Brucellosis and its public health impact

Brucellosis

1. Brucellosis is also known as…
In Humans
•Malta Fever
•Undulant Fever
•Mediterranean Fever
•Rock Fever of Gibraltar
•Gastric Fever
In Animals
•Bang’s Disease
•Enzootic Abortion
•Epizootic Abortion
•Slinking of Calves
•Ram Epididymitis
•Contagious Abortion

2. Sir David Bruce (1855-1931)
British Army physician
and microbiologist who
discovered Micrococcus
melitensis

3. Bernhard Bang (1848-1932
Danish physician and
veterinarian
Discovered Bacterium
abortus could infect cattle,
horses, sheep and goats

4. Brucella Taxonomy

5. Species Biovar/Serovar Natural Host Human
Pathogen
B. abortus 1-7 Cattle yes
B. melitensis1-3 Goats, Sheep yes
B. suis 1, 3 Swine yes
2 Hares yes
4 Reindeer, Caribouyes
5 Rodents yes
B. canis none Dogs, other canidsyes
B. ovis none Sheep no
B. neotomaenone Desert wood rat no
B. maris Marine mammals ?

6. Brucella spp.Source VirulenceInfective Dose
B. melitensis
Goats, sheep,
Cattle, Swine
++++ 1 – 10
B. suis Swine, Cattle+++1,000 – 10,000
B. abortusCattle ++ 100,000
B. canisDogs + 1,000,000

7. In nature, Brucella can survive for longer period as compared to
many other non-spore formers
 Heat
 Radiation
 Dust/Soil
 Water
 Frozen tissue
 Bovine feces
 Aborted foetus
 Uterine exudate
 Liquid mannure
 Drying
 Readily killed
 Killed by usual sterilizing dose
 Upto 10 weeks
 10-70 days depending on temperature
 For years
 Upto 120 days
 75 days
 200 days
 2½ years
 Survives for sometimes (longer in
protein-rich medium)
Physical
Resistance to Physico-Chemical agents

8.  Readily killed by most of
the disinfectants
 Organic matter interferes
phenol
 <15 Min (37
0
C)
 Most effective (<15
0
C)
 Xylene
 Calcium cyanamide
 >30 days
 >2 weeks
Chemical
Liquid Manure

9. HOST RANGE
  and TRANSMISSION
Brucella abortus: cattle, buffalo, select wildlife
Brucella melitensis: sheep, goats and bovines
 Main source of infection
 uterine discharge of infected animals
 aborted foetus
 infected semen
 Spreads mainly through
 ingestion of contaminated food and water
 Artificial Insemination
 tail splashing of urine - conjunctival route
 veterinary activities
 Rural poor zoo sanitary conditions

10. Species Reservoir Clinical Disease
B. melitensisSheep
Goat
Severe Acute
Complications [Common]
B. abortus Cattle Mild with Suppurative
Complications [Not common]
B. suis Pig Severe Chronic, Suppurative,
Destructive
B. canis Dog Mild with suppurative
Complications [Not common]
Human Brucellosis & Associated Species

11. Prominent animal hosts for Brucella in
Europe, Africa, Asia and Australia.
Countries declared brucellosis-free
Unknown
China
India
India
Spain
Portugal
France
Italy
Turkey
Iran
Sudan
Australia
New
Zealand
Algeria
Libya

12. Prominent animal hosts for Brucella
in North and South America.
Countries declared brucellosis-free
Unknown
Canada
[P]
USA
[B/P]
Mexico
[B/S/G/P/C]
Chile [B/S/G/P/C]
Argentina
[B/S/G/P/C]
Columbia
[B/P]
Venezuela
[C/S]
Guyana
[B/S/G/P/C]

13. Species Seropositivity Isolation
Cattle, Buffalo,
Sheep, Goat, Pig,
Equine
Yes Yes
Camel, Poultry,
Wild Animals
Yes No
Scenario in India
Isolation

14. Brucella sp. Biotype Animal
B. abortus 1,3,4,6 Cattle
1,3 Buffalo & Man
1 Sheep, Goat
# Equine
B. melitensis 1 Cattle
1,2,3 Sheep, Goat & Man
# Equine
B. suis 2 Pig, Goat
1 Dog
B. canis -- Dog
Contd.

