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EARLY DETECTION OF RIFT VALLEY FEVEREARLY DETECTION OF RIFT VALLEY FEVEREARLY DETECTION OF RIFT VALLEY FEVEREARLY DETECTION OF RIFT VALLEY FEVEREARLY DETECTION OF RIFT VALLEY FEVERANIMALS vs HUMANS vs VECTORS
EARLY DETECTION OF RIFT VALLEY FEVERANIMALS vs HUMANS vs VECTORS
EARLY DETECTION OF RIFT VALLEY FEVERANIMALS vs HUMANS vs VECTORS
EARLY DETECTION OF RIFT VALLEY FEVERANIMALS vs HUMANS vs VECTORS
R SWANEPOEL FOR:PETRUS VAN VUREN, CHANTEL LE ROUX, JANUSZ PAWESKA
SPECIAL PATHOGENS UNIT
R SWANEPOEL FOR:PETRUS VAN VUREN, CHANTEL LE ROUX, JANUSZ PAWESKA
SPECIAL PATHOGENS UNIT
R SWANEPOEL FOR:PETRUS VAN VUREN, CHANTEL LE ROUX, JANUSZ PAWESKA
SPECIAL PATHOGENS UNIT
R SWANEPOEL FOR:PETRUS VAN VUREN, CHANTEL LE ROUX, JANUSZ PAWESKA
SPECIAL PATHOGENS UNITSPECIAL PATHOGENS UNITNATIONAL INSTITUTE FOR COMMUNICABLE DISEASES
SOUTH AFRICA
SPECIAL PATHOGENS UNITNATIONAL INSTITUTE FOR COMMUNICABLE DISEASES
SOUTH AFRICA
SPECIAL PATHOGENS UNITNATIONAL INSTITUTE FOR COMMUNICABLE DISEASES
SOUTH AFRICA
SPECIAL PATHOGENS UNITNATIONAL INSTITUTE FOR COMMUNICABLE DISEASES
SOUTH AFRICA
PHASES OF RVF DETECTION:PHASES OF RVF DETECTION:
PREDICTIONREMOTE SENSING – RAINFALL ESTIMATES
NDVI
PREDICTIONREMOTE SENSING – RAINFALL ESTIMATES
NDVI- NDVI- ENSO PHENOMENA
SURVEILLANCE
- NDVI- ENSO PHENOMENA
SURVEILLANCEVETERINARY & MEDICAL ALERTNESSSENTINEL HERDS/FLOCKSVECTOR SURVEILLANCE
VETERINARY & MEDICAL ALERTNESSSENTINEL HERDS/FLOCKSVECTOR SURVEILLANCE
RECOGNITIONVETERINARY DISEASE
RECOGNITIONVETERINARY DISEASEHUMAN DISEASE(VECTORS)HUMAN DISEASE(VECTORS)
CONFIRMATIONLABORATORY METHODS
CONFIRMATIONLABORATORY METHODS
RECOGNITION OF RVF OUTBREAKS
E E E E E • SUDDEN OUTBREAK OF DISEASE INVOLVING DEATHS OF YOUNG RUMINANTS (ESPECIALLY LAMBS & CALVES) AND ABORTION IN PREGNANT ADULTS FOLLOWING THE OCCURRENCE OF HEAVY RAINS
• ACCOMPANIED BY REPORTS OF FEBRILE DISEASE IN HUMANS –OFTEN WITH SOME DEATHS
• OUTBREAKS OFTEN OCCUR IN AREAS WITH POOR ROADS AND SERVICES – ABORTIONS IN LIVESTOCK AT 5-15% LEVEL NOT REPORTED
• IN CONTRAST THE OCCURRENCE OF HUMAN DISEASE WITH FATALITIES IS USUALLY REPORTED BY MEDICAL SERVICES/NGO’s
CLINICAL SIGNS IN LIVESTOCKCLINICAL SIGNS IN LIVESTOCKCLINICAL SIGNS IN LIVESTOCKCLINICAL SIGNS IN LIVESTOCK
• YOUNG ANIMALS:• SUDDEN ONSET OF HIGH FEVER• ACUTE PROSTRATION, COLLAPSE & DEATH
• YOUNG ANIMALS:• SUDDEN ONSET OF HIGH FEVER• ACUTE PROSTRATION, COLLAPSE & DEATH,
• ADULTS:• ABORTIONS THE MOST IMPORTANT SIGN
,
