Rafael Monleon, DVM, MSpVM, ACPV, PASBusiness Unit Manager (Poultry)

16th January 2014Taiwan

Prevention and Control of MSWithout Vaccination

Contents

• Mycoplasma Synoviae• Diagnosis & Monitoring• Control

– Cases

Mycoplasma Synoviae(MS)

1. Introduction 1) Most frequently occurs as a subclinical upper

respiratory infection 2) Air sac lesions combined with ND, IB 3) Systemic infection with synovitis and CRD in chickens

and turkeys 4) Not a virus and real bacteria,

pleomorphic coccoid, approximately 0.2μm, fried egg type of colonial morphology

2. History and Incidence 1) 1954 – Enlarged joint condition in poultry caused by an

infectious agent(Olson et al) 2) 1964 – A respiratory form of MS infection occurs(Olson

et al) 3) 1972 – Air sacculitis in

broilers from MS combined with ND, IB(Kleven et al)

4) World wide distribution

3. Characteristics 1) Generally weak pathogen of poultry 2) Strains vary in virulence 3) Economically damaging disease A. Layers: 5-10 eggs loss per year (MG: 10-20 eggs) B. Egg drop in lay C. Decreased hatchability and increased condemnations D. May cause mortality with respiratory disease,

peritonitis and synovitis

4) Chickens will remain infected after clinical signs have disappeared

5) Survival of MS(Room temperature) A. feather: 3 days B. nasal cavity: 12 hours C. other materials: less than 1 days 6) Eggshell Apex abnormality (EAA,

since 2000) (South Africa, Japan, Holland, etc)

4. Host 1) Chickens, turkeys, partridge, pheasants, peafowl, quail,

guinea fowl, ducks and pigeons

5. Transmission 1) Vertical and horizontal infection 2) Faster spread than MG 3) Egg transmission rate appears to be highest during first

4-6 weeks after infection.

Diagnosis• Serology

– Plate agglutination test– ELISA test– HI test

• Molecular Tools• Isolation• Monitoring

1) Plate agglutination test(or RSA) A. simple equipment B. fast procedure and detection of

IgM (7DPI) C. become more difficult to get

antigen D. Many false positives: after oil vaccination frozen serum DOC serum(dehydrated blood) just after eating up(gelatinized, lipoid

change) hemolysis

2) ELISA test A. Most common due to easy test and cheaper cost B. MS single or MG/MS combo kits is available C. Low ammount false positives sometimes D. Advantage to get massive test (3-5 plates per time) E. Mostly detect IgG / some IgM F. Biochek detects from 7 dpi and some previously

undetectable variants

Mycoplasma Proficiency Results

MS Field Isolate in 4 Wk Old SPF Chickens , 7 DPI*

2010 2011

RPA Ms** 33% 17%

Mg/Ms(r) ELISA 100% 100%

Other Mg/Ms ELISAs** 17% 44%

% positivesAssay

*From the International PTS for Mg and Ms Report; GD B.V. Animal Health Services Ltd. , Netherlands ** various manufacturers

Proficiency testing conducted by Animal Health Institute suggested early detection using this recombinant based Ms antigen in the Ms/Mg ELISA

Results(MS WVU1853 Inoculated)

0 7 9 14 160

102030405060708090

100

Mg/Ms(r) ELISAMg/Ms (c) ELISARPA (1&2)RPA (3)

Days Post-Challenge

% P

ositi

ve

Results(MS K5664 Inoculated)

0 7 9 14 160

102030405060708090

100

Mg/Ms(r) ELISAMg/Ms (c) ELISARPA (all 3)

Days Post-Challenge

% P

ositi

ve

3) HI test A. used to be the golden standard to confirm positive of

RSA or ELISA B. HI test set up is rare

Need live antigen (mycoplasma) C. more labs are using PCR for confirmation of

serological test

2. PCR 1) Become more common in many countries 2) Not perfect, but more accurate than serological test 3) Be careful for cross contamination 4) Re-check with serological test after 3-4 weeks for re-

confirmation 5) Tracheal, orbital, choanal swabbing 6) Keep samples into PBS and deliver

to lab on the sampling day with ice

7) Sequencing for epidemiology A. If there is frequent infection,

sequencing will be very helpful

e.g. K1858(US origin), EsPk1UAF08(Pakistan), B45/04(UK), 94011(US), VlhA2.28.1, WVU1853, etc.

