Monitoring for Mycoplasma

Transcription

1 Monitoring for Mycoplasma in vaccinated and non-vaccinated Poultry flocks Dr. Bart van Leerdam, PhD

2 Prevention and Control Tactics of Mycoplasma spp. in Poultry 1. Maintaining flocks free of Mycoplasma through monitoring, strict Biosecurity, and Eradication 2. Antibiotic therapy, short term strategy and costly 3. Vaccination, long term strategy

3 Maintaining flocks free of Mycoplasma Non-vaccinated flocks, Serological monitoring is an effective tool to prevent spread of disease through early detection and control of infection. Only possible when starting with Mycoplasma negative flocks. Best chance for success is maintaining flocks on single age farms (all in-all out) with no other poultry farms nearby. Good biosecurity and an effective monitoring system are necessary for success.

4 Maintaining flocks free of Mycoplasma Serological monitoring ( ELISA, RPA) is commonly used for confirmation of negative status. Confirmatory testing of screening test positives (ELISA, RPA) done with PCR test Screening test used for monitoring should be both highly sensitive for early detection and highly specific for confirmation.

5 Specificity of BioChek MG MS kits 900 sera from US broiler breeder flocks were tested. Samples were collected at 10, 30, and 48 W of age. All samples were were confirmed negative for MG and MS by HI prior to testing with BioChek's MG/MS, MG, and MS ELISA kits. Table. Summary of BioChek Results Age No. Pos. Plate (weeks) No. Samples MG/MS MS MG No. Positive No. Tested Specificity Customers running more than 100,000 tests annually on the combination assay, MgMs, have confirmed that specificity of the test is 98.9% or better.

6 Sensitivity of BioChek MG MS kits The BioChek Mycoplasma assays will detect antibodies 7 14 days post-infection. Experimental data BioChek MG MG infected SPF layers % Positives Days P.I. HI MG MG/MS D D D D Samples Originated from PDRC (Dr Stan Kleven) HI testing done by PDRC (Athens, GA, USA) Data BioChek MS Data 2010 international PTS for Mycoplasma, AHS, Deventer, Holland % Laboratories Showing Positives Assay MS Field MS Field 7 d P.I 28 d P.I. BioChek MS 100% 100% BioChek MG/MS 100% 100% RPA MS 33% 100%

7 Results (MS WVU1853 Inoculated) % Positive Mg/Ms(r) ELISA Mg/Ms (c) ELISA RPA (1&2) RPA (3) Days Post-Challenge

8 Results (MS K5664 Inoculated) 100 % Positive Mg/Ms(r) ELISA Mg/Ms (c) ELISA RPA (all 3) Days Post-Challenge

9 Case History MS Hatching eggs (Yolk testing) and serum of parental BB flocks were found positive on BioChek MG/MS and BioChek MS ELISA. MG ELISA Negative. Shown are below are some examples of positive results

10 Case History MS False positives MGMS and MS ELISA? Confirmatory testing with RPA and another ELISA was performed on parental serum (80 samples). Results show that flocks test negative for Both RPA and another commercial MS ELISA see below: BioChek ELISA Brand X ELISA RPA MS POS NEG NEG Customer now contacts BioChek complaining on quality of BioChek tests showing massive false positives for MG/MS and MS ELISA on both yolk and serum samples.

11 Case History MS False positives MGMS and MS ELISA? Serum Samples (10 samples/house) and tracheal swabs (8 samples /house) were send to BioChek from MS BioChek positive houses for further testing. Serum samples were tested on BioChek ELISA and tracheal swabs tested on BioChek MGMS qpcr. Results shown on next slide

12 Case History MS Only BioChek ELISA and qpcr detected MS infection. RPA and other commercial ELISA tested negative!

13 MG: Vertical transmission PS - Broilers Case: Broiler chicks with acute Pneumonia and tracheal lesions. Embryo s also show tracheal lesions. Mortality during first week 10-15%. Parent stock (Non-vaccinated) also show positive serology from 28 weeks and onwards. Broilers at slaughter age display respiratory stress with air-sacculitis. H2 Broilers at 02D, 07D, 41D H3 Parent Stock 67W BR 02D PS 67W BR 07D BR 41D

14 Case of MS infection BB Case 1: fast seroconversion ELISA result (BioChek, Cutoff = 668) Phase 1: days(1 st Ab) Phase 2: 1-21days(5-10%) Phase 3: 7-32days(90-95%) Phase 4: 3-19days(100%) From 23 to 93days 39weeks old 42 weeks old House , , , , , , , , , , , , , , , , Positive 1/14 1/15 1/15 0/15 0/15 15/15 0/14 0/15 PCR Negative PCR Positive

15 2) Presumption of infection period Phase 1: days(1 st Ab) Phase 2: 1-21days(5-10%) Phase 3: 7-32days(90-95%) Phase 4: 3-19days(100%) From 23 to 93days From 11 to 72days after 1 st Ab Between Aug,21 and Sep,1? Infection 1 st Ab detection Less than 21days 100% Positive PCR +ve, Oct,8 EsPk1UAF08 Aug,28 Sep,4 Sep,11 Sep,18 Sep,25 Oct,4 Oct,11

16 Case of MS infection BB Case 2: Slow seroconversion BioChek MS ELISA result 22 weeks old 26 weeks old 30 weeks old 33 weeks old House , , , , ,056 4, , , , , ,731 7, , , ,126 4,243 6, , , ,149 2,012 1, , ,597 5, ,101 1, ,336 5,914 1, , , ,789 1, , ,193 1, ,107 3, ,513 1,167 12, , ,263 1,087 1,019 1,002 2, , ,990 1,807-2,864 Positive 0/15 1/15 0/15 0/15 8/15 1/15 7/15 12/15 7/15 11/15 13/14 9/15 % 0% 6.7% 0% 0% 53.3% 6.7% 46.7% 80.0% 46.7% 73.3% 92.8% 60.0%

