This document discusses Brucellosis, a zoonotic disease. It was first isolated 120 years ago by Sir David Bruce and Bernhard Bang. Brucellosis is caused by bacteria of the genus Brucella, which includes B. melitensis, B. suis, B. abortus, and others. It is transmitted from animals to humans through consumption of infected animal products. While treatment involves prolonged antibiotic use, control relies on vaccination programs and testing/culling infected herds. The document outlines the history, transmission, diagnosis, and strategies to control Brucellosis in animals and humans.

1. Brucellosis
Dr. RAJAT VARSHNEY
P-1772

2.  1st
spp isolated & characterized almost 120 years ago & recently the
complete nucleotide sequences of the genomes of a number of well-
characterized Brucella strains have been determined
 Sir David Bruce (1855-1931)
British Army physician & microbiologist
Discovered Micrococcus melitensis
 Bernhard Bang (1848-1932)
Danish physician & veterinarian
Discovered Bacterium abortus could infect cattle, horses, sheep, & goats
undulant fever in humans
 The zoonotic nature of the brucellosis was demonstrated in 1905 by Zammit
isolating Brucella melitensis from goat’s milk in Malta (Godfroid et al.,
2005)
HISTORYHISTORY

3.  Brucellosis is an important re-emerging zoonosis with a worldwide
distribution, in India was recognised first in 1942.
 It is still an uncontrolled serious public health problem in many developing
countries including India
 Brucellosis in India is yet a very common but often neglected disease
 The genus Brucella consist of 8 species according to antigenic variation &
primary host: Brucella melitensis (sheep & goats), B. suis (hogs), B. abortus
(cattle), B. ovis (sheep), B. canis (dogs), B. neotomae (wood rats), & B.
cetacaea and B. pinnipedialis (marine mammals),
 Biovar- carbon dioxide, H2S, thionine and digitonin sensitivity
 Susceptibility- age, sex , breed, pregnancy status (adult one more susceptible
regardless to sex)
INTRODUCTION

4. The Many Names of BrucellosisThe Many Names of Brucellosis
Human Disease
Malta Fever
Undulant Fever
Mediterranean Fever
Rock Fever of Gibraltar
Gastric Fever
Animal Disease
Bang’s Disease
Enzootic Abortion
Epizootic Abortion
Slinking of Calves
Ram Epididymitis
Contagious Abortion

5. MORPHOLOGYMORPHOLOGY
GENOMEGENOME
They are Gram-negative, partially acid fast, facultative intracellular
coccobacilli or short rods- Proteobacteriacea family, aerobic, Capnophilic,
Catalase positive, Oxidase positive , Urease positive, Not grow on
MacConkey agar (Garrity2001)
The genome contains two circular chromosomes of 2.1 Mb and 1.5
Mb
Eating food & drink with the bacteria
 Injured skin coming into contact with the bacteria
TRANSMISSIONTRANSMISSION

6. Species biotypes Species affected
B. abortus 1, 3, 4, 6
1
Cattle
Sheep, goat
B.melitensis 1
1, 2, 3
Cattle
Sheep, goat
B.suis 2 Pig, goat
B.canis - dog
B.ovis Not reported rams
B.neotome - Wood rats
BRUCELLA BIOTYPES ISOLATED
FROM INDIA

7.  There are two types of smooth lipopolysaccharide (SLPS) surface
antigens, designated A and M. A antigen predominates in B. abortus and B.
suis, while M is the major antigen in B. melitensis. Numerous outer and
inner membrane, cytoplasmic, and periplasmic proteins have also been
characterized
Other Virulence Factors
 Cyclic β-1,2-glucans (CbGs)
 Superoxide dismutase
 Catalase
 Urease
 Alkyl hydroperoxide reductase (ahpC&D)
 Cytochrome oxidase
 Nitric oxide reductase (norD)
 Brucella virulence factor A (BvfA)
 Brucella virulence factor B (Vir B) encode Type IV secretion system
ANTIGENIC DETERMINANTS

8. Phagosome
maturation
Phagosome
maturation
Oxidase killingOxidase killing
Preferentially localization of Gravid uterus (unknown factors – Allontoic
factors Erythritol)
PATHOGENESIS

9. Because of the deceptive nature, the disease may be easily
misdiagnosed or diagnosis may be delayed thereby making clinical
diagnosis a challenge
1. Clinical signs
2. Laboratory diagnosis
Diagnostic tools include
 Isolation and identification of Brucellae from clinical samples,
 Antigen detection
 Genome detection
 Antibodies detection
Diagnostic Technique For Brucellosis
in Cattle

