Dr.Kedar Karki 1 1. 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). 2. The importance of this contagious disease is the economic impact on livestock industry. 3. Causes sever hazard to human health, through either direct contact with infected animals or the consumption of contaminated 2 milk and dairy products. 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). 3 Table (1) show Brucella species : NO Species Biov ar Natura l host Other animal species affected Human disease 1. Brucella abortus 1-9 Cattle Wild animals, water buffalo, camels Less sever 2. Brucella melitensis Brucella ovis 1-3 Wild Sheep ruminant and cattle, Goat camels Sheep None (Ram) Various wild Sever 3. 1 None Sever4 ( except Morphology and staining 1. The bacteria are strictly parasitic and prefer the intracellular habit. 2. The species of the genus Brucella are small non motile, non spore forming, Gram negative rods and they do not produce true capsules. 3. They are somewhat resistant to decolonization by weak acids and thus stain red by the modified Ziehl 5 Neelsen method. Gram stain of Brucella (Gram – ve ) Coccobacilli 6 Antigenic Structure 7 The designation of the antigens in cultures composed of smooth and rough colonies are shown in the table 2. NO. 1. 2. Species Brucella abortus Brucella melitensis Type of colony Type of surface antigen Smooth Smooth Smooth A a A m M m ------- 3. Brucella suis Rough --- --R • 4. Production of monospecific antisera to A and M antigen The Brucella ovis can Brucella identificationRough of the Brucella species. 5. be used in thecanis --- --R • 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. 8 Cultural characteristics 1.Culture media There are two major types of media for cultivation of Brucella. A. Basal Medium • Direct isolation and culture performed on solid media. of Brucella are usually • 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, Serumdextrose 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 Farrell’s medium, which is prepared by 9 the addition of six antibiotics : • Polymyxin B sulphate,Bacitracin, Cycloheximide, Nalidixic 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, 10 glistening, translucent, and a pale • Brucella colonies on blood agar 11 Brucella colonies on blood agar 12 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, 13 inhalation of aerosols containing the bacteria and One of the most important route of animal to human transmission of Brucella 14 • 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 15 brucella melitensis. Brucellosis: Edema and swelling of scrotum 16 2.The economic losses of brucellosis 1. Losses due to abortion in the affected animal population. 2. Diminished milk production, mastitis and contamination of milk. 3. Cull and condemnation of infected animals due to breeding failure. 4. Human brucellosis causing reduced work capacity of the affected people. 5. Government costs on research and eradication schemes. 6. Losses of financial investments. 17 Collection of the samples A. For serological examinations: 1. Serum samples are collected for serological diagnosis. 2. Collect 5-10 ml of blood in plain tubes (with out EDTA). 3. Avoid shaking of the tubes (which contain blood) at transporting to prevent distraction of the RBCs and hemolysis. 4. Try to separate the serum from clotted blood as possible as you can. 5. The tubes are placed vertically at room temperature for 1 hour then refrigerated at (4 8 °C) for 1- 2 hour. Don’t refrigerate the whole blood immediately after collection. 6. Don’t freeze the whole blood. 7. After the serum was separated distribute it equally 18 in to two plastic tubes for serum and keep it in a B. For bacterial isolation 1. 2. 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. 3. In aborted and stillborn lambs the preferred culture sites are abomasal content and lung. Samples for culture should be 19 transported to the laboratory on ice as Classes of Immunoglobulin There are five major classes of antibodies which have diverse functions. 1. 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 20 protect the fetus and neonate. 3. 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 the surface of immunoglobulin found on many B lymphocytes;21 Types of antibody response A. 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 the22 memory B cells. Primary antibody response Secondary antibody response IgG Antibody Titer IgM 7 14 21 28 35 42 Days Diagram show the primary and secondary antibody response and the types of immunoglobulin 23 The most common serological test 1. Rose Bengal Test (RBT). 2. Complement Fixation Test (CFT). 3. Enzyme Linked Immuno Sorbent Assay (ELISA). 4. Milk Ring Test (MRT). 5. Standard Agglutination Test (SAT). 24 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 – – • Shake before use. • Do not freeze. • Store at 4 °C in a dark place. 