3. Isolation 1) Difficult and skillful procedure to

perform 2) Only few Mycoplasma researchers

have the set up 3) Most of confirmation is finished at

PCR with serological background

Monitoring Program

1) Monitor every 3-4 weeks life of flock 2) Seroconversion in general is not well developed before

6-7 weeks oldHowever if infections occurred before 6-7 weeks, further monitoring is required

3) 3 weeks interval will be matched to incubation period of embryo, so preventing distribution of positive DOC to customers

4) Example (weeks old): 1,7,10,14,18,22,26,30 (4 weeks interval),33,36,39,42,45,48,51,54,57,60,63 (3 weeks interval), 19 times

5) At least 18-23 sera from each house, if take sera from only one house, you may miss first infection

6) Always thinking about Cost-Effective

Case of infection1. Case 1: faster seroconversion 1) ELISA result (BioChek, Cutoff = 668)

39weeks old 42 weeks old House 1 2 3 4 1 2 3 41 207 1 50 128 330 11,742 55 642 63 4 260 82 414 8,407 99 1533 75 252 182 439 38 8,903 261 74 44 29 85 168 178 8,127 394 385 332 214 118 32 69 13,931 245 2396 69 101 75 47 47 12,608 93 847 172 10 172 85 108 5,464 354 938 394 66 60 145 72 4,757 297 1419 420 368 853 182 1 3,056 105 8710 391 53 168 32 5 14,314 344 52711 175 1,312 41 168 20 13,782 404 12012 38 158 186 214 47 6,626 1 113 688 18 1 175 330 13,875 102 22614 23 47 23 72 108 2,349 15 12915 214 179 114 20 6,674 384Positive 1/14 1/15 1/15 0/15 0/15 15/15 0/14 0/15

PCR Negative PCR Positive

Phase 1: 12-21 days(1st Ab)Phase 2: 1-21days(5-10%)Phase 3: 7-32days(90-95%)Phase 4: 3-19days(100%)From 23 to 93days

2) Presumption of infection period

Phase 1: 12-21 days(1st Ab)Phase 2: 1-21days(5-10%)Phase 3: 7-32days(90-95%)Phase 4: 3-19days(100%)From 23 to 93daysFrom 11 to 72days after 1st Ab

Infection

Aug,28 Sep,4 Sep,11 Sep,18 Sep,25 Oct,4 Oct,11

1st Ab detection

100% Positive

Less than 21days

Between Aug,21 and Sep,1 ? PCR +ve, Oct,8

EsPk1UAF08

3) Epidemiology A. PCR –ve when 1st

antibody detected B. Investigate visitor book

and disinfection record C. Contact with poultry

people D. Wild bird E. Repair procedure F. Sequencing: EsPk1UAF08

strain

< Visitor book >

4) Route of infection A. Failure to find source of infection in many case B. assuming very high dust in house #2 may facilitate

MS infection

5) Treatment A. Enrofloxacin application to postpone spread to other

houses B. Change order of visit from farm to farm

2. Case 2: Slower seroconversion 1) ELISA result

22 weeks old 26 weeks old 30 weeks old 33 weeks oldHouse 1 2 3 1 2 3 1 2 3 1 2 31 94 10 10 6 2,713 10 5,947 784 551 481 2,315 3732 10 10 10 10 2,063 10 4,056 4,003 336 507 755 9693 53 10 74 12 930 10 633 1,618 518 4,331 809 3414 75 10 10 167 407 10 374 944 138 1,503 350 6725 10 741 10 9 1,604 10 273 755 41 1,731 7,061 4296 10 203 10 38 1,136 10 5,185 485 1,126 4,243 6,700 3187 12 259 10 10 6 10 981 1,049 711 274 2,012 9448 10 10 10 4 569 86 148 792 198 1,149 2,012 1,0199 71 10 10 610 497 10 303 4,356 268 1,597 5,559 30410 113 10 10 10 10 10 1,101 1,980 10 2,336 5,914 1,16611 10 45 10 10 2,890 10 205 956 2,517 993 5,789 1,43512 10 29 10 10 6,382 10 237 842 804 186 6,193 1,19713 10 10 10 10 1,107 3,153 84 534 1,513 1,167 12,842 74514 10 10 59 10 4,758 99 2,263 1,087 1,019 1,002 2,418 35415 10 10 10 48 493 40 1,265 142 1,990 1,807 2,864Positive 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%

