Download Mycoplasma Diagnosis

Diagnostic Methods for
Avian Mycoplasmas
Mycoplasma , Hungaria
18.03.2003 H.R. Froin
Hendrik R. Froin
Mycoplasma Diagnosis
Mycoplasma , Hungaria
18.03.2003 H.R. Froin
Diagnosis is based on
the flock as a whole,
not individual birds
Criteria for Diagnosis
• Clinical signs
– Usually present in turkeys
– Inconsistent in chickens
• Detection of antibody
– Agglutination, HI, ELISA
Mycoplasma , Hungaria
18.03.2003 H.R. Froin
• Detection of the organism
– Culture, PCR, immunohistochemistry
Clinical Signs
Mycoplasma , Hungaria
18.03.2003 H.R. Froin
•
•
•
•
•
Respiratory signs
Synovitis
Egg production drops
Increased mortality?
Respiratory or leg problems in progeny
Purpose of Serology
• Assist in diagnosis when signs are
present
• Screening of breeding flocks in control
programs
Mycoplasma , Hungaria
18.03.2003 H.R. Froin
– Serology should help to identify flocks
which warrant a closer look
Utility of Serology
Mycoplasma , Hungaria
18.03.2003 H.R. Froin
• Serology can be definitive if positive and
associated with clinical signs or if a high
% of the flock is positive with high HI or
ELISA titers
• In most situations positive or suspicious
serology is confirmed by culture and/or
PCR
Sensitivity
Mycoplasma , Hungaria
18.03.2003 H.R. Froin
Ability of a test to detect
an infected animal or flock
(should not give false negative results)
Specificity
Mycoplasma , Hungaria
18.03.2003 H.R. Froin
Ability of a test to detect
birds or flocks which are negative
(should not give false positive results)
Characteristics of Agglutination
Mycoplasma , Hungaria
18.03.2003 H.R. Froin
• Cheap and fast
• Detects primarily IgM (the first class of
antibody produced)
• Sensitive (except in some cases of MS)
• Lacks specificity (tends to give false
positive results)
Characteristics of HI
Mycoplasma , Hungaria
18.03.2003 H.R. Froin
•
•
•
•
High degree of specificity
Low to medium sensitivity
Detects primarily IgG
Reacts about 1-2 weeks later than
agglutination
Characteristics of ELISA
• Detects primarily IgG, but also IgM
– ProFLOK® Elisa’s are detecting light and
heavy chain !
Mycoplasma , Hungaria
18.03.2003 H.R. Froin
• Highly sensitive
• Specificity of newer kits is quite good
– Expect about 5% of samples in suspect or low
positive category in young breeders
– These can be confirmed as negative by HI
Reasons for Difficulties in
Diagnosis
• Testing early after exposure
– No antibodies
• Medication
– Negative in culture, PCR…
Mycoplasma , Hungaria
18.03.2003 H.R. Froin
• Mild strains of MG or MS
– Weak immune response
• Strains which differ antigenically
Mycoplasma , Hungaria
18.03.2003 H.R. Froin
SYNBIOTICS ProFLOK®
Mycoplasma Elisa’s
• Chicken MS and MG
• Turkey MS, MG and MM
• USDA licensed
• Reliable, consistent results
• Optimized to specifically work with chicken or
turkey samples ( different cojugate)
• 18 month shelf-life
• Detects positive, negative and suspect titer levels
• High volume, easy-to-use format
ProFLOK® Specificity
Synbiotics Mg ELISA
Average S P1,2
Mycoplasma , Hungaria
18.03.2003 H.R. Froin
Reference S erum
Ratio Values S P Ranges3 Titer Values4
Infectious bursal disease (IBD)
0.011
-
0
Newcastle disease virus (NDV)
0.012
-
0
Avian reovirus (REO)
0.012
-
0
Infectious bronchitis virus (IBV)
0.006
-
0
Infectious laryngotracheitis (LT )
0.101
-
0
Avian encephalomyelitis (AE)
0.070
-
0
Pasteurella multocida (PM)
0.012
-
0
Staphylococcus aureus
0.055
-
0
Anti-tissue culture medium
0.017
-
0
Commercial inactivated vaccine
0.033
-
0
Mg negative control serum (NCS)
0.000
-
0
Mycoplasma meleagridis (Mm)
0.016
-
0
Mycoplasma synoviae : 40 HI (Ms)
0.000
-
0
Mycoplasma synoviae :160 HI (Ms)
0.059
-
0
Mycoplasma gallisepticum : 40 HI (Mg)
1.088
+
1781
Mycoplasma gallisepticum : 160 HI (Mg)
1.565
+
3032
1.
Values are the arithmetic mean of
15 replicate samples (3
replicates/5 ELISA plates)
2 SP Ratio =
OD Sample – Avg. OD NC
Avg. OD PC – Avg. OD NC
3.
Synbiotics Mg ELISA SP
threshold ranges:
Negative (-) < 0.199
Suspect (su) = 0.200 -0.599
Positive (+) > 0.600
4.
