jennifer giraldo gómez instituto de ciencia y tecnología animal...

Jennifer Giraldo Gómez

Instituto de Ciencia y Tecnología Animal (ICTA)

Joint Seminar of the Sub-Network on Production Systems & Sub-Network on Nutrition.

Innovation for Sustainability in Sheep and Goats. Vitoria-Gasteiz, Spain, 3-5 October 2017

Introduction

Joint Seminar of the Sub-Network on Production Systems & Sub-Network on Nutrition.

Innovation for Sustainability in Sheep and Goats. Vitoria-Gasteiz, Spain, 3-5 October 2017

Risk of antibiotic

residues

Therapeutic and prophylactic treatments

Mastitis (lactation/ dry period) and other (respiratory, reproductive,...)

Good farming practices (dosage, administration route, withdrawal

period,...)

Few drugs registered for small ruminants (“extra-label” use)

Veterinary Drugs in Sheep and Goat’s Livestock

Joint Seminar of the Sub-Network on Production Systems & Sub-Network on Nutrition.

Innovation for Sustainability in Sheep and Goats. Vitoria-Gasteiz, Spain, 3-5 October 2017

Maximum Residue Limit Control steps

European Normative

MRL CONTROL

Directive 23/96/EC

Decision 657/2002/EC

Regulation178/2002/EC

Regulations 852,853 and 854/2004/EC

Regulation 470/2009/EC

Commission Regulation 37/2010/EU

Technological

Inhibition of starter cultures

Effects on dairy product properties

Heat treatment stability

Economic

Immobilisation and destruction

of milk

Loss of income

CONSUMER HEALTH DAIRY INDUSTRY

Consequences of Antibiotics in Milk & Dairy Products

Toxicological

Ocurrence of allergic symptoms

Disorders in the intestinal flora

Development of bacterial resistance

Contamination

Contamination topsoil/ groundwater

Effects on microflora and microfauna

PRODUCERS (FARMS) ENVIRONMENT

Joint Seminar of the Sub-Network on Production Systems & Sub-Network on Nutrition.

Innovation for Sustainability in Sheep and Goats. Vitoria-Gasteiz, Spain, 3-5 October 2017

M é todos

cuantitativos

Espec í

No espec

M é todos

cualitativos

LC-UV, LC-MS, LC-MS/MS

Qualitative

methods

Quantification

Screening

Specific tests: receptor, immunoenzymatic

Non-specific tests: microbial inhibitor tests Farms and dairies

Milk quality laboratories

Official controls

National Monitoring Plan

Quantitative

Methods

Methods for Antibiotic Detection in Milk

Decision 657/2002/EC

Joint Seminar of the Sub-Network on Production Systems & Sub-Network on Nutrition.

Innovation for Sustainability in Sheep and Goats. Vitoria-Gasteiz, Spain, 3-5 October 2017

Inhibition of the growth of a specific microorganism

Geobacillus stearothermophilus var. calidolactis

Indicator system: pH, redox, bioluminescence,…

Advantages: cheap, easy, broad spectrum, high sample throughput,…

Disadvantages: non-specific, relative long incubation period (2-3 h)

Commercial microbial tests: BRT MRL, Delvotest SP-NT MCS, Eclipse 100,…

Developed in cow milk. Validation in milk from other species and matrixes needed

Principle of Microbial Inhibitor Tests

Joint Seminar of the Sub-Network on Production Systems & Sub-Network on Nutrition.

Innovation for Sustainability in Sheep and Goats. Vitoria-Gasteiz, Spain, 3-5 October 2017

Detection

capability

CC

Decision

limit

CCα

Trueness/

recovery Precision

Selectivity/

specificity

Applicability/

robustness/stability

Qualitative

methods

Screening

Methods + - - - + +

Confirmatory

Methods + + - - + +

Initial validation (originator lab):

- Detection capability (CCβ)

- Selectivity/specificity

- Applicability/robustness/stability

Guidelines for the validation of screening methods

Validation of Microbial Screening Tests

Abridged validation (receptor lab):

- Detection capability (CCβ)

- Selectivity/specificity

Equal performance characteristics

Decision 657/2002/EC

Joint Seminar of the Sub-Network on Production Systems & Sub-Network on Nutrition.

Innovation for Sustainability in Sheep and Goats. Vitoria-Gasteiz, Spain, 3-5 October 2017

Objective

Joint Seminar of the Sub-Network on Production Systems & Sub-Network on Nutrition.

