Diagnostic approach for E. coli presentation... · Diagnostic approach for E. coli ... How they cause disease in pigs . E. coli . with fimbriae Epithelial cell Blood ... Surveillance

Diagnostic approach for E. coli infections in pigs and control of post-weaning diarrhœa with a live oral vaccine

By John Morris Fairbrother Professor [email protected]

2

E. coli bacterium showing virulence factors and surface antigens used for bacterial classification by virotype and serotype

Escherichia coli

Flagella (H)

Production of toxins

Capsule (K)

LPS (O)

Fimbriae (F)

Cell membrane

3

E. coli population Sub-sets of clones differentiated by their ability to cause disease

Clone

Clone with resistance gene

Pathogenic

Potentially pathogenic

Commensal

4

Sub-sets of pathogenic E. coli

STEC

EPEC

ETEC

ExPEC

5

Virulence factors of pathogenic E. coli in pigs

STEC

EPEC

ETEC

ExPEC

Eae

Aero CNF

P Tsh

LT STa STb

Stx1 Stx2

6

ETEC: How they cause disease in pigs

E. coli with fimbriae

Receptor

Epithelial cell

Toxin

1

2 Colonisation

of the jejunum and ileum

3 Water Electrolytes

4 Diarrhœa

Weight loss Death Ingestion

of ETEC

7

ETEC diarrhœa

Newborn piglet with clinical signs of E. coli diarrhœa

Fluid distension and congestion of the small intestine of an unweaned piglet

with E. coli diarrhœa

8

ETEC diarrhœa

Post weaning pig with diarrhœa submitted for post-mortem examination

9

Intestinal colonisation by ETEC

Layers of E. coli bacteria (arrows) adhering to the mucosa of the ileum in a piglet with ETEC diarrhœa

10

STEC: How they cause disease in pigs


Epithelial cell

Blood vessel

Toxin

Receptor

2 Colonisation

of the jejunum and ileum 3

Transport of toxin to circulation

4

Œdema Ataxia Death

1 Ingestion of STEC

11

Œdema disease

Eyelid œdema in a pig with œdema disease

12

Œdema disease

Degenerative angiopathy of a small artery with swelling of endothelial cells (arrow), œdema, and haemorrhage in the ileum of a weaned pig with œdema disease

13

EPEC: How they cause disease in pigs

Receptor

Eae adhesin

Adherent E. coli

Epithelial cell

1 Ingestion of EPEC

2 Colonisation of small and large

intestine

3

4

Intimate attachment of epithelial cells + effacement of

microvilli

Diarrhœa

14

Extensive multifocal bacterial colonisation of the surface epithelium by a thin layer of dark-stained

coccobacilli oriented in a palisade pattern (arrows), in the colon of a weaned pig with diarrhœa

Intestinal colonisation by EPEC

15

Aetiology Risk factors E. coli

pathotype Host Environment

Œdema disease STEC:F18

-Some pigs resistant to F18 due to lack of receptor

-Up to 50% pigs may be resistant to F4 due to lack of receptor

-Earlier weaning age

-Stress

- Loss of specific antibodies from milk

Rapidly growing pigs

- High protein diet - Transportation - Mixing of pigs

Post-weaning

diarrhœa

ETEC:F4, F18, ETEC:AIDA, EPEC, mixed E. coli pathotypes

-Diet changes

- Diet constituants - Low level of milk and other products of animal source

- Certain ingredients such as soy beans

- Presence of other infections, such as PRRSv or pseudorabies virus

Risk factors for development of Escherichia coli disease

16

Direct smear from the colon of a healthy post weaning pig -

heterogeneous Gram-positive and gram-negative population

Postweaning pig with E. coli diarrhœa - predominance of

Gram-negative rods

Intestinal microflora

drastically altered in pigs with diarrhœa

17

Elimination of commensals and predominance of pathogenic E. coli

Healthy pig Pig with E. coli diarrhoea

Intestinal mucosa in disease

E. coli commensal clones

E. coli pathogenic clones E. coli potentially pathogenic clones

Other bacterial species

18

Transformation of the E. coli profile

in the pig intestinal ecosystem on the onset of disease

1 2 3 4 5 6 7 9 8

Viru

lenc

e

1 2 3 4 5 6 7 9 8

Viru

lenc

e

Bacterial count (log)

