STRTEGIES TO REDUCE THE USE OF

ANTIMICROBIALS IN LIVER ABSCESS

CONTROL

Tim McAllister1, Rob Gruninger1, Taylor Davedow1, Rahat Zaheer1, Leluo Guan2,

Greg Penner3 and Gabriel Ribeiro3

tim.mcallister@canada.ca

1 2 3

Antimicrobial use and resistance

� Raises concerns about

antimicrobial use in feed

and antimicrobial

resistance (AMR)

development in bacteria

PHAC, 2017

Spread in

environment

including via

contaminated water

and manure

Spread in

environment

including via

contaminated water

and manure

Spread

between

animals

Spread

between

animals

Spread

between

animals

and

humans,

including

via food

Spread

between

animals

and

humans,

including

via food

Spread

between

humans

Spread

between

humans

Introduction Cont.

Health Canada

Directive

Veterinary oversight

Vet prescription

for MIA

No growth promotion

claim

Responsible use

statement

Liver Abscesses

Human

Consumption

68%

Condemned

22%

Pet Food

10%

� Liver abscess prevalence

in cattle = 12-32% (Brink et al., 1990)

� 11% reduction in average

daily gain, 9.7% reduction in

feed efficiency (Brink et al., 1990)

BCRC, 2018

A+

severe

A+

severe

A

mild

A

mild

0

none

0

none

Identification of novel vaccine targets

The main reason for in-feed macrolide use in feedlots

• Increased prevalence from13% in 1999 to 23% in 2011

• Associated with consumption of high-grain diets

Fusobacterium necrophorum (1o)

Trueperella pyogenes (20)

Cattle liver abscesses: Elanco Liver Scoring System

0 A- A A+

Liver abscesses – identification of novel

vaccine targets

� Costs industry $61.2 M /yr

• Due to animal performance and liver condemnation

• A 10% reduction in both acidosis and liver abscesses would

save

the industry over $40M per year

• Prevalence is expected to reach 70% if in-feed

antimicrobials are banned

� Currently, vaccine efficacy is low

• Polymicrobial disease

���� Novel vaccine targets are needed

Bacteriology: culture-based approach

Genus Species Recovery %

Fusobacterium necrophorum 100

Trueperella pyogenes 49.8

Salmonella5 ≠ serotypes

Lubbock 60.8%25.3

Escherichia coli 32.4

Klebsiella pneumoniae 14.9

Enterobacter cloacae 14.4

Enterococcus faecalis 17.7

Lactococcus garviae 18.8

Bacillus cereus NS

Propionibacterium acnes NS

Corynebacterium falsenii NS

Streptococcus spp. 7.3

Acinetobacter spp. 5.5

Aeromonas spp. 5.5

Staphylococcus spp. 5.5

Citrobacter spp. 4.4

Pseudomonas spp. 2.4

Leclercia spp. 2.1

Proteus spp. 1.3

Bacteroides spp. 1

Micrococcus spp. 1

Shewanella spp. 1

Bacillus spp. 0.8

Peptoniphilus spp. 0.8

Wautersiella spp. 0.5

� Holstein steers vs Crossbred cattle

• More diversity in Holstein steers

• Fn in all samples

• No differences on % Salmonella

• Higher Tp cross breed

� Tyl vs no tylosin

• Tyl group higher Tp, lower

Salmonella

� 24 different genus

Amachawadi et al J. Anim. Sci. 95: 3425-3434

Liver abscesses – identification of

novel vaccine targets

Liver abscess microbiome – US cattle fed corn

(Weinroth et al. 2017).

