A presentation covering basic aspects regarding the prevention and control of Mycoplasma sinoviae (a poultry pathogen) without the use of vaccination. Presented at the 2014 Biochek Seminar in Taiwan by Dr. Rafael Monleon Contact me in LinkedIn for any question: www.linkedin.com/rafaelmonleon

1. Rafael Monleon, DVM, MSpVM, ACPV, PAS
Business Unit Manager (Poultry)
16th January 2014
Taiwan
Prevention and Control of MS
Without Vaccination

2. Contents
• Mycoplasma Synoviae
• Diagnosis & Monitoring
• Control
– Cases

3. 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

4. 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

5. 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

6. 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)

7. 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.

8. Diagnosis
• Serology
– Plate agglutination test
– ELISA test
– HI test
• Molecular Tools
• Isolation
• Monitoring

9. 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

10. 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

11. 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%
% positives
Assay
*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

12. Results
(MS WVU1853 Inoculated)
0
20
40
60
80
100
0 7 9 14 16
%Positive
Days Post-Challenge
Mg/Ms(r) ELISA
Mg/Ms (c) ELISA
RPA (1&2)
RPA (3)

13. Results
(MS K5664 Inoculated)
0
20
40
60
80
100
0 7 9 14 16
%Positive
Days Post-Challenge
Mg/Ms(r) ELISA
Mg/Ms (c) ELISA
RPA (all 3)

14. 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

15. 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

16. 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.

17. 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

18. Monitoring Program
1) Monitor every 3-4 weeks life of flock
2) Seroconversion in general is not well developed before
6-7 weeks old
However 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

19. 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

20. Case of infection
1. Case 1: faster seroconversion
1) ELISA result (BioChek, Cutoff = 668)
39weeks old 42 weeks old
House 1 2 3 4 1 2 3 4
1 207 1 50 128 330 11,742 55 64
2 63 4 260 82 414 8,407 99 153
3 75 252 182 439 38 8,903 261 7
4 44 29 85 168 178 8,127 394 38
5 332 214 118 32 69 13,931 245 239
6 69 101 75 47 47 12,608 93 84
7 172 10 172 85 108 5,464 354 93
8 394 66 60 145 72 4,757 297 141
9 420 368 853 182 1 3,056 105 87
10 391 53 168 32 5 14,314 344 527
11 175 1,312 41 168 20 13,782 404 120
12 38 158 186 214 47 6,626 1 1
13 688 18 1 175 330 13,875 102 226
14 23 47 23 72 108 2,349 15 129
15 214 179 114 20 6,674 384
Positive 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

21. 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 93days
From 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

22. 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 >

23. 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

24. 2. Case 2: Slower seroconversion
1) ELISA result
22 weeks old 26 weeks old 30 weeks old 33 weeks old
House 1 2 3 1 2 3 1 2 3 1 2 3
1 94 10 10 6 2,713 10 5,947 784 551 481 2,315 373
2 10 10 10 10 2,063 10 4,056 4,003 336 507 755 969
3 53 10 74 12 930 10 633 1,618 518 4,331 809 341
4 75 10 10 167 407 10 374 944 138 1,503 350 672
5 10 741 10 9 1,604 10 273 755 41 1,731 7,061 429
6 10 203 10 38 1,136 10 5,185 485 1,126 4,243 6,700 318
7 12 259 10 10 6 10 981 1,049 711 274 2,012 944
8 10 10 10 4 569 86 148 792 198 1,149 2,012 1,019
9 71 10 10 610 497 10 303 4,356 268 1,597 5,559 304
10 113 10 10 10 10 10 1,101 1,980 10 2,336 5,914 1,166
11 10 45 10 10 2,890 10 205 956 2,517 993 5,789 1,435
12 10 29 10 10 6,382 10 237 842 804 186 6,193 1,197
13 10 10 10 10 1,107 3,153 84 534 1,513 1,167 12,842 745
14 10 10 59 10 4,758 99 2,263 1,087 1,019 1,002 2,418 354
15 10 10 10 48 493 40 1,265 142 1,990 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%

25. 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 93days
From 11 to 72days after 1st Ab
Infection
Nov,7 Nov,17 Dec,8 Jan,5 Feb,2 Feb,23 Mar,24
1st Ab
detection
22wks old
92.8%
Positive
33wks old
77days
(11weeks)
Between
Nov,7 and 17 ?
PCR +ve,
B45/04

