vaccines and vaccination

• Vaccination increases animal resistance to
  systemic infection and in infected animals
  decreases the probability of placental infection,
  abortion and massive shedding of infections
  organisms.
• Live vaccines induce a long lasting immunity and,
  are normally administered to the young animals.
  Adults should also be vaccinated with reduced
  doses or by the conjunctival route in order to
  restrict the serological response.
• The following are the recommended vaccination
  procedures of the 3 live vaccines for B.
  melitensis and B. abortus.

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• 1. Brucella vaccines
• A. Brucella melitensis (Rev-1 Vaccine)
• Freeze-dried of live B. melitensis Rev-1 strain.
• For sheep and goats.
• At 3-6 months as a single S/C dose.
• Conjunctival route (reduced dose)
• 5. Disadvantages
• Persistent serological response.
• Abortion if given to pregnant animals.
• Human risk.

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• B. Brucella abortus (S19)
• Freeze-dried of live B. abortus strain 19.
• For cattle.
• At 3-6 months as a single S/C dose.
• Conjunctival route (reduced dose)
• Disadvantages
• Persistent serological response.
• Abortion if given to pregnant animals.
• Human risk.
• C. Brucella abortus (RB51)
• Freeze-dried of live B. abortus strain RB51.
• Lesser abortafecient effect than S19.
• At 4-12 months as a single S/C dose.
• Does not produce persistent Ab titers.

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• 2. Specifications of the Ideal Brucella Vaccine
• Effective (solid and durable immunity) but
  without inducing a long lasting vaccine
  infection.
• Not interfering with diagnostic tests (Compatible
  with eradication plans).
• With no limitation of its use, e.g. in pregnant
  animals.
• Safe for man when performing the vaccination.
• Not expensive and readily available world wide.

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• 3. Brucella vaccine quality control testing
• Identity test for the bacterial and colony
  morphology.
• Absence of contaminating organisms.
• Number of viable organisms.
• Dissociation test.
• Stability test.
• Virulence in guinea pigs and/ or mice.
• Safety.

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Diseases to be considered in the differential
diagnosis

• Vibriosis.
• Salmonellosis.
• Toxoplasmosis.
• Leptospirosis.
• Enzootic abortion of ewes.
• Rift Valley fever.
• Laboratory identifications of the
  causative organisms a requirement for the
  diagnosis.

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Thank you