Pig with sub-clinical infection Pig with E. coli diarrhoea

Commensal clones

Pathogenic clones Potentially pathogenic clones

Detection limit of diagnostic test

19 Antimicrobial resistance

on causative agents (Kirby Bauer)

Positive Negative 3 or more selected haemolytic

and/or lactose +ve colonies

Blood and/or selective (McConkey) Agar

Virotyping Multiplex PCR

Enrichment (overnight)

Multiplex PCR

Rapid evaluation of presence of E. coli pathotypes

Sample analysis Isolate analysis

Detection and identification of pathogenic E. coli Clinical sample

20

Clinical diagnosis

Added virulence factors tested in isolates from animals

ExPEC

ETEC

STEC

EPEC

Aero CNF

P Tsh

LT STa STb

Stx1 Stx2

Eae

F4 F5 F6

F41

F18

21

Cases (%)

associated with post-weaning diarrhœa in pigs according to age in Canada from 2008 to 2010

E. coli pathotypes

www.apzec.ca

22 Antimicrobial resistance

on causative agents (Kirby Bauer)

Positive Negative 3 or more selected haemolytic

and/or lactose +ve colonies

Blood and/or selective (McConkey) Agar

Virotyping, Multiplex PCR, and colony hybridisation

Enrichment (overnight)

Multiplex PCR

Rapid evaluation of presence of E. coli pathotypes

Sample analysis Isolate analysis

Detection and identification of pathogenic E. coli Clinical sample

22

23

Surveillance Added virulence factors tested by colony hybridisation in animals

Stx1 Stx2

F18

Eae

Tsh

Aero

LT STa STb

F4 F5 F6

F41

AIDA Paa

F17

CNF P

Afa

ExPEC

ETEC

STEC

EPEC

24

Prevalence of main E. coli pathotypes

Feedlot Nursery

Sow barns

% of cases

in pigs according to age in Canada from 2008 to 2010

24

25

Prevalence of E. coli pathotypes

Number (%) of positive cases in pigs of age: All 1-14 days 15-35 days

Colibacillosis – Causative agent

ETEC:F4 28 (30.1) 6 (15.8) 18 (56.3) ETEC:F5 8 (8.6) 4 (10.5) 3 (9.4) ETEC:F6 1 (1.1) 0 0

ETEC:F18 0 0 0 ETEC:F41 1 (1.1) 1 (2.6) 0 STEC:F18 1 (1.1) 0 1 (3.1)

Colibacillosis – Opportunistic agent

EPEC 10 (10.8) 1 (2.6) 5 (15.6) ETEC:AIDA 12 (12.9) 3 (7.9) 4 (12.5)

Colibacillosis – Possible agent

ETEC, others 8 (8.6) 1 (2.6) 3 (9.4) Negative 34 (36.6) 23 (60.5) 5 (15.6)

Total 93 38 32

in pig intestines or faeces of cases of diarrhœa at EcL in 2007

26






EPEC 10 (10.8) 1 (2.6) 5 (15.6) ETEC:AIDA 12 (12.9) 3 (7.9) 4 (12.5)



Total 93 38 32

Prevalence of E. coli pathotypes in pig intestines or faeces of cases of diarrhœa at EcL in 2007

27






EPEC 10 (10.8) 1 (2.6) 5 (15.6) ETEC:AIDA 12 (12.9) 3 (7.9) 4 (12.5)