17 2) Presumption of infection period Phase 1: days(1 st Ab) Phase 2: 1-21days(5-10%) Phase 3: 7-32days(90-95%) Phase 4: 3-19days(100%) From 23 to 93days From 11 to 72days after 1 st Ab Between Nov,7 and 17? 1 st Ab detection 22wks old 77days (11weeks) 92.8% Positive 33wks old PCR +ve, B45/04 Infection Nov,7 Nov,17 Dec,8 Jan,5 Feb,2 Feb,23 Mar,24

18 Vaccination as a control tactic Vaccination against MG or MS an option in situations where maintaining flocks free of infection is not feasible An option when antibiotic treatment is too costly or ineffective due to acquired resistance and residue control. multi-age commercial Layer and Breeder farms.

19 Mycoplasma (MG, MS) Vaccines Live Vaccines. Inact. Vaccines (Bacterins).

20 Inactivated Bacterins Must be injected - can cause local reactions Do not protect well against tracheal colonization Relatively expensive Will not eliminate field strain from farms

21 Live Vaccines Protect vs. tracheal colonization as well egg drop and airsacculitis (CRD) Less expensive than inactivated bacterins May eliminate field strain from farm

22 Live Mycoplasma Vaccines Mg Vaccines: F strain (MSD) Mostly used in USA and Latin America & Asia TS/11 (Bioproperties/ Merial) 6/85 (MSD) Ms Vaccines: MS-H VaxSafe MS (Bioproperties/ Merial)

23 Vaccines for Mg Killed F 6/85 ts-11 Route s/c or i/m Various Spray Eye-drop Safety +++ ± Persistence Antibodies ± Spread ± Displacement

24 Vaccines for Ms Killed MS-H Route s/c or i/m Eye-drop Safety Persistence - ++ Antibodies Spread - + Displacement - +

25 Mycoplasma Vaccination Live Mycoplasma vaccines must be applied before a flock becomes infected with a field strain. Testing prior to vaccination will confirm negative status of the flock Do not administer live vaccines within 4 wks of the onset of lay or to laying birds. A minimum interval of 2 wks must be allowed between the use of live vaccines and other vaccines against diseases of the respiratory tract (ex. NDV, IB..).

26 Mycoplasma Vaccination Do not medicate birds with any antibacterial drugs, during the 5 days prior to or 2 wks following vaccination. Good management practices are recommended to reduce the exposure to MG for at least 3 weeks following vaccination

27 Baselines Live Mycoplasma vaccines Differentiation of Vaccination Serology vs. Field Challenge Serology based on evaluation mean flock titers with baselines and evaluating % positives. Flocks are suspect of infection when mean titers > baseline and 100% positive.

28 Mycoplasma Vaccination Still a need for biosecurity to keep challenge low. Lack of serological response does not mean birds are not protected. Live vaccines induce mostly mucosal immunity that cannot be measured by ELISA. There is a cumulative effect from vaccination on multiage farms The longer the vaccine is used then the greater the effect. Progressive Loss of serological (flat lining) response, could be a loss of field challenge due to displacement. Serological flatlining could be a sign of success rather than failure

29 Mycoplasma Vaccination High rising titers do not equal vaccination failure Vaccination failure is the occurrence of clinical signs not serological response High rising titers above baseline, without clinical symptoms, indicates field challenge and successful resistance

30 Mycoplasma Vaccination Diagnosis of apparent vaccine failures After C. Morrow, 2012

31 0 % POS BB vaccinated with 6/85 At 06W and 14w with atomist spray At 48W birds negative as expected 91 % POS At 56W serology positive with Mean titer 4932 and 91 % pos Serology indicates wild challenge, but birds resistent to colonization by wild strain as no clinical symptoms present. Suspect titers also indicate the need for enhanced biosecurity

32 Application of Mycoplasma ELISA Mycoplasma control program (negative population) Matrix: Serum: Parents & Offspring (01D) Eggs: Egg Yolk testing wk1,7,10,14,18,22,26,30 (4 weeks interval before production) wk 33,36,39,42,45,48,51,54,57,60,63 (3 weeks interval start production) Mycoplasma vaccination a) 1-2 wks pre-vaccination b) 6-12 wks post-vaccination SPF Farm c) BioChek breeder monitoring program wk 0, 10, 18-24, 40, 60 wk 0, 20 (before production) once every 4 wks start/after production Sampling size is 5% of the flock

33 Conclusions For non-vaccinated flocks, Mycoplasma monitoring is an effective tool to prevent spread of disease through early detection and control of infection. BioChek system can be used to monitor the success of live Mycoplasma vaccinations and to differentiate vaccinated flocks from those flocks undergoing a Mycoplasma challenge Suspect serology can only indicate the presence of a field challenge. Suspect serology with absence of clinical symptoms, might just indicate that the vaccinated birds were challenged, but displayed successful resistance to colonization with a field strain of Mycoplasma

34 Conclusions Infections with field strains can only be confirmed by differential culture / PCR techniques for wild type Mycoplasma. live vaccines may be viable tools for displacing and eradicating Mycoplasma field strains on multi-age poultry farms. The loss and exclusion of field challenge is indicated by a decrease in serological response to normal baselines, following prolonged use of live vaccines.

35 Acknowledgements The following persons have contributed to the presentation: Dr Chris Morrow Dr Herman Bosman Dr Peter Scott Dr Janet Bradbury