• ADULTS:• ABORTIONS THE MOST IMPORTANT SIGN• ABORTIONS THE MOST IMPORTANT SIGN• DYSTOCIA, SOME TERATOLOGY, HYDROPS AMNII• ANOREXIA, DYSGALACTIA, NASAL AND LACHRYMAL
DISCHARGES
• ABORTIONS THE MOST IMPORTANT SIGN• DYSTOCIA, SOME TERATOLOGY, HYDROPS AMNII• ANOREXIA, DYSGALACTIA, NASAL AND LACHRYMAL
DISCHARGESDISCHARGES• SALIVATION, ‘VOMITING’, LYMPHADENITIS• COLIC, JAUNDICE, HAEMORRHAGIC ENTERITIS
DISCHARGES• SALIVATION, ‘VOMITING’, LYMPHADENITIS• COLIC, JAUNDICE, HAEMORRHAGIC ENTERITIS
MORBIDITY/MORTALITY IN MORBIDITY/MORTALITY IN LIVESTOCK
• SHEEP MOST SUSCEPTIBLE CATTLE GOATS
LIVESTOCK• SHEEP MOST SUSCEPTIBLE CATTLE GOATS • SHEEP MOST SUSCEPTIBLE CATTLE GOATS
CAMELS LEAST SUSCEPTIBLE (ABORTION ONLY)
CLINICAL DISEASE ESPECIALLY IN EXOTIC BREEDS
• SHEEP MOST SUSCEPTIBLE CATTLE GOATS CAMELS LEAST SUSCEPTIBLE (ABORTION ONLY)
CLINICAL DISEASE ESPECIALLY IN EXOTIC BREEDS• CLINICAL DISEASE ESPECIALLY IN EXOTIC BREEDS
• INDIGENOUS ANIMALS GENERALLY LESS U CEPTIBLE EXCEPT IN ARID ZONE
• CLINICAL DISEASE ESPECIALLY IN EXOTIC BREEDS
• INDIGENOUS ANIMALS GENERALLY LESS U CEPTIBLE EXCEPT IN ARID ZONESUSCEPTIBLE – EXCEPT IN ARID ZONES
• 20-90% MORBIDITY
SUSCEPTIBLE – EXCEPT IN ARID ZONES
• 20-90% MORBIDITY
• 40-60% MORTALITY IN YOUNG, 2-5% IN ADULTS• 40-60% MORTALITY IN YOUNG, 2-5% IN ADULTS
• PREGNANT ANIMALS ABORT• PREGNANT ANIMALS ABORT
RVF HUMAN DISEASERVF HUMAN DISEASE
INFECTED BY CONTACT WITH DISEASED ANIMAL TISSUES INFECTED BY CONTACT WITH DISEASED ANIMAL TISSUES ORMOSQUITO BITE – LESS COMMON IN SUB-SHAHARAN AFRICAWHERE VECTORS ARE SYLVATIC (DO NOT ENTER DWELLINGS)
ORMOSQUITO BITE – LESS COMMON IN SUB-SHAHARAN AFRICAWHERE VECTORS ARE SYLVATIC (DO NOT ENTER DWELLINGS)
INCUBATION PERIOD <1 WEEK
=80% INFECTIONS SUBCLINICAL OR MILD
INCUBATION PERIOD <1 WEEK
=80% INFECTIONS SUBCLINICAL OR MILD
<0.5% FATAL HEMORRHAGIC FEVER/ENCEPHALITIS
APPROXIMATELY 5% OCULAR SEQUELAE
<0.5% FATAL HEMORRHAGIC FEVER/ENCEPHALITIS
APPROXIMATELY 5% OCULAR SEQUELAEQQ
LABORATORY CONFIRMATIONOF CURRENT RVF INFECTIONLABORATORY CONFIRMATIONOF CURRENT RVF INFECTIONOF CURRENT RVF INFECTION
• ANATOMICAL PATHOLOGY
OF CURRENT RVF INFECTION• ANATOMICAL PATHOLOGY
• ANTIGEN DETECTION (AGID, ELISA, IF)
• ANTIGEN DETECTION (AGID, ELISA, IF)
• RT-PCR DETECTION OF VIRAL RNA
• VIRUS ISOLATION (MOUSE
• RT-PCR DETECTION OF VIRAL RNA
• VIRUS ISOLATION (MOUSE VIRUS ISOLATION (MOUSE INOCULATION, TC)
• ANTIBODY TESTS (HAI, NEUT, ELISA IgM, IgG)
VIRUS ISOLATION (MOUSE INOCULATION, TC)
• ANTIBODY TESTS (HAI, NEUT, ELISA IgM, IgG)ELISA IgM, IgG)
• HISTOPATHOLOGY, IMMUNOHISTOCHEMISTRY
ELISA IgM, IgG)
• HISTOPATHOLOGY, IMMUNOHISTOCHEMISTRY