Total 93 38 32


28






EPEC 10 (10.8) 1 (2.6) 5 (15.6) ETEC:AIDA 12 (12.9) 3 (7.9) 4 (12.5)



Total 93 38 32


29

Correlation of E. coli pathotypes

Number of cases

Examined by necropsy

With histopathological evidence of: Bacterial colonisation Submucosal

œdema stomach

Pathotype combination Intestine Colon

ETEC:F4 10 9 ETEC:F4 mixed 9 6 1

ETEC:F4+STEC:F18 1 1 ETEC:F5 5 2

ETEC:F6+EPEC 1 1 ETEC:F18 1 1 STEC:F18 4 2 4

STEC:F18+EPEC 1 1 EPEC 13 4 1

ETEC:AIDA 14 4 1 EPEC+ETEC:AIDA 2 2

ETEC, others 11 1 Negative 67 3 3

and histopathological findings for submitted pigs in 2007

29

30

Number of cases



œdema stomach








Correlation of E. coli pathotypes and histopathological findings for submitted pigs in 2007

30

31

Number of cases



œdema stomach








Correlation of E. coli pathotypes and histopathological findings for submitted pigs in 2007

31

32

Sample

Enrichment

Multiplex PCR APZEC pathotype • Virulence genes

Positive isolates colonies • Virotype • O serotype

• Antimicrobial resistance

APZEC strain collection

APZEC database

Data presentation • Geographical location • Age

• Disease • Animal species • Time

APZEC Approach

33

E. coli profile

Virulence genes

Antimicrobial resistance and

genes

O type, phylogenetic

type, pulse type

Demographic data

Animal species

Time of collection

Animal age

Geographical location

Diagnosis

Clinical signs

Histopatho-logical lesions

Global APZEC Project

34

www.apzec.ca

35

ETEC: F4 virotypes by year in pigs in Canada

36

ETEC: F4 virotypes by age in pigs in Canada

37

STEC: F18 virotypes by year in pigs in Canada

38

STEC: F18 virotypes by age in pigs in Canada

39

Trends in resistance to antimicrobials in pathogenic E. coli from diseased pigs in Canada with time from 2008 to 2010

40

Trends in resistance to antimicrobials in pathogenic E. coli from diseased pigs in Canada with time from 2008 to 2010

41

Resistance to antimicrobials in pathogenic E. coli in intestinal samples from diseased pigs in Quebec from 2008 to 2010 in relation to age

42

Multidrug resistance by patho-virotype in pigs in Canada

43

E. coli patho-virotypes in pigs by country

44

Sampling kit Shipped to you with detailed protocol

Sampling at the farm

Canadian F4-ETEC Diagnostic Survey Join national survey to evaluate the presence of F4-enterotoxigenic E. coli on Canadian swine farms Criteria: Farms with enteric cases (soft faeces, scours, anorexia, etc.), with sudden death, or facing suboptimal growth performance in the first 3 weeks post-weaning. Please refer to the protocol for more information.

Shipping box

Swabs

Ice pack FedEx waybill

Cooler

Rectal swabbing

Swab in the tube

Tube identification

Swabs stored on ice or refrigerated

2

1

45

Shipping of samples

Results Communicated confidentially to veterinarians via email within 7 days of sample reception

Detection of F4-ETEC at the EcL Laboratory

+

Confirmation of F4-ETEC by PCR Enrichment of swab Isolation of E. coli

Pre-paid shipment

3

4

5

Canadian F4-ETEC Diagnostic Survey

46

Strategies which result in: Reduced number of pathogenic E. coli

Increased resistance of animals to infection

- Increase weaning age - Warmth - Diet - Highly digestible - Milk-based protein - Restricted feed intake - Hygiene - Water additive - Organic acids - Feed supplements - Organic acids - ZnO - Spray-dried plasma - Probiotics