2) Presumption of infection period

Phase 1: 12-21 days(1st Ab)Phase 2: 1-21days(5-10%)Phase 3: 7-32days(90-95%)Phase 4: 3-19days(100%)From 23 to 93daysFrom 11 to 72days after 1st Ab

Infection

Nov,7 Nov,17 Dec,8 Jan,5 Feb,2 Feb,23 Mar,24

1st Ab detection22wks old

92.8% Positive

33wks old77days

(11weeks)Between

Nov,7 and 17 ?PCR +ve,

B45/04

3) Epidemiology A. No relationship with previous strain (new strain) B. 12 visits from out of farm C. Less visitors and disinfection than laying farm

4) Route of infection A. Failure to find a source of infection

5) Treatment A. Enrofloxacin application to postpone spread to other

houses B. Change order of visit from farm to farm

Control• Biosecurity• Treatment

– Cases• Vaccination

1.- Biosecurity 1) Visit Authorization 2) Shower-in, shower-out 3) Fumigation 4) Vehicle disinfection unit 5) Feed transporting system 6) Wild bird control

Vehicle disinfection Hand sanitation Authorization

1) Authorization

Change shoes and hand disinfection

Spraying disinfectant mist and boots disinfection

Take off private clothes

Shower

Put on working clothes

Inside of farm

2) Shower-in, shower-out

All things from outside to inside of farm, must be disinfected with peracetic acid through procedure of spray of 20um particle-> disinfection -> exhausting for 30 minutes

3) Fumigation

4) Vehicle disinfection unit

5) Feed transporting system

6) Wild bird control A. Remove all trees inside of farm B. Remove all vegetation inside of farm C. Remove feed spilt

2.- Treatment 1) Generally antibiotics treatment is totally successful 2) But, we can find several success story with antibiotics A. Cost a lot B. Cage system in laying period C. Floor system in rearing period D. Floor system in laying period (Fiorentin et al, 2003)

Eradication in cage laying system

A. History - open-sided house, 4 lines of A type cage - 6 houses can hold 10,000 birds respectively - multi-age, artificial insemination

B. Multi-aged flocks - New flock is replaced every 4~6 month at the age of 12 to 14 weeks old. - Jun 2002 flock* => Dec 2002 => Mar 2003 => July 2003** => Mar 2004

=> Jun 2004 => Oct 2004

*Red colored flocks are MS positive ** Black colored flocks are MS negative

< House arrangement and flock held situation>

4th: Dec 2002 flock => Mar 2004 flock

3rd: Dec 2002 flock => Mar 2004 flock

2nd: Mar 2003 flock => Jun 2004 flock

1st: Mar 2003 flock => Jun 2004 flock

5th : Jun 2002 =>

July 2003 =>

Oct 2004 flo

ck

6th : Jun 2002 =>

July 2003 =>

O

ct 2004 flock

C. Treatment - Feed additives : 10mg/kg of Chlortetracycline - Drinking water : 10mg/kg Doxycycline 10mg/kg Enrofloxacin

7.5mg/kg Tilmicosin - Tilmicosin : every 6 weeks administration program - Others: between Tilmicosins

D. Results

WeekFlock

2003 2004

15 20 25 26 29 34 38 42 47 51 2 5 7 14 18 20 22 24 26 28 31

Jun, 2002 + - - -Dec, 2002 + - + + + - + + + - - + -

Mar, 2003 - - - + + + - - - - - - -Jul, 2003 + + - - - - - - - - - - - -Mar, 2004 - - - -