Innovation for Sustainability in Sheep and Goats. Vitoria-Gasteiz, Spain, 3-5 October 2017

To study the performance and applicability of the

Eclipse Farm test in combination with an e-Reader for

the detection of antimicrobials in raw sheep and goat’s

milk and in goat’s cheese whey

Joint Seminar of the Sub-Network on Production Systems & Sub-Network on Nutrition.

Innovation for Sustainability in Sheep and Goats. Vitoria-Gasteiz, Spain, 3-5 October 2017

Material & Methods

Joint Seminar of the Sub-Network on Production Systems & Sub-Network on Nutrition.

Innovation for Sustainability in Sheep and Goats. Vitoria-Gasteiz, Spain, 3-5 October 2017

Sheep and goat’s milk samples:

- Milk quality control laboratories in Spain

- Antibiotic-free: Eclipse 100 (Zeulab, S.L.) and TwinSensor (Unisensor)

- Milk quality analysis

Cheese whey samples:

- Experimental goats flock UPV: Murciano-Granadina breed

- No antibiotic treatment

- Milk quality analysis

Milk and Cheese Whey Samples

1. Heat at 33°C, coagulation

by rennet (30 min)

2. Cutting curd and

shaking for 15 min

3. Centrifugation: 3000 rpm-10 min

Whey separation

Cheese making at lab-scale

Joint Seminar of the Sub-Network on Production Systems & Sub-Network on Nutrition.

Innovation for Sustainability in Sheep and Goats. Vitoria-Gasteiz, Spain, 3-5 October 2017

Microbial Inhibitor Test

Adding 100 µL of sample

1 hour at room temperature

Cleaning with distilled water (3 times)

Pressing start and incubation 64 ± 1ºC; 2.5 - 3 hours

Results on screen

TEST PROCEDURE

Eclipse Farm 3G coupled to an e-Reader (Zeulab S.L., Zaragoza, Spain)

Microbial qualitative test (Geobacillus sterathermopnilus var. calidolactis)

Sensitive broad-range of antibiotics (multi-residue system)

Incubation period about 2 hours 30 min

Autonomous system (optimal test endpoint)

Interprets results and saves data in memory

Easy to use and economical

CHARACTERISTICS

Joint Seminar of the Sub-Network on Production Systems & Sub-Network on Nutrition.

Innovation for Sustainability in Sheep and Goats. Vitoria-Gasteiz, Spain, 3-5 October 2017

Cheese Whey Samples: Preliminary Study

1. Centrifugation (3000 rpm, 10 min)

2. Heating up (80ºC, 10 min)

3. Centrifugation and heating up (under the same conditions)

4. Pre-diffusion for one hour at room temperature

Color gradients: inaccurate reading

Goat’s chesse whey samples

Previous treatment of whey samples (n=10)

Correct intepretation of results

Optimal test endpoint

Joint Seminar of the Sub-Network on Production Systems & Sub-Network on Nutrition.

Innovation for Sustainability in Sheep and Goats. Vitoria-Gasteiz, Spain, 3-5 October 2017

180 individual sheep milk samples

123 individual goat’s milk samples

75 whey samples from individual goat’s milk

CUT-OFF = Mean ± 3xSD

(Mata et al., 2016)

Eclipse Farm Test Cut-Off

The Cut-Off Level is the response or signal from a screening test

which indicates that a sample contains an analyte at or above the

Screening Target Concentration (CRLs, 2010)

Joint Seminar of the Sub-Network on Production Systems & Sub-Network on Nutrition.

Innovation for Sustainability in Sheep and Goats. Vitoria-Gasteiz, Spain, 3-5 October 2017

Mean

3xSD

Cut-off

0102030405060708090

100

0 25 50 75 100 125

Colo

r (A

u)

Samples

Eclipse Farm Test Specificity (False-Positive rate)

250 individual sheep milk samples

150 individual goat’s milk samples

130 whey samples from individual goat’s milk

Ability of a method to distinguish between the analyte being

measured and other substances (EC, 2002)

Joint Seminar of the Sub-Network on Production Systems & Sub-Network on Nutrition.