10.  Samples- aborted fetuses (stomach contents, spleen and lung), fetal
membranes, vaginal secretions (swabs), milk, semen and arthritis or
hygroma fluids
a) Staining methods
 Stamps’s method- modified zeihl neelson method
 Brucella organisms stain red against a blue background
 A fluorochrome or peroxidase-labelled antibody conjugate based technique
could also be used
b) Culture
 Basal media- tryptose (or tryptone) soy agar (TSA)
 B. abortus biovar 2, such as blood agar base or Columbia agar
Serum–dextrose agar (SDA) or glycerol dextrose agar
 Biphasic medium, known as Castañeda’s
 Selective Medium - Farrell’s medium
IDENTIFICATION OF THE AGENT

11.  Serum agglutination test (SAT)
 The complement fixation text (CFT)
 ELISA (CELISA) , Recombinant OMP28 ELISA
 RBPT, MRT , 2-mercaptoethanol test (2ME)
 Agar gel immunodiffusion test (AGID)
 Fluorescence polarization assay (FPA) etc.
SEROLOGYSEROLOGY
MOLECULAR
METHODS
MOLECULAR
METHODS
PCR,
 RFLP and Southern blot
 Pulse-field gel electrophoresis has been developed that allows the
differentiation of several Brucella species

12. Tube Agglutination Test
Also known as the standard agglutination test or serum
agglutination test (SAT)
Test serum is diluted in a series of tubes (doubling
dilutions)
Constant defined amount of antigen is then added to each
tube and tubes incubated for ~20h @37°C
Particular antigen clumps at the bottom of the test tube
Test is read at 50% agglutination
Quantitative

13. 13
Tube Agglutination Test

14. No agglutinationAgglutination
1/10 1/20 1/40 1/80 1/160 1/320 Neg. ctrl
In this case, the titre is 1/40
Tube Agglutination Test

15. RBPT and MRT

16. Blood
Serum
BrucellaBrucella
serologyserology
Blood Culture
Medium
Isolate
Environmental
Sample Isolation
Biochemical Tests
Dye Tolerance
Slide Agglutination
Gel Formation
Requirement of CO2
Tb Phage Lysis
H2S test
Molecular Tests
Real Time PCR
Multiplex PCR
Sequencing of conserved
genes
 16S rRNA
 RecA
 RpoB
MLVA: Strain Id
MLSA: Subtyping
Whole genome sequencing:
Genetic relatedness
Contd..
16

17. AMOS
• Set of 8 primers used
• For B. abortus, B. melitensis, B. ovis and B. suis
• Could detect
 Biovars 1, 2, and 4 of B. abortus
 All 3 biovars of B. melitensis
 Biovar 1 of B. suis
 Biovar 1 of B. ovis
• Showed 100% agreement with conventional biotyping methods
• Able to differentiate vaccine strain S19 and RB51 from field
strain isolates
Multiplex PCRs
(Yu and Nielsen, 2012)17

18. MLVA
Multiplex PCR using 8 multi-locus variable number
tandem repeat analysis
Able to distinguish B. melitensis from other Brucella
species and allowed strain typing
Bruce-ladder
A mPCR to identify 9 Brucella sp. at genus level,
including 6 terrestrial species, the marine species of
Brucella, and the vaccine strains S19, RB51and Rev. 1
(Yu and Nielsen, 2012)18

19. Loop-mediated isothermal amplification
(LAMP) test
 Highly specific
 Rapid (60 min.)
 Works at constant temp. 61
.
C
 Sensitive 100 fold than PCR
19(Karthik et al., 2014)

20. 20
(Karthik et al., 2014)

21. Brucella
Ab tests
21(ICAR – NIVEDI, 2014)

22. Guinea pig - most susceptible to brucella. (García-Carrilo 1977).
Infection - non progressive and may recover spontaneously
Inoculation - into thigh
Examination - animal is killed after 6-8 weeks.
Lymph node in the region of inoculation may be
enlarged and should be cultured.
Spleen - should be cultured by rubbing the cut surface on the
surface of SD agar containing bacitracin 10 units/ml ,
polymyxin B 4 uints/ml to suppress the contaminants.
Blood from JV - tested for presence of antibodies to brucella
Guinea pig inoculation

23. Contd……
Guinea pigs (Cavia porcellus) are probably the animals that are
most susceptible to Brucella spp infection. As few as 11 cells of
Brucella spp are sufficient to cause infection (García-Carrilo
1977).
 They are very sensitive to infection by any route: subcutaneous,
conjunctival, intraperitoneal, intranasal, intravenous, vaginal, oral
or cutaneous scarification (García-Carrillo, 1990).
Female guinea pigs have generally been preferred for Brucella
spp. studies, even though sensitivity to Brucella spp. Infection is
practically the same for both sexes (García-Carrillo, 1978).