• Samples of abscess

fluid in corn-fed cattle

• Canada has a higher

incident rate

compared to the US

- may be due to

different diets

• Tissue-adherent

population in barley-

fed cattle

- using a 16S

rRNA sequencing

as a targeted

approach for

identification of

novel vaccine

candidates

T. pyrogenes

F. necrophorum

Alternative Strategies

Prebiotics

VaccinesNutritional Management

53.3

33.3

20.0 20.0 20.0

6.7

0

10

20

30

40

50

60

70

80

0 12 15 18 eXPC ANT

Ab

sce

ss P

erc

en

tag

e (

%)

Treatments

Total Severe

Figure 1. Effect of Saccharomyces cerevisiae fermentation product (SCFP;

NaturSafe®), encapsulated XPC (eXPC) or antibiotics (TYL) on total and

severely abscessed livers (%) in finishing steers

Total (P = 0.97); Severe (P = 0.10).

TYL

In press

Objectives

15

3 Cattle performance

2 AMR development in enteric enterococci populations

1 Liver abscess prevalence and severity

Determine effect of shorter duration tylosin administration

(FIRST-78, LAST-75) compared to continuous feeding (CTRL) on:

Hypotheses

1

• Cattle in the two shorter duration tylosin feeding regimens (FIRST-78, LAST-75), will have no significant difference on liver abscess prevalence or growth performance.

2

• The proportion of macrolide resistant enterococci will decrease only for cattle that were fed tylosin in the FIRST-78 treatment

0 20 40 60 80 100 120 140 160

CTRL

LAST-75

FIRST-78

Days on feed

Tre

atm

en

ts

TYLOSIN NO TYLOSIN

Experimental Design

Fecal Collection:

� Beef cattle randomly assigned to 1 of 3 treatments

� Allocated in replicates: 1 pen/ group/rep

� 250 head/pen� 10 reps

n = 2526 n = 2525 n = 2525

FIRST-78 LAST-75 CTRLTylosin phosphate in-feed: 11 ppm

Materials and Methods:

Fecal Composite

Bile esculin azide (BEA)

agar

BEA + erythromycin (8 �g/mL; BEAE)

% EryR Enterococci = ��������� ��������� ������������

������������� ������������ �����% EryR Enterococci =

��������� ��������� ������������

������������� ������������ �����

Sample Processing

Enterococci Isolation & Enumeration

Enterococcus spp. Characterization

• Duplex PCR of groES-EL intergenic region& mur2 gene to target E. hirae

Enterococcus spp. Characterization

• Duplex PCR of groES-EL intergenic region& mur2 gene to target E. hirae

Antibiotic Susceptibility Testing: Disc Diffusion

• Panel of 12 antibiotics with medium or high importance in human health

Antibiotic Susceptibility Testing: Disc Diffusion

• Panel of 12 antibiotics with medium or high importance in human health

Resistant Gene Determinant PCR

• Macrolide resistant genes erm(B) & msrC

• Tetracycline resistant genes tet(L,M & O)

Resistant Gene Determinant PCR

• Macrolide resistant genes erm(B) & msrC

• Tetracycline resistant genes tet(L,M & O)

Sample Analysis

RESULTS & DISCUSSION

Results

Proportion of erythromycin resistant fecal enterococci isolates.

Method of tylosin administration, P = 0.34; Tylosin administration X

Days on feed, P =0.37; Days on feed, P < 0.01.

0

20

40

60

80

100

0 81 160

Pro

po

rtio

n o

f m

acr

olid

e

resi

sta

nce

(%

)

Days on feed

FIRST-78 LAST-75 CTRL

Results

Enterococcus species distribution of characterized isolates from

feedlot cattle. Isolates are pooled across all pens, experimental

groups and media type.

0

20

40

60

80

100

0 81 160

Spe

cie

s p

reva

len

ce (

%)

Days on feed

E. hirae E. villorum E. faecium E. durans

Antibiograms of enterococci (n=176) isolated from feedlot

cattle feces from cattle fed tylosin during the feeding period1

1 Isolates pooled across all treatments, sampling days, and isolation media types2 There were 16 antibiogram profiles in total3 Doxycycline, DOX; Erythromycin, ERY; Linezolid, LIN; Nitrofurantoin, NIT; Quinupristin-

dalfopristin, Q-D; Streptomycin, STR; Tigecycline, TIG; Tylosin, TYL. Upper case denotes

complete resistance and lower case denotes intermediate resistance.