15. Scenario of BrucellosisScenario of Brucellosis
 Incidence
World wide in animals & man
Endemic in 86 of 175 countries, i.e., 49%
5% of total animals affected
500,000 (4%) of human cases/year in
world
More than 3.3% cases of PUO in Indian
hospitals

16.  Increasing concern for Public Health in
developing countries
 Urbanization
 Lack of hygienic measures
 Animal husbandry
 Food handlers
 Expansion of animal industry
 Expansion of international traveling
Contd.

17.  Barrier to international trade on
animals and animal products
 Profound impact on socio-economic
development
 A potent BIOLOGICALBIOLOGICAL warfare agent
Contd.

18. Brucella as a Bio-Weapon
Aerosolized B. melitensis
City of 100,000 people
Inhale 1,000 cells
Results
82,500 cases requiring extended therapy
413 deaths
$477.7 million in economic impact

19. Annual incidence of brucellosis
Per 1,000,000 population
>10-50
>500
>50-500
>2-10
<2
Possibly endemic, No data
Non-endemic/No data
Annual incidence of Human brucellosis

20. Milk,
Slaughter,
Vet. care
Vaccination or
Laboratory
accidents
Slaughter
or Veterinary
care
Veterinary
care
Goat & Sheep
Cattle Swine
Dogs
B. abortus B. suis
B.canisB.melitensis

21. Brucella enters through different portals in the host body
Infects the macrophages in host immune system
Avoids endolysosomal fusion
Transits in intracellular compartment resembling
autophagosomes
Autocrine stimulation of
macrophages is impaired
Inhibits the TNF-α
production
Impairment of NK
cell activation
Th1 type specific immunity is
abolished
Resistance to programmed
cell death
Brucella infection pathway

22. Human Disease
Can affect any organ or organ system
All patients have a cyclical fever
Variability in clinical signs
Headache, weakness,
arthralgia, depression,
weight loss, fatigue,
liver dysfunction

23. Human Disease
20-60% of cases
Osteoarticular complications
Arthritis, spondylitis, osteomyelitis
Hepatomegaly may occur
Gastrointestinal complications
2-20% of cases
Genitourinary involvement
Orchitis and epididymitis most common

24. Human Disease
Neurological
Depression, mental fatigue
Cardiovascular
Endocarditis resulting in death
Chronic brucellosis is hard to define
Length, type and response to treatment variable
Localized infection
Blood donations of infected persons should
not be accepted

25. Human Disease
Congenitally infected infants
Low birth weight
Failure to thrive
Jaundice
Hepatomegaly
Splenomegaly
Respiratory difficulty
General signs of sepsis (fever, vomiting)
Asymptomatic

26. Diagnosis in Humans
Isolation of organism
Blood, bone marrow, other tissues
Serum agglutination test
Four-fold or greater rise in titer
Samples 2 weeks apart
Immunofluorescence
Organism in clinical specimens
PCR

27. Treatment of Choice
Combination therapy has the best
efficacy
Doxycycline for six weeks in combination
with streptomycin for 2-3 weeks or rifampin
for 6 weeks
CNS cases treat 6-9 months
Same for endocarditis cases plus surgical
replacement of valves

28. Prognosis
May last days, months, or years
Recovery is common
Disability is often pronounced
About 5% of treated cases relapse
Case-fatality rate: <2% ( untreated)
Endocarditis or Meningitis caused by B.
melitensis

30. Clinical Signs: Cattle &
Bison
Third trimester abortions
with B. abortus
Retained placenta
Once expelled will have a
leathery appearance
Endometritis
Birth of dead or weak calves
Respiratory distress and lung infections
Low milk yield