Mean ELISA titer of each flock

1

10

100

1000

10000

Jun-03

Jul-0

3

Aug-03

Sep-

03

Oct-03

Nov-03

Dec-03

Jan-04

Feb-04

Mar-04

Apr-04

May-04

Jun-04

Jul-0

4

Date

Tite

r

Jun, 2002 Dec, 2002Mar, 2003 July, 2003Mar, 2004

Positive cutoff 594

E. Summary

2003Antibiotics(for 3days, mg/kgB.W)

Flock

DateJun,2002 Dec,2002 Mar,2003 July,2003 Mar,2004

age PCR ELISA age PCR ELISA age PCR ELISA age PCR ELISA age PCR ELISA

15wks Til. 7.5 43 + 18 + 4/1420wks Til. 7.5 48 - 22 - 5/1922wks CTC 10 50 9/10 24 10/10 6/225wks CTC 10(14d) 53 - 27 + 13 - 6/23

26wksEnro. 10(5d)

54 - 10/10 28 + 10/10 14 - 0/10 6/30Doxy. 10(5d)

29wks 31 + 9/10 17 0/10 7/2134wks Depleted 36 - 10/10 22 - 0/10 8/2536wks Doxy. 10(5d) 9/838wks Enro. 7(5d) 40 + 10/10 26 + 1/10 9/2242wks Til. 7.5 44 + 10/10 30 + 3/10 15 + 10/2047wks Til. 7.5 49 + 10/10 35 + 7/10 20 + 0/10 11/2451wks 53 - 10/10 39 - 7/10 24 - 0/10 12/252004

1wks Til. 7.5 56 42 26 1/7

2wks Doxy. 10 57 - 10/10 43 - 9/10 27 - 0/10 1/145wks Enro. 10 60 + 46 - 30 - 2/47wks Til. 7.5 62 - 10/10 48 - 10/10 32 - 0/10 2/189wks 64 50 34 3/213wks Til. 7.5 Depleted 54 10/10 38 0/10 3/3114wks Doxy. 10 55 - 10/10 39 - 0/10 4/718wks Enro. 10 59 - 43 - 5/519wks Til. 7.5 60 44 5/1220wks 61 - 8/10 45 - 0/10 5/1922wks Depleted 47 - 6/224wks 49 - 0/10 14 - 0/10 6/1626wks 51 - 16 - 6/3028wks 53 - 18 - 7/1431wks 56 - 0/10 21 - 0/10 8/5

A. History - Transovarian infection is suspected (GP flock is seroconverting) - 3 houses of 30,000 birds - closed house

Eradication in floor systemCase #1

B. Treatment - Drinking water: 3 days/week, 5.0mg/kg BW - From 1 week to 20 weeks old

C. Result - Mother flock’s ELISA: 34.1% Positive

Seroconversion is in progress

Age(W) House 1 2 3 PCR

1 5/15(33%) -ve

5 0/15 0/15 0/15 -ve

7 0/15 0/15 1/15

10 0/15 0/15 0/15 -ve

14 0/15 0/15 0/15 -ve

18 0/15 0/15 0/15 -ve

22 0/15 0/15 0/15 -ve

D. Comments - 2 flocks successful in a row

- 3rd flock was failed(isolate same strain with mother flock)- 0.3USD/bird

Eradication on floor rearing Case #2

• Day 0 – MS positive• Tylosin @ 3d for 5 days• Tylosin @ 3 wks every 4 weeks• Results seems to have eliminated serological evidence

by 3 & 10 weeks

Eradication on floor rearing #2DOC

Eradication on floor rearing #23wks

Eradication on floor rearing #210wks

Serological ProfileFollowing Treatment

Serological ProfileFollowing Treatment

Serological ProfileFollowing Treatment

Control

3.- Vaccination 1) Killed and live vaccine are licensed A. Killed vaccine can’t protect infection 2) MSH Live vaccine is from

Bioproperties now registered in more and more countries

A. No vertical transmission evidence B. Not pathogenic for chickens C. Chemical mutagenesis treated

Contents• Biochek Software

– Field Interface– Multiuser-Cloud Database

• VDP• Collaborative Research Projects

Acknowledgements• Dr. Young Ho Hong• Ms. Wendy Wu

THANK YOUrafaelmonleon@biochek.com