VIROLOGICAL DIAGNOSIS OF RVFVIROLOGICAL DIAGNOSIS OF RVF
ANTIGEN DETECTIONANTIGEN DETECTIONANTIGEN DETECTIONRecombinant NP sandwich ELISAANTIGEN DETECTIONRecombinant NP sandwich ELISA
NUCLEIC ACID DETECTIONRT-PCR NUCLEIC ACID DETECTIONRT-PCR Taqman Real-Time PCRLAMP RT-PCRTaqman Real-Time PCRLAMP RT-PCR
VIRUS ISOLATIONMiceVIRUS ISOLATIONMiceMiceCell culturesMiceCell cultures
ELISA DETECTION OF RVF ANTIGEN (NUCLEOCAPSID PROTEIN)
ELISA DETECTION OF RVF ANTIGEN (NUCLEOCAPSID PROTEIN)
ELISA DETECTION OF RVF ANTIGEN (NUCLEOCAPSID PROTEIN)
120
140
120
140
120
140
120
140
80
100
valu
e
103.6
87.1 88.0
102.9
82.180
100
valu
e
103.6
87.1 88.0
102.9
82.180
100
valu
e
103.6
87.1 88.0
102.9
82.180
100
valu
e
103.6
87.1 88.0
102.9
82.1
60
80
ELIS
A PP
v
60
80
ELIS
A PP
v
60
80
ELIS
A PP
v
60
80
ELIS
A PP
v
20
40
E
28.5
0 5 0 6 1 2
20
40
E
28.5
0 5 0 6 1 2
20
40
E
28.5
0 5 0 6 1 2
20
40
E
28.5
0 5 0 6 1 2
Liver Kidney Heart Liver Kidney Brain Liver Kidney Brain0
0.5 0.6 1.2
RVF-infected buffalo fetus Infected mouse Non-infected mouse
Liver Kidney Heart Liver Kidney Brain Liver Kidney Brain0
0.5 0.6 1.2
RVF-infected buffalo fetus Infected mouse Non-infected mouse
Liver Kidney Heart Liver Kidney Brain Liver Kidney Brain0
0.5 0.6 1.2
Liver Kidney Heart Liver Kidney Brain Liver Kidney Brain0
0.5 0.6 1.2
RVF-infected buffalo fetus Infected mouse Non-infected mouseRVF infected buffalo fetus Infected mouse Non infected mouseRVF infected buffalo fetus Infected mouse Non infected mouseRVF infected buffalo fetus Infected mouse Non infected mouse
ELISA DETECTION OF ANTIGENEMIAELISA DETECTION OF ANTIGENEMIA
7
8
300
350
7
8
300
350
7
8
7
8
300
350
5
6
50/m
l
200
250
valu
e5
6
50/m
l
200
250
valu
e5
6
5
6
50/m
l
200
250
valu
e
3
4
Log1
0 TC
ID
150
ELIS
A P
P v
3
4
Log1
0 TC
ID
150
ELIS
A P
P v
3
4
Log1
0
3
4
Log1
0 TC
ID
150
ELIS
A P
P v
1
2
50
100
1
2
50
100
1
2
1
2
50
100
01 2 3 4 5 6 7 8 9 10
Days post inoculation
001 2 3 4 5 6 7 8 9 10
Days post inoculation
001 2 3 4 5 6 7 8 9 10
Days post inoculation
01 2 3 4 5 6 7 8 9 10
Days post inoculation
0
LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP)
AMPLIFIES TARGET NUCLEIC ACID UNDER ISOTHERMAL CONDITIONS
LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP)
AMPLIFIES TARGET NUCLEIC ACID UNDER ISOTHERMAL CONDITIONS AMPLIFIES TARGET NUCLEIC ACID UNDER ISOTHERMAL CONDITIONS (60 - 65ºC) USING SIMPLE EQUIPMENT: HEATING BLOCK OR WATER BATH (NOT THERMPCYCLER)
B E G P E E E B
AMPLIFIES TARGET NUCLEIC ACID UNDER ISOTHERMAL CONDITIONS (60 - 65ºC) USING SIMPLE EQUIPMENT: HEATING BLOCK OR WATER BATH (NOT THERMPCYCLER)