Log10 titer = (1.464 x Log10 SP)
+ 3.197
ProFLOK® Sensitivity
Mycoplasma , Hungaria
18.03.2003 H.R. Froin
Log 2
Dilution
Ratio
Mg HI Titer Values
Synbiotics Mg ELISA
SP Values 1
Titer Value 1,4
SP
Range 1,2
Lab A3
Lab B 1
-1
1:2
3.371
+
9326
186.7
146.7
-2
1:4
3.005
+
7881
66.7
80.0
-3
1:8
2.218
+
5062
66.7
60.0
-4
1:16
1.600
+
3132
53.3
40.0
-5
1:32
1.000
+
1574
26,7
33.3
-6
1:64
0.560
su
674
20.0
20.0
-7
1:128
0.320
su
297
13.3
13.3
1. Values are the arithmetic mean of 6 replicate samples
3. Synbiotics Mg ELISA SP threshold ranges:
Negative (-) < 0.199
Suspect (su) = 0.200 -0.599
Positive (+) > 0.600
4. Log10 titer = (1.464 x Log10 SP) + 3.197
ProFLOK® Reproducibility
Mycoplasma , Hungaria
18.03.2003 H.R. Froin
Plate
Average Optical
Density A
CV (%)
1
.642 ± .007
7,49
2
.687 ± .033
4,81
3
.575 ± .040
6,95
4
.605 ± .005
8,73
5
6
.590 ± .005
.686 ± .005
6,05
4,44
7
.703 ± .006
5,28
8
.617 ± .005
4,75
9
.634 ± .005
4,68
10
.621 ± .006
5,68
Avg. Plate CV AValues are the arithmetic mean ±
SEM of one Mg positive chicken
(%) B
5,88
serum assayed 84 times per Mg
ELISA plate.
BValue
is the mean % Coefficient of
Variation (CV%) of one Mg positive
chicken serum assayed 84 times
per ELISA plate on each of ten Mg
ELISA plates.
Comparitive Study of
Agglutination, HI and Elisa
Mycoplasma , Hungaria
18.03.2003 H.R. Froin
S. H. Kleven
Department of Avian Medicine
University of Georgia
Data are from a lecture
given during the IPE congress in Atlanta,
USA in January 2003
An Easy Example:
Birds were challenged by aerosol by MG,
MS, or negative controls
ELISA’s used:
• ProFLOCK® Mg or MS Elisa
Mycoplasma , Hungaria
18.03.2003 H.R. Froin
– Using a suspect range
• Commercial Elisa’s (C –Kit)
– Only positive / negative !
MS Serology / Controls
14
12
10
8
Positive
Suspect
Negative
6
4
2
Mycoplasma , Hungaria
18.03.2003 H.R. Froin
0
Plate
HI
Synbiotics
C - Kit
MG serology / Controls
14
12
10
8
Positive
Suspect
Negative
6
4
2
Mycoplasma , Hungaria
18.03.2003 H.R. Froin
0
Plate
HI
Synbiotics
C - Kit
MG Serology / MG Infected
18
16
14
12
Positive
Suspect
Negative
10
8
6
4
2
Mycoplasma , Hungaria
18.03.2003 H.R. Froin
0
Plate
HI
Synbiotics
C - Kit
Birds were challenged by aerosol by MG
MG Serology / MS Infected
14
12
10
8
Positive
Suspect
Negative
6
4
2
Mycoplasma , Hungaria
18.03.2003 H.R. Froin
0
Plate
HI
Synbiotics
C-Kit
Birds were challenged by aerosol by MS
MS Serology / MS Infected
14
12
10
8
Positive
Suspect
Negative
6
4
2
Mycoplasma , Hungaria
18.03.2003 H.R. Froin
0
Plate
HI
Synbiotics
C-Kit
Birds were challenged by aerosol by MS
A Difficult Example:
Mycoplasma , Hungaria
18.03.2003 H.R. Froin
Birds were challenged by contact by MG,
MS, or negative controls
-> Spread of infection was
slow and inconsistent
MS Serology / Controls
25
20
15
Positive
Suspect
Negative
10
5
Mycoplasma , Hungaria
18.03.2003 H.R. Froin
0
Plate
Control Birds
HI
Synbiogics
C- Kit
MG Serology / Controls
25
20
15
Positive
Suspect
Negative
10
5
Mycoplasma , Hungaria
18.03.2003 H.R. Froin
0
Plate
Control Birds
HI
Synbiotics
C - Kit
MS Serology / MG Infected
18
16
14
12
Positive
Suspect
Negative
10
8
6
4
2
Mycoplasma , Hungaria
18.03.2003 H.R. Froin
0
Plate
HI
Synbiotics
C - Kit
Birds were challenged by contact by MG
MG Serology / MG Infected
16
14
12
10
Positive
Suspect
Negative
8
6
4
2
Mycoplasma , Hungaria
18.03.2003 H.R. Froin
0
Plate
HI
Synbiotics
C - Kit
Birds were challenged by contact by MG
MG Serology / MS Infected
Mycoplasma , Hungaria
18.03.2003 H.R. Froin
20
18
16
14
12
10
8
6
4
2
0
Positive
Suspect
Negative
Plate
HI
Synbiotics
C - Kit
Birds were challenged by contact by MS
MS Serology / MS Infected
16
14
12
10
Positive
Suspect
Negative
8
6
4
2
Mycoplasma , Hungaria
18.03.2003 H.R. Froin
0
Plate
HI
Synbiotics
C - Kit
Birds were challenged by contact by MS
Conclusions
Mycoplasma , Hungaria
18.03.2003 H.R. Froin
The ProFLOK® MG and MS Elisa are
showing:
• Superior sensitivity ( No false negative !)
• Excellent specificity
• Better interpretation due to suspect range
• High reproducibility