25 Important notes: Rose Bengal Antigen. Positive and Negative controls. C. Equipments 1. Precision pipettes calibrated to 30 μl. 2. Enamel plate or disposable agglutination cards. 3. Disposable stirring sticks. 26 D. Procedure 1. Allow reagents and serum samples to reach room temperature (18-25 °C). 2. Gently shake the reagents. 3. Check the reagent against positive and negative controls (as follows). 4. Place a drop (30 μl) of undiluted serum onto a circle of the slide. 5. Add a drop of the reagent (Rose Bengal Brucella antigen) next to the drop of the serum. 6. Mix both drops by the disposable stirring stick, spreading them over the full surface of the circle. 7. Rotate the plate (card) manually or with mechanical rotator at (80- 100) R.P.M 27 E. Reading 1. The reading must be carried out exactly 4 minutes from the beginning of shacking. 2. Beyond this time nonspecific reaction may occurs (False Positive). 3. No agglutination = (- Ve) Negative result. 4. Any visible agglutination (even slight) = (+Ve) Positive results (Presence of specific antibodies). 28 Positive and negative results for RBT (Rose Bengal test) Positive Negative 29 F. Important Notes 1. The test is very sensitive especially in vaccinated animals. 2. Positive samples should be retested by confirmatory test such as CFT or ELISA. 3. False Negative reaction may occur and can be detected by retesting animals at intervals over a period of 30 • 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. Complement Fixation Test (CFT) • 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 31 of A. Principle 1. In the complement fixation reactin an antigen-antibody reaction is demonstrated by binding of complement. 2. The reaction can be visualized with the aid of an indicator – reactin (hemolytic system). 3. The hemolytic system consist of sheep erythrocytes and antiserum to sheep erythrocytes (amboceptor). 4. If no antigen-antibody reaction develops with the consumption 32 of complement, the erythrocyte will be lysis 33 B. Reagents Standardized 1. antigen 2. Complement Hemolytic system 3. (Sensitized RBC) Brucella abortus strain 99 or Brucella abortus strain 1119-3 Guinea pig complement 3% suspension of sheep red blood cells (SRBC) sensitized with rabbit anti-Sheep RBC (amboceptor). 0.1% NaCl + (0.1% Complement MgCl2+CaCl2) 4. fixation buffer C. Results pH =7.3 The test is read by the eye : Sedimentation of the sensitized RBC = Positive result. Complete lysis of the sensitized RBC = Negative result. 34 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. Milk Ring Test (MRT) 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 Brucella35 abortus S 99 inactivated by heat and C. Procedure 1. Keep the antigen for 1 hour at room temperature, and shake it gently before the beginning of the test. 2. Carefully mix the tested milk, and put 1 ml test tube. in a 3. Add 50 µl of the antigen and mix carefully. 4. 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 1. Ring of cream equal or more colored than the underlying milk = Positive result. 2. Ring of cream less colored than the 36 • 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 37 Types of Brucella vaccines and vaccination • • 1. Brucella vaccines A. Brucella melitensis (Rev-1 Vaccine): 1. Freeze-dried of live B. melitensis Rev-1 strain. 2. For sheep and goats. 3. At 3-6 months as a single S/C dose. 4. Conjunctival route (reduced dose) 5. Disadvantages: – – – Persistent serological response. Abortion if given to pregnant animals. Human risk. 38 B. Brucella abortus (S19): 1. Freeze-dried of live B. abortus strain 19. 2. For cattle. 3. At 3-6 months as a single S/C dose. 4. Conjunctival route (reduced dose) 5. Disadvantages: – Persistent serological response. – Abortion if given to pregnant animals. – Human risk. C. Brucella abortus (RB51): 1. Freeze-dried of live B. abortus strain RB51. 2. Lesser abortafecient effect than S19. 3. At 4-12 months as a single S/C dose. 39 2. Specifications of the Ideal Brucella Vaccine 1. Effective (solid and durable immunity) but without inducing a long lasting vaccine infection. 2. Not interfering with diagnostic tests (Compatible with eradication plans). 3. With no limitation of its use, e.g. in pregnant animals. 4. Safe for man when performing the vaccination. 5. Not expensive and readily available40 world wide. 3. Brucella vaccine quality control testing 1.Identity test for the bacterial and colony morphology. 2.Absence of contaminating organisms. 3.Number of viable organisms. 4.Dissociation test. 5.Stability test. 6.Virulence in guinea pigs and/ or mice. 7.Safety. 41 Diseases to be considered in the differential diagnosis 1. 2. 3. 4. 5. 6. Vibriosis. Salmonellosis. Toxoplasmosis. Leptospirosis. Enzootic abortion of ewes. Rift Valley fever. Laboratory identifications of the causative organisms a requirement for the diagnosis. 42 43