B E G P E E E B BASED ON AUTOCYCLING STRAND DISPLACEMENT DNA SYNTHESIS BY Bst DNA POLYMERASES AND 4-6 PRIMERSBASED ON AUTOCYCLING STRAND DISPLACEMENT DNA SYNTHESIS BY Bst DNA POLYMERASES AND 4-6 PRIMERS
Courtesy of Prof Morita, University of Nagasaki, JapanCourtesy of Prof Morita, University of Nagasaki, Japan
LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (L MP)
LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (L MP)(LAMP)
P b d d lProducts visible by naked eye fluorescence
(LAMP)
P b d d lProducts visible by naked eye fluorescence LAMP turbidimeter connected to laptop
for real-time monitoring
1 2 3 4 5 6 7 8 9
Products visible by naked eye, fluorescence,agarose gel electrophoresis, or turbidity
LAMP turbidimeter connected to laptop
for real-time monitoring
1 2 3 4 5 6 7 8 91 2 3 4 5 6 7 8 9
Products visible by naked eye, fluorescence,agarose gel electrophoresis, or turbidity
1 2 3 4 5 6 7 8 91 2 3 4 5 6 7 8 91 2 3 4 5 6 7 8 9
Courtesy of Prof Morita, University of Nagasaki, JapanCourtesy of Prof Morita, University of Nagasaki, JapanCourtesy of Prof Morita, University of Nagasaki, Japan
RT-LAMP ASSAY & Taqman RTD-PCR EQUALLY SENSITIVE:DETECTION LIMIT 0.065 TCID50/REACTION VOLUME
RT-LAMP ASSAY & Taqman RTD-PCR EQUALLY SENSITIVE:DETECTION LIMIT 0.065 TCID50/REACTION VOLUME
0.5
0.6
0.5
0.6
0.5
0.6
0.1
0.2
0.3
0.4
Turb
idity
(400
nm)
RT-LAMP0.1
0.2
0.3
0.4
Turb
idity
(400
nm)
0.1
0.2
0.3
0.4
Turb
idity
(400
nm)
RT-LAMP 00 10 20 30 40 50 60
Time (min)Virus concentration - TCID50 per reaction volume
65000 6500 65065 6.5 0.65
RT LAMP 00 10 20 30 40 50 60
Time (min)Virus concentration - TCID50 per reaction volume
65000 6500 65065 6.5 0.65
00 10 20 30 40 50 60
Time (min)Virus concentration - TCID50 per reaction volume
65000 6500 65065 6.5 0.65
RT LAMP
1 5
2
2.5
3
nce
0)
0.065 0.0065 Negative control
1 5
2
2.5
3
nce
0)
0.065 0.0065 Negative control0.065 0.0065 Negative control
-0.5
0
0.5
1
1.5
1 5 9 13 17 21 25 29 33 37 41 45
Cycles
Fluo
resc
en(5
30/6
4
TaqmanRT-PCR
-0.5
0
0.5
1
1.5
1 5 9 13 17 21 25 29 33 37 41 45
Cycles
Fluo
resc
en(5
30/6
4
TaqmanRT-PCR
CyclesVirus concentration - TCID50 per reaction volume
65 000 6 500 65065 6.5 0.650.065 Negative control
CyclesVirus concentration - TCID50 per reaction volume
65 000 6 500 65065 6.5 0.650.065 Negative control
COMPARISON OF RT-LAMP, Taqman-PCR & VIRUS ISOLATIONFOR DETECTION OF RVFV IN CLINICAL SPECIMENSFOR DETECTION OF RVFV IN CLINICAL SPECIMENS
RT-LAMP -RTD-PCR -Isolation +
RT-LAMP +RTD-PCR +Isolation -
