Brucellosis in humans and domestic ruminants in Bangladesh

1
Epidemiology of brucellosis in humans and
domestic ruminants in Bangladesh

• A. K. M. Anisur RAHMAN

Promoters Professor Dr. Claude SAEGERMAN, ULg,
Liege Professor Dr. Dirk BERKVENS, ITM, Antwerpen
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Talk plan
3

• General Introduction
• Objectives
• Studies Brief methods, Results and Discussion
• Brucellosis in cattle
• Brucellosis in small ruminants
• Species of Brucella prevalent in man and
  ruminants
• Brucellosis in occupationally exposed people
• Brucellosis in patients with pyrexia of unknown
  origin
• General conclusions
• General recommendations for prevention and
  control

4
Brucellosis is a zoonotic (bacterial) disease
Introduction
5
Smooth colony
Bruclla
Photograph CODA-CERVA
Brucella melitensis from aborted ewes (FAO, 2006)
Slow-growing, transparent and honey coloured
Stamp staining pale red Brucella
Gross and microscopic morphology of Brucella spp.
6
History of brucellosis
7
Vertebral epiphysitis in anterosuperior corner
Pedro-Pons sign
2.3-2.5 million years old bone remains of
australopiths, South Africa (Danastasio et al.,
2011)
Australopith
Human skeletal remains brucellosis 3550-4100
years ago Palestine and Jordan (Danastasio et
al., 2011)
Carbonized burried cheese Herculaneum, Italy
A.D. 79
Brucella-like organism in scanning electron
microscope x 25 000) Capasso, 2002
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Aborted fetus
1897
liver of diseased soldiers
Figure Moreno, 2014
Type a quote here.
1887
Johnny Appleseed
1914
zoonotic Brucella spp. with preferred hosts
9
Brucella has conquered oceans also
Figure Moreno, 2014
Brucella inopinata Breast implant
Now considered zoonotic as isolated from wild
boar hunters
10
Brucellosis in animals and humans
11
Figure Dey et al., 2013
Abortion
Retention of placenta
Metritis
Drop in milk yield 25
Infertility
Epididymitis in ram (FAO, 2006)
Epididymitis in bull (FAO, 2006)
Major manifestations of brucellosis in animals
12
Headache
Fever debilitating
Arthralgia
Muscle pain
Endocarditis
Neurobrucellosis
Fatal (lt1)
Back pain
Weight loss
Major manifestations of brucellosis in humans
13
Transmission among animals and humans
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Transmission in animals

• Ingestion Brucella contaminated feed or water
• Licking infected placenta, calf or fetus,
  genitalia of an infected animal after abortion or
  delivery
• Breeding Artificial insemination or natural
  service

15
Transmission in humans

• Ingestion Unpasteurized milk or milk products
• Skin lesions and aerosol prolonged contact with
  animals occupational
• Venereal Also occurs but not usual

16
Treatment and vaccination

• Ruminants Treatment not recommended, culled, but
  vaccines available
• Humans WHO recommendations no safe and
  effective vaccine
• Doxycycline (orally six weeks) streptomycin
  (Injection) for two to three weeks
• Doxycycline rifampicin six weeks orally

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World status of brucellosis in ruminants

• Brucellosis free Australia, Canada, Denmark,
  Finland, New Zealand, The United Kingdom, etc.
• High risk areas Mediterranean Sea Basin, South
  and Central America, Asia, Africa, the Caribbean
  and Near East
• Prevalence 2.0 to 42.1 cattle level
• Prevalence 0.5 to 30.6 Goats 2.5-37.6
  Sheep