- Live oral non-toxigenic F4 and F18 E. coli vaccines

- Oral powdered egg yolk from F4 and

F18 immunised hens - Stx toxoid vaccine (œdema disease) - Selection of F4 and F18-resistant

animals

of enteric E. coli infections in post-weaning diarrhœa and œdema disease

Strategies for control

47

Strategy should specifically target pathogenic E. coli Maintain beneficial E. coli Minimal effect on protective normal intestinal flora

Safe Positive effect on animal health would be an added

value Environmentally friendly Economical Practical

Desirable properties of intervention strategies

to control E. coli post-weaning diarrhœa in pigs

48

Desirable outcome of control strategy E. coli colonise and persist in a complex intestinal ecosystem in healthy animals

Pathogenic E. coli Commensal E. coli Other bacterial commensals

49

Undesirable outcome of control strategy Pathogenic E. coli may build up and colonise the intestinal mucosa


50

Growth promoting in feed or oral therapy Not specific for pathogenic E. coli Development of antimicrobial resistance Upset of intestinal microflora and possible

overgrowth and increased faecal shedding of ETEC or STEC

Induce emergence of new pathogenic and possible zoonotic E. coli such as O104:H4

Antimicrobials

51

Effect on the intestinal ecosystem

After Before

Worst case scenario

Antimicrobials


52

Experimental design & sampling 80 pooled faecal samples (rectal and floor) were collected from pigs receiving a diet with or without 2% Clinoptilolite

Days after weaning

0 2 7 14 28 168 piglets in 6 pens

CTC PG CTC & PG 0 7 14 28

Antimicrobials used as growth promoter in diet:

53

Antimicrobial resistance profiles in E. coli isolates from pigs receiving or not clinoptilolite in the feed as detected by disk diffusion method (* P< 0.05)

Time Group Total No. of

isolates

No. of isolates by No. of antimicrobial classes in the

resistance pattern

No. of resistant Isolates by antimicrobial class and antimicrobial(%)

Aminoglycosides β-lactams Sulfonamides Phenicols Quinolones Tetracyclines

0 1-2 3-4 5-6 GEN KAN STR AMP AMC CRO FOX TIO FIS SXT CHL CIP NAL TET

Day 0 C- 10 1 4 5 0 3 4 5 5 2 0 0 0 6 6 0 0 1 8 C+ 10 2 3 4 1 0 1 4 4 1 0 0 0 6 6 2 0 0 6

Day 7 C- 10 0 1 5 4 2 4 9 9 7 0 0 0 9 8 4 1 1 10 C+ 10 0 0 4 6 2 2 10 10 9 4 4 4 10 10 6 0 1 10

Day 14 C- 10 0 0 7 3 3 0 8 10 7 0 0 0 7 7 6 0 0 10 C+ 10 0 0 5 5 3 3 10 10 10 3 3 3 10 9 4 0 1 10

Day 28 C- 10 0 1 5 4 1 1 7 9 8 5 5 5 7 7 6 0 0 10 C+ 10 0 2 5 3 1 3 9 10 8 4 4 4 9 9 4 0 0 10

Total 80 3(3.75) 11(13.75) 40(50) 26(32.5) 15(18.75) 18(22.5) 62(77.5) 67(83.7) 52(65) 16(20) 16(20) 16(20) 64(80) 62(77.5) 32(40) 1(1.25) 4(5) 74(92.5)

All isolates were susceptible for AMK All ceftiofur-cefoxitin-resistant E.coli isolates were co-resistant to AMC,

AMP, STR, SXT, FIS, CHL and TET

AMC: Amoxicillin/Clavulanic acid AMK: Amikacin AMP: Ampicillin CHL: Chloramphenicol CIP: Ciprofloxacin

C-: Control group C+: Treatment group

CRO: Ceftriaxone FIS: Sulfisoxazole FOX: Cefoxitin GEN: Gentamicin KAN: Kanamycin

NAL: Nalidixic acid STR: Streptomycin SXT: Trimethoprim-Sulphamethaoxazole TET: Tetracycline TIO:Ceftiofur