RT-LAMP -RTD-PCR -Isolation -
RT-LAMP +RTD-PCR +Isolation +
ResultsResultsResultsResultsNumber testedSourceSpecimenRT-LAMP -RTD-PCR -Isolation +
RT-LAMP +RTD-PCR +Isolation -
RT-LAMP -RTD-PCR -Isolation -
RT-LAMP +RTD-PCR +Isolation +
ResultsResultsResultsResultsNumber testedSourceSpecimen
11323165HumanSerum00066SheepPlasma00101020SheepSerumIsolation +Isolation -Isolation -Isolation +
11323165HumanSerum00066SheepPlasma00101020SheepSerumIsolation +Isolation -Isolation -Isolation +
11425397Total00033BuffaloKidney00033BuffaloLiver
11425397Total00033BuffaloKidney00033BuffaloLiver
ELISA ANTIBODY TESTSELISA ANTIBODY TESTS
1. INACTIVATED MOUSE LIVER ANTIGEN, ANTI-SPP CONJUGATES:
HUMAN
1. INACTIVATED MOUSE LIVER ANTIGEN, ANTI-SPP CONJUGATES:
HUMANSandwich ELISA: anti-RVFV IgG in humansCapture ELISA: anti-RVFV IgM in humans
LIVESTOCK
Sandwich ELISA: anti-RVFV IgG in humansCapture ELISA: anti-RVFV IgM in humans
LIVESTOCKLIVESTOCKSandwich ELISA: anti-RVFV IgG in cattleSandwich ELISA: anti-RVFV IgG in sheep and goatsCapture ELISA: anti-RVFV IgM in sheep, goats and cattle
LIVESTOCKSandwich ELISA: anti-RVFV IgG in cattleSandwich ELISA: anti-RVFV IgG in sheep and goatsCapture ELISA: anti-RVFV IgM in sheep, goats and cattle
2. RECOMBINANT ANTIGEN, ANTI-PROTEIN G CONJUGATE:Recomb NP indirect ELISA: anti-RVFV IgG in sheep, goats and cattleRecomb NP indirect ELISA: anti-RVFV IgG in wild ruminants
2. RECOMBINANT ANTIGEN, ANTI-PROTEIN G CONJUGATE:Recomb NP indirect ELISA: anti-RVFV IgG in sheep, goats and cattleRecomb NP indirect ELISA: anti-RVFV IgG in wild ruminantsRecomb NP indirect ELISA: anti RVFV IgG in wild ruminantsRecomb NP indirect ELISA: anti-RVFV IgG antibody in humans
3. CELL-LYSATE ANTIGEN, DETECTION: RABBIT ANTI-RVF & CONJUGATE:I hibiti ELISA ti RVFV i h d ti & ild i l
Recomb NP indirect ELISA: anti RVFV IgG in wild ruminantsRecomb NP indirect ELISA: anti-RVFV IgG antibody in humans
3. CELL-LYSATE ANTIGEN, DETECTION: RABBIT ANTI-RVF & CONJUGATE:I hibiti ELISA ti RVFV i h d ti & ild i lInhibition ELISA: anti-RVFV in humans, domestic & wild animalsInhibition ELISA: anti-RVFV in humans, domestic & wild animals
IgG Experimental sheep
140
IgM Experimental sheep300
IgG Experimental sheep
140
IgM Experimental sheep300
60
80
100
120
SA P
P va
lue
150
200
250
A P
P va
lue
60
80
100
120
SA P
P va
lue
150
200
250
A P
P va
lue
0
20
40
60
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 21 42
ELIS
0
50
100
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 21 28 42 49 58 65 72
ELIS
A
0
20
40
60
