Anderson Rincón Artunduaga Alejandro Giraldo Giraldo Wilson Galvis Torres.
Jennifer Giraldo Gómez Instituto de Ciencia y Tecnología Animal...
Transcript of 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