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Type a quote here.
Johnny Appleseed
The global map of human brucellosis Pappas et
al., 2006
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Context of this Research
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2,400-mile border between Bangladesh and India
150 million people
23.4 million cattle
32 million households
25.2 million goats
3.1 million sheep
Border between Myanmar and Bangladesh
19 million livestock farmers
Temperature 10-40?
Annual rainfall 2600 mm
Relative humidity 74.1
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Mymensingh district
Gayal
Central Cattle Breeding and Dairy Farm
Thirteen districts of Bangladesh covered through
this research
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Indigenous cattle under subsistence management
system Mymensingh district
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Mosquito net for cattle
Housing and feeding in subsistence management
systems
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Cattle, buffalo, goats and sheep graze together
the definition of a herd is different
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Cut and carry system of feeding straw feeding
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Tethering system of feeding both in goats and
cattle, rare in sheep
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Holstein Friesian
Holstein Friesian
Sahiwal breed
Central Cattle Breeding and Dairy Farm (CCBDF)
The largest government farm herd size gt2000
28
Liquid nitrogen reserve
Artificial Insemination (AI)
AI wing of CCBDF where Breeding bulls are reared
for the production of frozen semen
29
Mostly Holstein Friesian crossbred
Waiting for help?
Concentrate feed
Hand milking
Sirajgonj district A potential dairy zone in
Bangladseh only milk sample tested
30
Not happy with suspension?
Straw reserve
Chittagong district another dairy zone only
milk sample tested
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Portable machine milking
Packaging raw milk
Hand milking
Chittagong district another dairy zone only
milk sample tested
32
Black Bengal goats
Prolific
Asset for poor farmer
Delicious meat
Jamunapari goats
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together with goats
Indigenous sheep
34
Gayal Bos frontalis Figure from Web

• They are semi-domestic ruminants
• Have some similarity with the Indian bison or
  gaur

35

• Humans are extremely close to animals in
  Bangladesh

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Ropes with the bed stand to tie at night
Cattle house very close to human house
Goats and humans share same house at night
37
Professional butcher no protective clothes
38
Occasional participation of general people during
festival
39
Animals and humans bath in the same pond
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Children are also very close to ruminants
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Status of brucellosis in Bangladesh
42
2013
2012
2011
2010
2009
2006
2005
2004
1992
1970
0
5
10
15
20
Prevalence
43
Tests used
Brucellosis in small ruminants
2014
113
RBT, iELISA, SAT, MAT, PAT
2012
Performed in Korea
cELISA, FPA
2011
Sample size
2.9 (2.1-4.1) Overall apparent prevalence
except 1988
2010
2007
1988
PAT, TAT
350
0
5
10
15
Prevalence
Reported prevalence of brucellosis in goats in
Bangladesh1988-2014
44
60
2014
Brucellosis in small ruminants
2012
Sample size
Overall prevalence 5.4 (3.9-7.1)
2011
206
2007
9
0
3
6
Prevalence
Reported prevalence of brucellosis in sheep in
Bangladesh 2007-2014
45
2010
50
2009
Sample size
Overall prevalence 9.9 (7.6-12.7)
1988
Overall prevalence 6.9 (4.7-9.6)Excluding
study of 1988
1983
210
0
20
30
10
Prevalence
Prevalence of human brucellosis in Bangladesh
1983-2010
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What are unknown with regard to brucellosis in
Bangladesh?

• The performance of diagnostic tests
• True exposure prevalence and acute infection
• Prevalent species of Brucella
• Risk factors of brucellosis in humans and animals
• Brucellosis in patients with pyrexia of unknown
  origin

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Objectives Study 1

• To evaluate the performance RBT, SAT, iELISA for
  the diagnosis of bovine brucellosis and to
  estimate the true prevalence of brucellosis in
  cattle

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Bovine brucellosis
Submitted
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• Test evaluation Most tests are imperfect
  (Selt100 Splt100)
• Gold standard test Se100 Sp100(Rare in
  Practice) Culture for brucellosis
  Sensitivity30-90
• In the absence of gold standard Latent class
  evaluation
• Frequentist approach Hui-Walter, 1980
• Assumptions not always true
• Rogan-Gladen estimator True prevalence sometimes
  negative or more than 100

Rogan-Gladen, 1978
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Acute brucellosis SAT ELISA