54

Trends in presence of blaCMY-2 gene in fecal samples from weaned pigs over time, as detected by PCR (* P< 0.05)

0

20

40

60

80

100

0 2 7 14 28

% o

f pos

itive

sam

ples

Days

Control Treatment

*

*

*

55

0,0

2,0

4,0

6,0

8,0

10,0

12,0

14,0

16,0

18,0

0 2 7 14 28

% o

f pos

itive

isol

ates

Days

blaCMY-2

Control

Treatment

* *

*

Trends in isolates positive for iucD, tsh or blaCMY_2 genes in fecal samples from pigs as detected by HGMF method (* P<0.05)

56

0 0 0

2

0 0 0

3

0

4

3 3

0

1

0

1

2

3

4

5

6

C- C+ C- C+ C- C+ C- C+

Day 0 Day 7 Day 14 Day 28

No.

of b

laC

MY-

2 po

sitiv

e is

olat

es

phylotype B1

phylotype A

40%

60%

75%

25%

100%

100%

Association between blaCMY-2-positive isolates and Phylogenetic grouping over time

C-: Control group C+: Treatment group

57

Certain strains of Enterococcus faecalis , Lactobacillus spp

Daily oral treatment starting before susceptible period Gradual onset of effect

Stimulate beneficial microorganisms Competitive exclusion Non-specific

Many products on the market Increased weight gain Variable efficacy

Probiotics

58


After Before

Probiotics


59

Feed supplementation with egg yolk antibodies from immunised chickens specific for F4 and F18

Specific inhibition of intestinal colonisation of ETEC

No effect on normal intestinal microflora

Rapid onset of action, lasts as long as in feed

Controlled efficacity studies show promise

Passive immunotherapy

60

Administration in the feed of powdered egg yolk from hens immunised with F4 or F18 results in blocking of bacterial adherence to epithelial cells of the intestinal mucosa due to the presence of specific anti-F4 or F18 antibodies

Passive immunisation of young pigs

61

with an injectable killed whole-cell bacterin or purified fimbrial subunit vaccine leads to protection of young pigs from ETEC infection

Maternal immunisation

62

After Before

Passive immunotherapy Effect on the intestinal ecosystem


63

Breed to increase prevalence of F4 and F18 resistance loci

Possible co-selection of unwanted traits due to linkage of resistance locus with these traits

Susceptibility is genetically dominant Resistant sows do not produce or transfer F4-

specific antibodies to colostrum or milk Additional F4 resistance loci need to be

identified Availability of techniques for large-scale

selection of resistant animals is lacking

Breeding for resistance

64


Receptor

Toxin

Selection of F4 and F18 resistant animals

After Before

Epithelial cell

Epithelial cell

65

Oral vaccination

Specific mucosal

immunity: Anti-F4 antibodies

Specific antibodies block

bacterial adherence

Vaccines

66

Immunisation results in production of antibodies Specific inhibition of intestinal colonisation of

ETEC F4 Live non-toxigenic F4-positive E. coli strain

No effect on normal intestinal microflora

One or more doses, gradual onset of effect, more long-lasting

Decreased faecal shedding in controlled and field studies

Vaccines

67


Vaccines

After Before


68

Specific Safe Positive effect on animal health Environmentally friendly Economical Practical

Strategies for control Summary of features

-

- -

- - -

69

Oral live vaccine

Containing naturally avirulent culture of E. coli: Positive for F4 Negative for toxins and other virulent factors

Recommended for the vaccination of weaned pigs as an aid in the prevention of post-weaning diarrhœa (PWD) caused by F4-positive ETEC

Lyophilized multi-doses formats: 200 doses 500 doses

The Coliprotec™ vaccine

70

Experimental design

Weaning (17-day old pigs)

Coliprotec/Placebo

Challenge (F4+ETEC)