Profile2 Phenotype3 n = 176 (%)

A1 No Resistance 21 11.9

A5 ERY-TYL 81 46.0

A7 dox-ERY-TYL 42 23.9

Treatment

No. Positive (%)1

Macrolide Tetracycline

n erm(B) msrC Negative n tet(L) tet(M) tet(O) Negative

FIRST-78 51 49 (96.1) 1 (2.0) 2 (3.9) 17 13 (61.9) 13 (61.9) 4 (19.0) 0 (0)

LAST-75 51 48 (94.1) 3 (5.9) 3 (5.9) 21 18 (85.7) 19 (90.5) 2 (9.5) 0 (0)

CTRL 51 47 (92.2) 2 (3.9) 4 (7.8) 16 10 (62.5) 10 (62.5) 5 (31.3) 1 (6.3)

Total 153 144 (94.1) 6 (3.9) 9 (5.9) 54 41 (75.9) 42 (77.8) 11 (20.4) 1 (6.3)

Distribution of enterococci isolates grouped according to macrolide

(n = 153) and tetracycline (n= 54) resistance genes and by treatment

1Isolates pooled across all media types and sampling days

Liver abscesses of feedlot cattle from cattle fed tylosin for

the FIRST-78%, LAST-75%, or continuously (CTRL) during the

feeding period.

1 Severely abscessed (A+), total liver abscesses (A and A+), and no liver

abscesses (none = 0). 2 Reference group = CTRL; Comparison groups = FIRST-78 and LAST-75

Treatment

Liver Abscesses (%)1 Risk Ratio2

Abscessed Severe None Total Abscessed Severe

FIRST-78 61.00 23.47 39.00 100.00 0.99 1.18

LAST-75 64.15 22.95 35.85 100.00 1.04 1.16

CTRL 61.88 19.83 38.12 100.00 1.00 1.00

Growth performance of feedlot cattle.

1 DMI = dry matter intake; ADG = average daily gain; F:G = feed-to-gain ratio2 Based on live weight

Item1

Treatment P – values

FIRST-78 LAST-75 CTRL SEMFIRST-78

vs CTRL

LAST-75

vs CTRL

No. of Cattle 2525 2526 2525

Initial BW (kg) 393.5 395.2 393.6 5.49 0.99 0.22

Final BW (kg) 681.0 680.0 677.5 9.25 0.25 0.40

DMI (kg/d) 11.9 11.9 11.8 0.14 0.80 0.22

ADG (kg/d)2 1.8 1.8 1.7 0.03 0.25 0.69

F:G2 6.7 6.8 6.8 0.07 0.23 0.70

Composition of Capsule microbiome was similar to interior of abscess

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Fusobacterium BacteroidesEscherichia-Shigella Campylobacter

Rela

tive A

bundance (

%)

Capsule Interior

� Levels of liver abscesses can be very high – even with contol measures

�Undeniably a diseased state even if it does not always impact growth performance

�Solutions:• Vaccine??

• More forage less concentrate??

• Grain processing?

• Balanced protein levels?

• Genetic predisposition?

�A clear problem that needs a solution.

Conlusions:

Conclusions� E. hirae a species that seldom causes disease in people was the

predominant species in beef cattle

� Removal of tylosin did not alter levels of macrolide resistant

enterococci

� Removal of tylosin tended to result in more severe abscesses

� Overall total number of liver abscesses not affected by reduced

tylosin

� Removal of tylosin did not impact growth performance or health

� Mileage in letting it be known that antimicrobial use has been

reduced in beef??

Conlusions:

Thank you!

�Abscess microbiome had low diversity and was

dominated by Fusobacterium

�Small differences in the relative abundance of taxa

between inner abscess wall and pus

�Treatment did not have a significant effect on abscess

microbiome however small shifts in taxonomy were

detected.

�Need to validate whether these shifts were

statistically significant