31. Clinical Signs: Sheep &
Goats
B. melitensis causes late term abortions
Retained placenta
Birth of dead or weak lambs/kids
Goats - articular and periarticular hygroma
localizations
B. ovis causes abortions,
fertility problems
Orchitis, epididymitis
Abnormal breeding soundness exam
Organisms present in semen

32. Clinical Signs: Swine
B. suis
Prolonged bacteremia
Abortion, early or late gestation
Fertility problems
Sows temporary
Boars, unilateral or bilateral orchitis
Lameness, posterior paralysis, spondylitis,
metritis, abscesses

33. Clinical Signs: Horses
B. abortus most common
Susceptible to B. suis
Fistulous Withers or Poll Evil
Inflammation of the
supraspinous bursa
Exudative process
Fills with clear viscous liquid
Can eventually rupture

34. Clinical Signs: Dogs
Susceptible to
B. melitensis, B. abortus, and B. suis
B. canis causes abortions
Last trimester of pregnancy
Prolonged vaginal discharge
Bacteremia
Failure to conceive, stillbirths, prostatitis,
epididymitis

35. Clinical Signs: Wildlife
Elk
Abortions, no retained
placenta
Moose
Debilitated, death
Predators not clinical, but are vectors
Coyotes, crows, vultures, bears
Aid in disease spread by carrying infected
tissues away from abortion site

36. Diagnosis in Animals
Isolation of organism
Blood, semen, other tissues
Serology
Brucellosis card test, ELISA
Brucella milk ring test
Demonstration by fluorescent antibody
of organism in clinical specimen
Placenta, fetus

37. Laboratory Identification of Brucella spp.
Blood
Serum
BrucellaBrucella
serologyserology
(BMAT)(BMAT)
Blood Culture
Medium
Isolate
Environmental
Sample Isolation
Biochemical TestsBiochemical Tests
DyeDye ToleranceTolerance
Slide AgglutinationSlide Agglutination
Gel FormationGel Formation
Requirement of CORequirement of CO
22
Tbilisi Phage LysisTbilisi Phage Lysis
HH
22S testS test
Molecular TestsMolecular Tests
Real Time PCRReal Time PCR
Multiplex PCRMultiplex PCR
Sequencing of conserved Sequencing of conserved
genesgenes
16S rRNA16S rRNA
RecARecA
RpoBRpoB
MLVA: Strain IdMLVA: Strain Id
MLSA: SubtypingMLSA: Subtyping
Whole genome sequencing: Whole genome sequencing:
Genetic relatednessGenetic relatedness

38. Identification of Brucella spp.
Standard phenotypic identification
Manual of Clinical Microbiology
Identification of Unusual Pathogenic Gram-Negative Aerobic
and Facultatively Anaerobic Bacteria
Molecular characterization
Conventional Multiplex & Real-Time PCR assays to identify
Brucella at the species level
Sequencing of highly conserved genes-16S rRNA, rpoB, recA
and omp2a/2b for phylogenetic analysis with close relatives;
Ochrobactrum spp.
Multi-Locus Sequence Analysis (MLSA) for molecular subtyping
of the genus Brucella
Multi-Locus Variable-Number Tandem Repeat (VNTR) Analysis
(MLVA) to identify Brucella strains in epidemiologic
investigations
Whole genome sequence analysis to study the genetic
relatedness of Brucella spp. and gain insight into virulence

39. Control and Eradication:
A. Test and Slaughter:
 Most effective
 Not feasible if incidence >2%
 Not applicable in small ruminants
B. Mass Vaccination:
 Only way to bring down incidence
 Calfhood vaccination to cover >80% population
 Initial vaccination of adult animals

40. Requirements of an ideal vaccine
for brucellosis
No disease in vaccinates
Protect both sexes against infection at any age
Provide long-term protection against infection and
abortion
Life-long immunity with single vaccination
No induction of antibodies interfering with diagnosis
Not be transmitted to other animals in case of latent
infection