0 1 2 3

4 5

6

7 8

9 (DPC 1)

10 (DPC 2)

11 (DPC 3)

Day

pos

t-wea

ning

Necropsy (peak of clinical signs)

- Confirmation of the absence of F4-ETEC strain from the source of pigs

- Individual weight (growth and DWG) - Excretion of F4+ETEC (CFU/g of faeces)

- Diarrhœa scoring (0=normal to 4=watery)

Controlled challenge studies

71

- Colonisation of F4+ETEC in the intestines (CFU/g of tissue)

- Scoring for accumulation of fluid in the intestines (0=normal to 3=mainly liquid)

Weaning (17-day old pigs)

Coliprotec™/Placebo

Challenge (F4+ETEC)

0 1 2 3

4 5

6

7 8

9 (DPC 1)

10 (DPC 2)

11 (DPC 3)

Day

pos

t-wea

ning

Necropsy (peak of clinical signs)

Experimental design

Controlled challenge studies

72

Proportion of pigs excreting >1 x 108 CFU/g of faeces or being colonised by >1 x107 CFU/g of jejunum

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Challenge DPC 1 DPC 2 DPC 3 Necropsy (DPC3)

Fecal excretion of F4-ETEC Colonisation of jejunum by F4-ETEC

Prop

ortio

n of

pig

s

High infectious pressure

Coliprotec™ reduced F4+ETEC colonisation and excretion

Coliprotec™

Placebo

73

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Challenge DPC 1 DPC 2 DPC 3 Jejunum Ileum Caecum Colon Rectum

High infectious pressure

Severe diarrhoea Accumulation of fluid

Coliprotec™ reduced fluid in the intestines and diarrhœa

Proportion of pigs with severe diarrhœa (score >2) and fluid in the intestines (score >1)

Prop

ortio

n of

pig

s

Coliprotec™

Placebo

74

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Diarrhea Fluid accumulation Diarrhea Fluid accumulation

High infectious pressure Low infectious pressure

Proportion of pigs with diarrhœa (cumulative score >4) and fluid in the intestines (cumulative score >7)

Prop

ortio

n of

pig

s

Coliprotec™

Placebo

Coliprotec™ reduced fluid in the intestines and diarrhœa

75

Susceptibles Tous Susceptibles Résistants

Pression forte Pression faible

37

308

200

395 393 410

370

421

High infectious pressure Low infectious pressure

All pigs Susceptible Resistant

Coliprotec™ improved DWG after F4+ETEC infection

Daily weigh gain (g) during the post-challenge period

Coliprotec™

Placebo

76

Overall production performance and mortality during the nursery phase

Room Weaning groups

Vaccinated with

Coliprotec

# Pigs

Moderate to severe diarrhoea

Total weight gain

per animal*1

Daily weight gain *1

Daily Feed

Intake*1

Feed conversion

Mortality at exit

3 4 and 5 No 200 30% 35.9 Kg 463.6 g 81.7 Kg 2.04 11.0%

4 6 and 7 Yes 219 1.4% 37.9 Kg 489.1 g 101.5 Kg 2.00 4.1%

Improvement +2 Kg +5% +24% -2% -6.9%

*1 Average for the 2 weaning groups of the room

Effect of oral administration of Coliprotec™

77

Conclusion statement for Coliprotec™

Coliprotec™ significantly reduced: Intestinal colonisation and excretion of F4-ETEC Accumulation of fluid in the intestines Diarrhœa Weight loss

Coliprotec™ significantly improved weight gain resulting in normal growth rates after F4-ETEC infection compared to the rapid loss of growth observed after the challenge for unvaccinated pigs.

Thank you!

www.ecl-lab.ca www.apzec.ca

Documents

Diagnostic approach for E. coli presentation... · Diagnostic approach for E. coli ... How they cause disease in pigs . E. coli . with fimbriae Epithelial cell Blood ... Surveillance