• Bayesian approach Can combine observed field
  data with existing external information
  (Prior/expert opinion)
• Se and Sp are random variables Not fixed
• Verification prior and data conflict Bayesian-p
  value and DIC
• Multiple tests RBT, SAT and iELISA
• Both conditional dependence and independence
  assumptions
• Stage of infection Concomitant presence of IgM
  and IgGacute brucellosis

Ig
IgG
IgM
SAT cut-off
ELISA cut-off
Time
Kinetics of immune response against Brucella
infection (Godfroid et al., 2010)
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iELISA 2IU/ml 5 IU/ml
RBT
SAT 30 IU/ml

• Random blood sample from Mymensingh district
  1020
• Random blood sample from largest Government farm
  (CCBDF) 340
• Three tests applied in parallel
• Priors on Se and Sp (Meta analysis) Beta
  distribution
• Two latent class models examined Conditional
  independence and (Hui-Walter, 1980) and
  Conditional dependence with covariances (Gardner
  et al., 2000).

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Results and Discussion
Locality Prevalence Se_RBT Sp_RBT Se_SAT Sp_SAT Se_iELISA Sp_iELISA
Mymensingh 0.003 0.81 0.99 0.635 0.986 0.905 0.993
CCBDF 0.205 0.761 0.956 0.797 0.953 0.913 0.992
CCBDF Acute brucellosis 15.6
Mymensingh Acute brucellosis 0.29
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Locality Prevalence Sensitivity Specificity PPV_iELISA NPV_iELISA
Mymensingh 0.3 90.5 99.3 28.0 99.9
CCBDF 20.5 91.3 99.2 96.7 97.8
Mymensingh
CCBDF
Black line Negative
Blue line False positive
Red line True positive
iELISA serological class in function of age
Prediction from ordered logistic regression
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Bovine brucellosis discussion

• Low prevalence in Mymensingh
• Large-scale slaughter all the year round 3.5
  million/year
• Average age of cattle 3.7 years puberty 2-3
  years
• Infrequent abortion due to brucellosis
• More indigenous cattle 86.3
• High prevalence in CCBDF import, artificial
  insemination, slow or no culling of infected
  cattle, large herd size, more cross-bred does
  not represent commercial herds

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Objectives Study 2

• To evaluate the performance of RBT, SAT and
  iELISA for the diagnosis brucellosis in small
  ruminants along with estimation of true prevalence

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Brucellosis in Small Ruminants
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Methods

• Random blood sample 636 goats
• Random blood sample 1044 sheep
• Bayesian latent class evaluation conditional
  dependence and independence
• Priors on Se and Sp Metanalysis uniform
  distribution

RBT
iELISA 2IU/ml in goats 6 IU/ml in sheep
SAT 30 IU/ml
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Species Prevalence Se_RBT Sp_RBT Se_SAT Sp_SAT Se_iELISA Sp_iELISA
Goats 0.01 0.802 0.996 0.571 0.993 0.929 0.965
Sheep 0.012 0.828 0.983 0.72 0.986 0.92 0.995
True exposure prevalence, Sensitivity and
Specificity of three serological tests for the
diagnosis of brucellosis in goats and sheep
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Dependence coefficient between RBT and iELISA
among infected sheep 18.0
Dependence coefficient between RBT and SAT among
infected sheep 53.0
Acute infection in sheep 0.77 and 0.31 in goats
Positive Predictive Value of iELISA in sheep
69.0 and that in goats 21.1
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Brucellosis in small ruminants Discussion

• Low prevalence in both species
• Large scale slaughter all the year round 15
  million goats/year
• Average age of goats 1.6 years puberty 6.5
  months
• Average age of sheep 2.1 years puberty 8.2
  months
• Infrequent abortion due to brucellosis
• Presence of Brucella abortus only
• iELISA and SAT in series may be applied for
  diagnosis culling maximize Sp and PPV

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Objectives Study 3

• To identify the species of Brucella prevalent in
  humans and animals in Bangladesh

