mycoplasma, hungaria 18.03.2003 h.r. froin diagnostic methods for avian mycoplasmas hendrik r. froin

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Diagnostic Methods for Diagnostic Methods for Avian MycoplasmasAvian Mycoplasmas

Hendrik R. FroinHendrik R. Froin

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Mycoplasma DiagnosisMycoplasma Diagnosis

Diagnosis is based on Diagnosis is based on

the flock as a whole, the flock as a whole,

not individual birdsnot individual birds

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Criteria for DiagnosisCriteria for Diagnosis

• Clinical signsClinical signs– Usually present in turkeysUsually present in turkeys– Inconsistent in chickensInconsistent in chickens

• Detection of antibodyDetection of antibody– Agglutination, HI, ELISAAgglutination, HI, ELISA

• Detection of the organismDetection of the organism– Culture, PCR, immunohistochemistryCulture, PCR, immunohistochemistry

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Clinical SignsClinical Signs

• Respiratory signsRespiratory signs

• SynovitisSynovitis

• Egg production dropsEgg production drops

• Increased mortality?Increased mortality?

• Respiratory or leg problems in progenyRespiratory or leg problems in progeny

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Purpose of SerologyPurpose of Serology

• Assist in diagnosis when signs are Assist in diagnosis when signs are presentpresent

• Screening of breeding flocks in control Screening of breeding flocks in control programsprograms– Serology should help to identify flocks Serology should help to identify flocks

which warrant a closer lookwhich warrant a closer look

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Utility of SerologyUtility of Serology

• Serology can be definitive if positive and Serology can be definitive if positive and associated with clinical signs or if a high associated with clinical signs or if a high % of the flock is positive with high HI or % of the flock is positive with high HI or ELISA titersELISA titers

• In most situations positive or suspicious In most situations positive or suspicious serology is confirmed by culture and/or serology is confirmed by culture and/or PCRPCR

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SensitivitySensitivity

Ability of a test to detect Ability of a test to detect

an infected animal or flock an infected animal or flock

(should not give false negative results)(should not give false negative results)

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SpecificitySpecificity

Ability of a test to detect Ability of a test to detect

birds or flocks which are negative birds or flocks which are negative

(should not give false positive (should not give false positive results)results)

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Characteristics of AgglutinationCharacteristics of Agglutination

• Cheap and fastCheap and fast

• Detects primarily IgM (the first class of Detects primarily IgM (the first class of antibody produced)antibody produced)

• Sensitive (except in some cases of MS)Sensitive (except in some cases of MS)

• Lacks specificity (tends to give false Lacks specificity (tends to give false positive results)positive results)

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Characteristics of HICharacteristics of HI

• High degree of specificityHigh degree of specificity

• Low to medium sensitivityLow to medium sensitivity

• Detects primarily IgGDetects primarily IgG

• Reacts about 1-2 weeks later than Reacts about 1-2 weeks later than agglutinationagglutination

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Characteristics of ELISACharacteristics of ELISA

• Detects primarily IgG, but also IgMDetects primarily IgG, but also IgM– ProFLOKProFLOK®® Elisa’s are detecting light and Elisa’s are detecting light and

heavy chain !heavy chain !

• Highly sensitiveHighly sensitive

• Specificity of newer kits is quite goodSpecificity of newer kits is quite good– Expect about 5% of samples in suspect or low Expect about 5% of samples in suspect or low

positive category in young breederspositive category in young breeders– These can be confirmed as negative by HIThese can be confirmed as negative by HI

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Reasons for Difficulties in Reasons for Difficulties in DiagnosisDiagnosis

• Testing early after exposureTesting early after exposure– No antibodies No antibodies

• MedicationMedication– Negative in culture, PCR…Negative in culture, PCR…

• Mild strains of MG or MSMild strains of MG or MS– Weak immune responseWeak immune response

• Strains which differ antigenicallyStrains which differ antigenically

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SYNBIOTICS ProFLOKSYNBIOTICS ProFLOK®® Mycoplasma Elisa’sMycoplasma Elisa’s

• Chicken MS and MGChicken MS and MG• Turkey MS, MG and MMTurkey MS, MG and MM• USDA licensed USDA licensed • Reliable, consistent resultsReliable, consistent results• Optimized to specifically work with chicken or Optimized to specifically work with chicken or turkey samples ( different cojugate)turkey samples ( different cojugate)• 18 month shelf-life18 month shelf-life• Detects positive, negative and suspect titer levelsDetects positive, negative and suspect titer levels• High volume, easy-to-use formatHigh volume, easy-to-use format

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ProFLOKProFLOK®® SpecificitySpecificity

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 NC3. Synbiotics Mg ELISA SP threshold ranges:Negative (-) < 0.199Suspect (su) = 0.200 -0.599Positive (+) > 0.6004. Log10 titer = (1.464 x Log10 SP) + 3.197

Average SP1,2

Ratio Values SP 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

Synbiotics Mg ELISA

Reference Serum

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ProFLOKProFLOK®® Sensitivity Sensitivity