24. Mice
Susceptibility to Brucella infection varies according to genetic
constitution of mice (García-Carrillo, 1990).
Bosseray et al. (1984) suggest mice as the animal model for
titration of biological activity and quality control of anti-Brucella
spp. vaccines.

25.  There is no practical treatment for infected cattle or pigs, but long-term
antibiotic treatment is sometimes successful in infected dogs.
 Prolonged treatment with clinically effective antibiotics are necessary to
penetrate these facultative, intracellular pathogens
 Brucellae are inaccessible to antibiotics - The treatment recommended by
the WHO for acute brucellosis in adults is rifampicin and doxycyclin
 However, the use of more than one antibiotic is needed for several weeks,
because the bacteria incubate within cells
 Since the treatment of animal brucellosis is very expensive, one should
encourage the mass vaccination of livestock
TREATMENT

26.  OIE/FAO Reference Laboratory for Brucellosis in France
 Building of laboratory technical staff as well as of strengthening of national
laboratories and good epidemiological knowledge
 Since the treatment of animal brucellosis is very expensive, one should encourage
the mass vaccination of livestock
 Standard operating procedures for diagnostic tests --RBT, CFT and iELISA
 Human brucellosis is best prevented by controlling the infection in animals.
Pasteurisation of milk from infected animals
 Also, the Government of India has made it mandatory to regularly screen all the
breeding bulls from artificial insemination centres for brucellosis and to use
brucellosis free bulls for semen production ( Renukaradhya et al 2002)
Major OIE Recommendations

27.  Education about risk of transmission
 Farmer, abattoir worker, butcher, consumer, hunter, public
 Wear proper attire if dealing with infected animals/ tissues
 Veterinarian- Gloves, masks, goggles
 Avoid consumption of raw dairy products
 Immunize in areas of high prevalence
 Young goats and sheep with Rev-1
 No human vaccine
 Eradicate reservoir
 Identify, segregate, and/or cull infected animals
contd

28.  Japan, Canada, some European countries, Australia, New Zealand, and
Israel, has been eradicated
 B. abortus persists in wildlife hosts in some regions, including the Greater
Yellowstone Area of North America
 It is a notifiable disease
 Australia declare itself free of bovine brucellosis in 1992 (Bunn, 2002)
 Canada declared their cattle herd brucellosis-free on September 19, 1985
and Ireland was declared free of brucellosis on 1 July 2009
 The nation of Trinidad and Tobago was classified “free of bovine
brucellosis” by the (OIE) in 1984 (Blajan and Melendez, 1984)
Eradication of Bovine
Brucellosis
Eradication of Bovine
Brucellosis

29. Strategies to Fight BrucellaStrategies to Fight Brucella
Control the infection :
 Source of Infection or Transmission of infection, restricting Movement of
animals, Natural service by bulls ,Transmission by carnivores animals and
through milk
Test and slaughter method :
 No effective treatment, so diagnose, if +ve kill the animals until no reactor
animal for three consecutive tests, carried out at three-month interval is found
(Mathur et. al., 1974) Financial compensation to farmers
Vaccination Programme :
 Vaccination Programme Increases resistance and decreases the source of
infection. Different vaccine against the B. abortus are Live B. abortus Strain-
19 vaccine, Killed adjuvant B. abortus 45/20 vaccine, B. abortus vaccine
RB51.
 Make calfhood vaccination compulsory and avoid vaccination of adult
animals

30. contdcontd
Live B. abortus Strain-19 vaccine :
 Calves are vaccinated once at the age of 4-8 month
 Disadvantages: Induces abortion in pregnant animals Excreted in milk
Killed adjuvant B. abortus 45/20 vaccine :
 Not giving lasting immunity
 Two initial vaccinations 1st at 3-4 month of age and then repeat after 6 month
and an annual booster (Plommet, 1991)
B. abortus strain RB51 vaccine :
 Compared with S-19 vaccine, RB51 vaccine causes less abortion (Cheville et
al., 1996) Protective effect is similar to that of S-19
B. melitensis strain Rev 1 vaccine :
 Partially attenuated vaccine for sheep and goat
B. melitensis H38 vaccine :
 Killed vaccine for sheep and goat

31. CONTROLCONTROL
Control of brucellosis is difficult
due to absence of defined control
strategies, long life span of
infected animals, uncertainty of
epidemological reservoir and
bacteria species involved.