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Brucella abortus is dominant in humans and
ruminants in Bangladesh
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Brucella abortus is dominant in Bangladesh
Had clinical symptoms seropositive
Placenta
Vaginal swabs
Bull Semen (cattle)
Human sera
Ring Test positive milk
Cow (05), goats (10), sheep (08) 23
Cow (04), goats (10), sheep (03) 17
13
Cow (14), goats (2), gayal (1) 17
05

• Stamp staining Microscopy
• Culture of animal samples Isolation in Farrells
  medium

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Brucella abortus is dominant in Bangladesh

• BCSP31 Brucella cell surface protein genus
  specific real time (rt) PCR screening of humans
  and animal samples
• BCSP31 rt PCR positive samples Further tested by
  IS711 Brucella abortus and Brucella melitensis rt
  PCR
• No Brucella-like organism in stained smears
• No Brucella colony in culture

Cut-off ct 40
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Brucella abortus is dominant in Bangladesh

• Brucella abortus DNA detected from six animal
  samples
• Three cow milk, one goat milk, one gayal milk and
  one bull semen orchitis
• Brucella abortus DNA was also detected from 13
  acutely infected human sera Treated and
  recovered
• Dairy workers 7, livestock farmers 5, butcher
  1
• Out of 13, seven dairy workers originated from
  CCBDF

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Brucella abortus is dominant in Bangladesh

• No Brucella melitensis DNA was detected from
  either animal or human samples
• Isolation failure old sample, repeated freezing
  and thawing, competing microflora with
  antimicrobial resistance, infrequent abortion
  due to brucellosis

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Brucella abortus is dominant in Bangladesh

• Brucella abortus in goat
• As Brucella melitensis is not endemic
• Brucella abortus in gayal
• Transboundary route gayals from Mizoram and
  Tripura, India cross into Bangladesh contiguous
  habitat
• Cross-breeding with cattle Bulls infected with
  brucellosis

Gayal X Holstein Friesian cross-bred calf (left)
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Objectives Study 4

• To study the prevalence and risk factors of
  brucellosis in a high-risk group of people in
  Bangladesh

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Brucellosis in a high-risk group of humans
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Brucellosis in a high-risk group of humans
Veterinary Practitioner 19
Livestock farmer 386
Butcher 40
Milker 55
iELISA 20IU/ml
RBT
STAT 1160

• Total sample 500, tested by three tests in
  parallel
• Statistical analysis Random effect logistic
  regression
• Brucella genus specific rt PCR using three test
  positive sera

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Brucellosis in a high-risk group of humans

• Overall apparent prevalence 4.4 based on
  parallel interpretation
• Contact with livestock for more than 26 yrs
  (comparison with 5 yrs) significant risk factor
• Contact with goats increases the risk further
• Brucella genus specific DNA was amplified from
  all of the 13 seropositive patients had clinical
  symptoms, treated and recovered

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Brucellosis in a high-risk group of humans

• High apparent prevalence in previous reports
• Small size of the sample, type and cut-off of
  test, non-random sample.
• Exposure prevalence 4.4 but acute infection 2.6
  13/500

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Brucellosis in a high-risk group of humans

• Probable reasons for low human prevalence
• Food habit proper boiling, drinking raw milk
  rare
• Abortion less frequent so long time contact with
  infected animals required to become infected
  e.g. 26 yrs
• Only Brucella abortus less pathogenic in humans
  in animals prevalence in humans 1-5
• Low prevalence in domestic ruminants in
  Bangladesh
• Education about personal hygiene and risk factor
  will decrease this disease further in humans

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Objectives Study 5

• To estimate the prevalence of brucellosis in
  patients with pyrexia of unknown origin in
  Bangladesh

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Brucellosis in pyrexic patients
Manuscript in preparation
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Brucellosis in PUO patients

• Malaria, typhoid, tuberculosis are endemic in
  Bangladesh
• Randomly collected 300 sera from PUO patients