1. Values are the arithmetic mean of 6 replicate samples 3. Synbiotics Mg ELISA SP threshold ranges:Negative (-) < 0.199Suspect (su) = 0.200 -0.599Positive (+) > 0.6004. Log10 titer = (1.464 x Log10 SP) + 3.197

Dilution

Log 2 Ratio SP Range 1,2

Titer Value 1,4 Lab A3 Lab B1

-1 1:2 + 9326 186.7 146.7

-2 1:4 + 7881 66.7 80.0

-3 1:8 + 5062 66.7 60.0

-4 1:16 + 3132 53.3 40.0

-5 1:32 + 1574 26,7 33.3

-6 1:64 su 674 20.0 20.0

-7 1:128 su 297 13.3 13.3

1.000

0.560

0.320

3.371

3.005

2.218

1.600

Synbiotics Mg ELISA Mg HI Titer ValuesSP Values1

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ProFLOKProFLOK®® Reproducibility Reproducibility

PlateAverage Optical

DensityA CV (%)Avg. Plate CV

(%)B

1 .642 ± .007 7,49 5,88

2 .687 ± .033 4,81

3 .575 ± .040 6,95

4 .605 ± .005 8,73

5 .590 ± .005 6,056 .686 ± .005 4,44

7 .703 ± .006 5,28

8 .617 ± .005 4,75

9 .634 ± .005 4,68

10 .621 ± .006 5,68

AValues are the arithmetic mean ± SEM of one Mg positive chicken 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.

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Comparitive Study of Comparitive Study of Agglutination, HI and ElisaAgglutination, HI and Elisa

S. H. KlevenS. H. Kleven

Department of Avian MedicineDepartment of Avian Medicine

University of GeorgiaUniversity of Georgia

Data are from a lecture Data are from a lecture

given during the IPE congress in Atlanta, given during the IPE congress in Atlanta, USA in January 2003USA in January 2003

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An Easy Example:An Easy Example:

Birds were challenged by aerosol by MG, Birds were challenged by aerosol by MG, MS, or negative controlsMS, or negative controls

ELISA’s used:ELISA’s used:• ProFLOCKProFLOCK® Mg or MS Elisa® Mg or MS Elisa

– Using a suspect rangeUsing a suspect range

• Commercial Elisa’s (C –Kit)Commercial Elisa’s (C –Kit)– Only positive / negative !Only positive / negative !

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MS Serology / ControlsMS Serology / Controls

0

2

4

6

8

10

12

14

Plate HI Synbiotics C - Kit

PositiveSuspectNegative

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MG serology / ControlsMG serology / Controls

0

2

4

6

8

10

12

14



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MG Serology / MG InfectedMG Serology / MG Infected

0

2

4

6

8

10

12

14

16

18



Birds were challenged by aerosol by MGBirds were challenged by aerosol by MG

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MG Serology / MS InfectedMG Serology / MS Infected

0

2

4

6

8

10

12

14

Plate HI Synbiotics C-Kit


Birds were challenged by aerosol by MSBirds were challenged by aerosol by MS

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MS Serology / MS InfectedMS Serology / MS Infected

0

2

4

6

8

10

12

14

Plate HI Synbiotics C-Kit


Birds were challenged by aerosol by MSBirds were challenged by aerosol by MS

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A Difficult Example:A Difficult Example:

Birds were challenged by contact by MG, Birds were challenged by contact by MG, MS, or negative controlsMS, or negative controls

-> -> Spread of infection was Spread of infection was

slow and inconsistentslow and inconsistent

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MS Serology / ControlsMS Serology / Controls

0

5

10

15

20

25

Plate HI Synbiogics C- Kit


Control BirdsControl Birds

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MG Serology / ControlsMG Serology / Controls

0

5

10

15

20

25



Control Birds Control Birds

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MS Serology / MG InfectedMS Serology / MG Infected

0

2

4

6

8

10

12

14

16

18



Birds were challenged by contact by MGBirds were challenged by contact by MG

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MG Serology / MG InfectedMG Serology / MG Infected

0

2

4

6

8

10

12

14

16



Birds were challenged by contact by MGBirds were challenged by contact by MG

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MG Serology / MS InfectedMG Serology / MS Infected

0

2468

101214161820



Birds were challenged by contact by MSBirds were challenged by contact by MS

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MS Serology / MS InfectedMS Serology / MS Infected

0

2

4

6

8

10

12

14

16



Birds were challenged by contact by MSBirds were challenged by contact by MS

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ConclusionsConclusions

The The ProFLOKProFLOK®® MG and MS Elisa are MG and MS Elisa are showing:showing:

• Superior sensitivity ( No false negative !)Superior sensitivity ( No false negative !)

• Excellent specificityExcellent specificity

• Better interpretation due to suspect rangeBetter interpretation due to suspect range

• High reproducibilityHigh reproducibility

mycoplasma, hungaria 18.03.2003 h.r. froin diagnostic methods for avian mycoplasmas hendrik r. froin

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