26. 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

27. Control
• Biosecurity
• Treatment
– Cases
• Vaccination

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

29. Vehicle disinfection Hand sanitation Authorization
1) Authorization

30. 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

31. 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

32. 4) Vehicle disinfection unit

33. 5) Feed transporting system

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

35. 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)

36. 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

37. 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

38. 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

39. D. Results
Week
Flock
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-03
Aug-03
Sep-03
O
ct-03
Nov-03
Dec-03
Jan-04
Feb-04
M
ar-04
Apr-04
M
ay-04
Jun-04
Jul-04
Date
Titer
Jun, 2002 Dec, 2002
Mar, 2003 July, 2003
Mar, 2004
Positive
cutoff
594

40. E. Summary
2003
Antibiotics
(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 + 4/14
20wks Til. 7.5 48 - 22 - 5/19
22wks CTC 10 50 9/10 24 10/10 6/2
25wks CTC 10(14d) 53 - 27 + 13 - 6/23
26wks
Enro. 10(5d)
54 - 10/10 28 + 10/10 14 - 0/10 6/30
Doxy. 10(5d)
29wks 31 + 9/10 17 0/10 7/21
34wks Depleted 36 - 10/10 22 - 0/10 8/25
36wks Doxy. 10(5d) 9/8
38wks Enro. 7(5d) 40 + 10/10 26 + 1/10 9/22
42wks Til. 7.5 44 + 10/10 30 + 3/10 15 + 10/20
47wks Til. 7.5 49 + 10/10 35 + 7/10 20 + 0/10 11/24
51wks 53 - 10/10 39 - 7/10 24 - 0/10 12/25
2004
1wks Til. 7.5 56 42 26 1/7
2wks Doxy. 10 57 - 10/10 43 - 9/10 27 - 0/10 1/14
5wks Enro. 10 60 + 46 - 30 - 2/4
7wks Til. 7.5 62 - 10/10 48 - 10/10 32 - 0/10 2/18
9wks 64 50 34 3/2
13wks Til. 7.5 Depleted 54 10/10 38 0/10 3/31
14wks Doxy. 10 55 - 10/10 39 - 0/10 4/7
18wks Enro. 10 59 - 43 - 5/5
19wks Til. 7.5 60 44 5/12
20wks 61 - 8/10 45 - 0/10 5/19
22wks Depleted 47 - 6/2
24wks 49 - 0/10 14 - 0/10 6/16
26wks 51 - 16 - 6/30
28wks 53 - 18 - 7/14
31wks 56 - 0/10 21 - 0/10 8/5

41. A. History
- Transovarian infection is suspected
(GP flock is seroconverting)
- 3 houses of 30,000 birds
- closed house
Eradication in floor system
Case #1

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

43. 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

44. D. Comments
- 2 flocks successful in a row
- 3rd flock was failed(isolate same strain with mother
flock)
- 0.3USD/bird

45. 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

46. Eradication on floor rearing #2
DOC

47. Eradication on floor rearing #2
3wks
National Chiayi University
Report: Histogram/Blockdiagram
Dr. Kuo Lab
ResultSample ID Well Raw OD S/P Ratio Titer Titer Group
Reason :
Housenumber :
smallCode :
Type :
21 Day(s)Age :
2013/10/30Samplingdate :
Company :
G.P.Customer-­Name :
2013/10/30 07:24:37Lab code :
Assay:
Bleedingdate:
Lotnumber:
Testdate:
0
2013/10/30
Ms
2013/10/30
10/0/0Neg/Sus/Pos:
10Mean Titer:
Min-­Max Titer:
GMT:
%CV:
50
1 -­ 138
23
88
No. Samples:
Target Titer:
Target %CV:
VI Index: 0
Target Range VI:
Interpretation VI:
RF10 (2910)Meantiter Ref.Controls
RF10 (1500-­4500)Titer Range Ref.Controls
Positive Cuttoff S/P: 0.5>=
0.584D01+
0.608C01+
0.125B01-­
0.104A01-­
01 F01 0.191 0.159 138 0 neg
02 G01 0.157 0.088 65 0 neg
03 H01 0.164 0.103 79 0 neg

48. Eradication on floor rearing #2
10wks

49. Serological Profile
Following Treatment

50. Serological Profile
Following Treatment

51. Serological Profile
Following Treatment

52. 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

53. Contents
• Biochek Software
– Field Interface
– Multiuser-Cloud Database
• VDP
• Collaborative Research Projects

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

55. THANK YOU
rafaelmonleon@biochek.com