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 21 42
ELIS
0
50
100
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 21 28 42 49 58 65 72
ELIS
A
Days post infection0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 21 28 42 49 58 65 72
Days post infection
IgG Vaccinated sheep Smithb rn IgM Vaccinated sheep Smithburn
Days post infection0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 21 28 42 49 58 65 72
Days post infection
IgG Vaccinated sheep Smithb rn IgM Vaccinated sheep SmithburnIgG Vaccinated sheep- Smithburn
100
120
140
alue
IgM Vaccinated sheep- Smithburn
200
250
300
alue
IgG Vaccinated sheep- Smithburn
100
120
140
alue
IgM Vaccinated sheep- Smithburn
200
250
300
alue
20
40
60
80
ELIS
A P
P va
50
100
150
200EL
ISA
PP
va
20
40
60
80
ELIS
A P
P va
50
100
150
200EL
ISA
PP
va
00 1 2 3 4 5 6 7 8 9 10 11 12 13 14 20 28 34
Days post vaccination
00 1 2 3 4 5 6 7 8 9 10 11 12 13 14 20 28 34
Days post vaccination
00 1 2 3 4 5 6 7 8 9 10 11 12 13 14 20 28 34
Days post vaccination
00 1 2 3 4 5 6 7 8 9 10 11 12 13 14 20 28 34
Days post vaccination
OPTICAL FIBER IMMUNSENSOR (OFIS) = biosensor with detection by chemiluminescenceOPTICAL FIBER IMMUNSENSOR (OFIS) =
biosensor with detection by chemiluminescenceos nsor w th t ct on y ch m um n sc nc(Sorbazo et al 2007)
Human anti-RVF IgG - more sensitive and quicker than ELISA
os nsor w th t ct on y ch m um n sc nc(Sorbazo et al 2007)
Human anti-RVF IgG - more sensitive and quicker than ELISAHuman anti-RVF IgG - more sensitive and quicker than ELISAHuman anti-RVF IgG - more sensitive and quicker than ELISA
Ai d l t f ‘BIOPEN’ Ai d l t f ‘BIOPEN’ Aim: development of ‘BIOPEN’ for detection of viral antigens and antibodies
Aim: development of ‘BIOPEN’ for detection of viral antigens and antibodies
LESIONS IN LIVESTOCK (COETZER)LESIONS IN LIVESTOCK (COETZER)
LIVER – NECROTIC FOCIHAEMORRHAGE INTO ABOMASUM/
INTESTINES
LIVER – NECROTIC FOCIHAEMORRHAGE INTO ABOMASUM/
INTESTINES
PATHOGNOMONIC LIVER NECROSISWITH PRIMARY FOCI PATHOGNOMONIC LIVER NECROSISWITH PRIMARY FOCI
LIVER – INTRANUCLEAR INCLUSIONSLIVER – INTRANUCLEAR INCLUSIONS
PREVENTION/CONTROL EIN LIVESTOCK
• ROUTINE VACCINATION – ESPECIALLY EXOTIC BREEDS - WEANERS
• RAINFALL RSSD PREDICTIONS TO DRIVE COST-EFFECTIVE VACCINATION STRATEGIES???
• SMITHBURN MLVV – LIFELONG IMMUNITY BUT ONLY PARTIALLY ATTENUATED – SOME ABORTIONS –THEREFORE VACCINATE WEANERS – ANNUALLY
• INACTIVATED VACCINE EXPENSIVE AND REQUIRES 2 DOSES – PLUS BOOSTERS
• NEED FOR SAFE AND POTENT NEW VACCINES –NEED FOR SAFE AND POTENT NEW VACCINES HUMAN AND VETERINARY!