Innovation for Sustainability in Sheep and Goats. Vitoria-Gasteiz, Spain, 3-5 October 2017

False-positive rate = positive samples / total of samples analyzed x 100

Statistical analysis

(Statgraphics, version 5.1)

Characteristics of Milk and Whey

Average, Standard deviation, Maximum and Minimum

CCβ is the smallest content of the analyte that may be detected,

identified and/or quantified in a sample with an error probability of β.

For screening tests the β error should be < 5% (EC, 2002)

Evaluation in 3 steps (Mata et al., 2016)

1) Initial setting: 5 levels + Negative Control-NC (1 replicate)

Lowest concentration with a positive result: step 2

2) Detection Limit (DL): (ISO/IDF, 2002) 1 level (5 replicates) + NC

3) Detection Capability (CCβ): (CRLs, 2010): 20 to 60 replicates at the

LD level. Only 1 substance per antibiotic group (representative

molecules)

Sheep and goat’s milk: 4 β-lactams; 2 Aminoglycosides;

2 Macrolides; 2 Tetracyclines & 2 Sulfonamides

Goat’s cheese whey: 3 β-lactams & 1 Tetracycline - ANTIBIOTICS Sigma Aldrich

Joint Seminar of the Sub-Network on Production Systems & Sub-Network on Nutrition.

Innovation for Sustainability in Sheep and Goats. Vitoria-Gasteiz, Spain, 3-5 October 2017

Eclipse Farm Test Detection Limit (DL) &

Detection Capability (CCβ)

Results & Discussion

Joint Seminar of the Sub-Network on Production Systems & Sub-Network on Nutrition.

Innovation for Sustainability in Sheep and Goats. Vitoria-Gasteiz, Spain, 3-5 October 2017

J. Giraldo, R. Cabizza, D. Sanz, L. Mata and M.P. Molina

Performance of Eclipse Farm test coupled with e-Reader for

Screening Antibiotics in Sheep and Goat’s milk

Joint Seminar of the Sub-Network on Production Systems & Sub-Network on Nutrition.

Innovation for Sustainability in Sheep and Goats. Vitoria-Gasteiz, Spain, 3-5 October 2017

0

10

20

30

40

50

60

70

80

90

100

0 20 40 60 80 100 120 140 160 180

Co

lor

(AU

)

Samples

Eclipse Farm Test Cut-Off

MEAN 42.6

SD 7.8

3xSD 63.4

Sheep milk

CUT-OFF ≥ 66 AU

Joint Seminar of the Sub-Network on Production Systems & Sub-Network on Nutrition.

Innovation for Sustainability in Sheep and Goats. Vitoria-Gasteiz, Spain, 3-5 October 2017

Mean

3xSD

Cut-off

0

10

20

30

40

50

60

70

80

90

100

0 20 40 60 80 100 120

Colo

r (A

u)

Samples

Mean

3xSD

Cut-off

CUT-OFF ≥ 66 AU

Goat’s milk

MEAN 40.1

SD 7.7

3xSD 63.2

Parameters

Individual Sheep Milk

(n=250)

Individual Goat’s Milk

(n=150)

Mean SD Min Max Mean SD Min Max

pH 6.66 0.09 6.46 6.83 6.73 0.04 6.68 6.76

Fat (%) 5.14 1.44 3.10 8.27 4.15 0.45 3.10 4.76

Protein (%) 5.53 0.61 4.78 7.13 3.60 0.14 3.31 3.81

Lactose (%) 5.00 0.32 4.45 5.54 4.82 0.09 4.69 5.01

Total solids (%) 11.37 0.49 10.47 12.48 9.74 0.09 9.54 9.83

Log SCC 5.25 0.52 4.41 6.00 5.48 0.64 4.20 6.08

Log BC 4.46 0.52 4.00 6.19 5.17 0.34 4.45 5.72

False-positive rate = 0.9 %

Eclipse Farm Test Specificity (False-Positive rate)

Joint Seminar of the Sub-Network on Production Systems & Sub-Network on Nutrition.

Innovation for Sustainability in Sheep and Goats. Vitoria-Gasteiz, Spain, 3-5 October 2017

False-positive rate = 0 %

Antimicrobial MRL

(µg/kg)

Sheep Milk Goat’s Milk

DL

(µg/kg)

CCβ

(µg/kg)

DL

(µg/kg)

CCβ

(µg/kg)

Amoxicillin 4 4 4 4 4

Benzilpenicillin 4 3 4 3 3

Cephalexin 100 100 100 100 100

Cloxacillin 30 >30 - 30 -

Gentamycin 100 250 250 >250 300

Neomycin 1,500 600 - 600 -

Lincomycin 150 150 - 150 -

Tylosin 50 25 35 25 35

Sulfathiazole 100 80 80 80 80

Sulphametazine 100 100 - 100 -

Oxytetracycline 100 >150 300 >150 250

Tetracycline 100 200 - 200 -

Joint Seminar of the Sub-Network on Production Systems & Sub-Network on Nutrition.