STAT 1160
iELISA 20IU/ml
RBT

• Firths logistic regression analysis
• Brucella genus and species specific rt PCR

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Brucellosis in PUO patients

• Overall apparent prevalence in PUO patients 2.7
  based on parallel interpretation acute infection
  6/3002.0
• Risk factors and symptoms age, residence
  rural, type of patient inpatient, contact
  with animals, type of animal handled goats,
  arthralgia and backache
• Only Brucella abortus DNA was detected from six
  three test positive PUO patients had clinical
  symptoms, treated and recovered

80
Brucellosis in PUO patients

• Brucellosis in PUO patients is not uncommon
• Based on performance, simplicity and cost RBT is
  recommended as a screening test in humans
• PUO patients with the history of animal contact
  should be referred by physicians for routine
  screening by RBT

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General Conclusions
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General conclusions

• The true exposure prevalence and acute infection
  of brucellosis is very low domestic ruminants
  under subsistence management system.
• The true exposure prevalence and acute infection
  in the largest government dairy farm is very high
  but does not represent commercial herds.
• The SAT and iELISA simultaneously may be applied
  to know the stage of brucellosis infection in
  domestic ruminants both in high and low
  prevalence scenarios.

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General conclusions

• The apparent prevalence of brucellosis in a
  high-risk occupationally exposed people and in
  pyretic patients are also low.
• Based on performance, simplicity and cost, the
  RBT is recommended as screening test in humans
  having signs and symptoms of brucellosis along
  with the history of animal contact.
• Only Brucella abortus is dominant in humans and
  animals in Bangladesh.

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General recommendations for prevention and control
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General recommendations for prevention and control

• The Department of Livestock Services should take
  some initiative
• Animal or their herd identification system
• Legislative support like human birth
  registration act.
• Veterinary services should be tagged with
  animal/herd registration certificate
• Abortion should be notifiable and tested
• Legislative support needed

86
General recommendations for prevention and control

• Further research on brucellosis in dairy rich
  zones
• True exposure prevalence and acute infection IgM
  and IgG detecting tests
• Isolation of Brucella species Milk ring test
  positive samples should be preferred over
  abortion specimens
• Initially, animal inoculation (Guinea pigs)
  technique should be preferred than culture

87
General recommendations for prevention and control

• Immediate control measures in CCBDF
• Culling of remaining infected cattle and
  breeding bulls
• Vaccination of female calves
• Import from brucellosis free countries and herds
  (local)
• Regular testing of breeding and pre-breeding
  bulls for brucellosis once in a year

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General recommendations for prevention and control

• One health approach to prevent and control
• Enhancing activities of the existing One Health
  Hub, Bangladesh priority list Avian influenza,
  Nipah virus disease, Rabies and Anthrax
• Establishing one Reference laboratory for the
  diagnosis of brucellosis in humans and animals
• Educating people on personal hygiene and risk
  factors Posters, SMS in cell phone, video, etc.

89
General recommendations for prevention and control

• Regional cooperation
• Control of illegal import of animals from
  neighbouring countries
• Quarantine of imported animals
• Cooperation among traders, suppliers and
  regulatory bodies

Boundary is the river
Almost 40 8 million of the total cattle
slaughtered annually 20 million in Bangladesh
come from India illegally
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Final General Figure
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Acknowledgement

• Promoters Prof. Claude Saegerman and Prof. Dirk
  Berkvens
• Members of the committee Prof. Jacques Mainil
  and Dr. David Fretin
• Institutions
• Farmers and other participants
• Family members and well-wishers

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Thanks for your attention
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SAT titer based on the degree of agglutination
Dilution serum Agglutination Titre (UI/ml)
1/12.5 0 0
1/12.5 gt0 to lt75 lt25
1/12.5 75 25
1/25 25 30
1/25 50 40
1/25 75 50
1/50 25 60
1/50 50 80
1/50 75 100
Cut-off
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Positive control
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