41. Biologically stable
Free from reversion to original form in vivo & in
vitro
Non-pathogenic to human
Not contaminate animal products
Readily culturable for large scale production
Have specific genotypic or phenotypic
marker(s)
Be cheap
No adverse local reaction

42. Human:
i. Safe and effective
No disease
No hypersensitivity
Minimal local or systemic reaction
ii. Induce life-long immunity with single vaccination

43. Vaccines available:
A. For Animals:

i. Live-attenuated
a) B. abortus S19 d) B. suis S2
b) B. melitensis Rev.1 e) B. melitensis M111
c) B. abortus RB51

ii. Killed:
a) B. abortus 45/20
b) B. melitensis H38

44. A. For Human:
i. Live attenuated:
a) B. abortus 19BA
b) B. melitensis 104M
ii. Killed:
a) PI-SDS from B. abortus and B. melitensis
b) LPS-Protein conjugate
c) Acid extract

45. B. abortus S19 & B. melitensis Rev.1:
 Low pathogenicity
 High immunogenicity
 Stable attenuation
 Easy to grow and use
 Low loss of viability during lyophilization
Advantages of available vaccines

46. B. abortus RB51:
 Stable on in vivo and in vitro passage
 No O-side chain
 Non-pathogenic to human – potent live vaccine for man ??
 Immunity similar to that provided by B. abortus S19
 Safe for pregnant cattle ?
 Protects swine against B. suis
 Protects goats against B. melitensis
 Candidate for oral vaccination – wild animals ??

47.  Able to differentiate “wild type”
exposure from immunization
 Safe during pregnancy
 Can be used in adult
Lacks LPS-O antigen that causes antibody
response on serologic or milk tests

48. B. suis
S2:
Live attenuated
As stable as B. abortus S19
Virulence similar to B. abortus S19
Satisfactory protection in cattle
Suitable for oral administration (in
drinking water), particularly in sheep
& goats

49. Drawbacks of available vaccines for brucellosis
B. abortus S19 & B. melitensis Rev.1:
 abortion in pregnant animals
 interference with diagnosis
 infective to human
 use not allowed in countries free from B. melitensis

50. B. abortus
RB51
Immunity weak ??
Only CMI active
Immunosuppressive ??
Zoonotic potential ???
Infectious to humans
Serologically negative upon testing exposure
CDC registry of human exposures
32 documented exposures as of 1998
May loose viability
unacceptable antibiotic resistance

51. B. Suis S2:
Suitable for all species ???
Only CMI active ??
No marker for identification
Zoonotic potential not known
Low protection in sheep (B. melitensis & B. ovis)

52. Strategies for raising
Brucellosis-free
Herd

54. Permanently mark all eligible cattle
Serologically test all eligible cattle
Remove/cull all reactors from the herd
Retest the herd after 30-60 days, remove/cull
reactors, if found
If no reactors found, retest herd after 6 months
Consider two consecutive negative tests at
interval of 6 months as freedom from brucellosis
Prohibit sale of females over one year of age from
infected herd
Strategies for raising brucellosis-free herd

55. In case of high prevalence (>2%), vaccinated all
animals at 3-6 months of age
Once the prevalence is <0.2%, discontinue
vaccination
Monitor herd for brucellosis 3-4 times a year
Enact necessary legislation
● compulsory ◘ vaccination
◘ testing
◘ culling
◘ compensation
● strict control on importation of animals
Under exceptional circumstances vaccinate all eligible cattle initially
and follow it up with calfhood vaccination at 3-6 months of age

56. Remove the calf from the dam as soon as
possible after birth
Minimize the contact between calf and cow
Place the calf in a clean, dry pen that has been
properly sanitized. This is CRITICAL
Feed colostrum (2 to 4 L) as soon as possible
Make sure that the colostrum used comes
from test negative cows
If there is any doubt, DO NOT USE THE COLOSTRUM