Innovation for Sustainability in Sheep and Goats. Vitoria-Gasteiz, Spain, 3-5 October 2017

Eclipse Farm Test Detection Limit (DL) &

Detection Capability (CCβ)

J. Giraldo, R. Cabizza, L. Mata and M. P. Molina.

Validation of a Microbiological Inhibition System based on

Eclipse Farm 3G coupled with an e-Reader to Screen β-lactam

and Tetracycline Antibiotics in Goat’s Cheese Whey

Joint Seminar of the Sub-Network on Production Systems & Sub-Network on Nutrition.

Innovation for Sustainability in Sheep and Goats. Vitoria-Gasteiz, Spain, 3-5 October 2017

Cheese Whey Samples: Preliminary Study

n = 10 Control

Centrifugation

(3000 rpm, 10 min)

Heating up

(80°C, 10 min)

Centrifugation

+ Heating up

Pre-diffusion

1 h/Room T

e-Reader

value (Au) 74 66 47 52 42

Pre-diffusion for one hour at room temperature

Optimal test endpoint ≈ 40 Au

Correct intepretation of results

Joint Seminar of the Sub-Network on Production Systems & Sub-Network on Nutrition.

Innovation for Sustainability in Sheep and Goats. Vitoria-Gasteiz, Spain, 3-5 October 2017

Eclipse Farm Test Cut-Off

MEAN 41.2

SD 7.0

3xSD 62.1

CUT-OFF ≥ 62 AU

Joint Seminar of the Sub-Network on Production Systems & Sub-Network on Nutrition.

Innovation for Sustainability in Sheep and Goats. Vitoria-Gasteiz, Spain, 3-5 October 2017

Mean

3xSD

Cut-off

Parameters Mean SD Min Max

pH 6.45 0.22 5.46 6.69

Fat (%) 0.68 0.16 0.33 1.21

Protein (%) 1.09 0.15 0.83 1.48

Lactose (%) 4.91 0.10 4.63 5.10

Total solids (%) 6.69 0.16 6.36 7.12

Eclipse Farm Test Specificity (False-Positive rate)

False-positive rate = 2.3 %

Joint Seminar of the Sub-Network on Production Systems & Sub-Network on Nutrition.

Innovation for Sustainability in Sheep and Goats. Vitoria-Gasteiz, Spain, 3-5 October 2017

Antimicrobial MRL

(µg/kg)

DL

(µg/kg)

CCβ

(µg/kg)

Amoxicillin 4 4 5

Benzylpenicillin 4 3 3

Cephalexin 100 >50 60

Oxytetracycline 100 >60 100

Joint Seminar of the Sub-Network on Production Systems & Sub-Network on Nutrition.

Innovation for Sustainability in Sheep and Goats. Vitoria-Gasteiz, Spain, 3-5 October 2017

Eclipse Farm Test Detection Limit (DL) &

Detection Capability (CCβ)

Conclusions

Joint Seminar of the Sub-Network on Production Systems & Sub-Network on Nutrition.

Innovation for Sustainability in Sheep and Goats. Vitoria-Gasteiz, Spain, 3-5 October 2017

1) Eclipse Farm test coupled to an e-Reader presents a low

percentage of false-positive results for the detection of

antibiotics in sheep and goat’s milk and most of the detection

capabilities were at or below EU-MRL level.

2) Eclipse Farm test coupled to an e-Reader allows the detection

of benzylpenicillin, cephalexin and oxytetracycline at

concentrations below or equal to their MRLs in cheese whey,

with a minimum rate of false-positive results.

Eclipse Farm coupled to an e-Reader test is suitable to be included

in sheep and goat's milk quality programs on farms and dairies.

This method could also be a good option to screen β-lactam and

tetracycline residues in cheese whey.

Joint Seminar of the Sub-Network on Production Systems & Sub-Network on Nutrition.

Innovation for Sustainability in Sheep and Goats. Vitoria-Gasteiz, Spain, 3-5 October 2017