The efficacy of IgG-ELISA/IEA as a new
diagnostic method in dogs with adverse
food reactions
S. Smits*
* solisID: 3546179, Utrecht University, Faculty of Veterinary Medicine, Yalelaan 1, 3584 CL Utrecht, the Netherlands.
Supervisior: E.A. Hagen-Plantinga. Date: 11-2015
Abstract
Nowadays the best reliable diagnostic method to diagnose adverse food reactions (AFR) is an
elimination-challenge diet trial. Since this cost a lot of time and effort, an easier diagnostic
method is of interest. The IgG-ELISA/IEA test might be a new diagnostic method. To test the
reliability of this IgG-ELISA as a diagnostic method in AFR, blood was taken from 40 dogs, of this
group 16 dogs belonged to the healthy control group and 24 dogs belonged to the test group,
suffering from adverse food reactions. This blood was send, in duplo, to the laboratory, for an
IgG-ELISA/EIA on 39 food antigens. The results showed that the ELISA is not able to detect a
significant difference between the outcome of the control and test group. Significant correlations
were found between the different antigen groups, suggesting that the ELISA is not able to
differentiate between all 39 ingredients. This study demonstrated that the IgG-ELISA/IEA of this
laboratory does not seem to be an effective diagnostic method for diagnosing AFR in dogs.
Samenvatting
Momenteel wordt de diagnose voedselovergevoeligheid veelal gesteld door middel van een
eliminatie dieet. Omdat dit erg veel tijd en moeite kost is het van belang dat er een eenvoudigere
diagnostische tool ontwikkeld wordt. De IgG-ELISA/IEA is een potentieel nieuwe diagnostische
methode. Om de betrouwbaarheid van deze IgG-ELISA/IEA te testen is bloed afgenomen van 40
honden. Van deze groep behoorden 16 honden tot de gezonde controlegroep en 24 honden tot de
testgroep, welke waren gediagnosticeerd met voedselovergevoeligheid. Dit bloed is, in duplo,
verzonden naar een laboratorium voor een IgG-ELISA/IEA voor 39 voedselallergenen. De
resultaten laten zien dat de ELISA geen significant verschil kon aantonen tussen de uitslagen van
de test- en controlegroep. Ook is er een significant verband gezien tussen de verschillende
antigeen groepen. Het is hierdoor waarschijnlijk dat de ELISA test niet gevoelig genoeg is om
onderscheid te kunnen maken tussen de 39 verschillende ingrediënten. Deze studie uitslagen
tonen aan dat de onderzochte IgG-ELISA/IEA geen effectieve diagnostische methode is voor het
aantonen van voedselovergevoeligheid bij honden.
1
Introduction
Adverse food reactions
An adverse food reaction (AFR) is an abnormal response to an ingested food ingredient. AFR is
considered as an abnormally high sensitivity to harmless environmental substances, in this
case food or food additives.1, 2
The prevalence of adverse food reactions in the dog population in 2012 was estimated to be up
to 8%. 7-25% of all allergic skin diseases are cutaneous adverse food reaction (CAFR). The
literature is spread whether there is any breed or sex predisposition. Veenhof reports that there
is no breed or sex predisposition,1, 3 but Hensel reported that there is a higher risk for breeds
such as Boxers, West Highland White Terriers, Retrievers and Cocker/Springer Spaniels.4
CAFR often occurs at young age, especially at dogs under the age of 3.1, 5 Phillip Roudebush
states that there is not a clear conspectus about the prevalence of adverse food reactions,
because AFR looks similar to other diseases, like pruritic dermatoses, pyoderma,
ectoparasitism, folliculitis2 and atopic dermatitis.5
The classic clinical signs of cutaneous adverse food reactions are: nonseasonal pruritic
dermatitis2, 6, pruritus of the ears, axillae, distal limbs and the inguinal area4, 5 sometimes
accompanied with gastrointestinal signs.1, 2, 5, 6 Secondary infections may occur and impede the
diagnosis.5
Immune reactions
There are different types of immune reactions which may influence an adverse food reaction:
type I, type III and type IV.1, 4, 7 Type I hypersensitivity is mediated by IgE antibodies,1, 7, 8 and
gives an acute hypersensitivity reaction.2 Although food allergy in human is in most cases IgEmediated5, 8 it is unlikely to be the basis of AFR, especially in chronic cases.2 There is no
evidence that IgE is involved in CAFR,1 even though there are some cases reported which
suggest the influence of IgE in dogs with CAFR.5 Type III and type IV hypersensitivity are
delayed types of hypersensitivity (DTH), of which type III is mediated by IgG antibodies. 7-9 The
prevalence of DTH to food is unknown in the canine population but the clinical picture of dogs
with AFR and the chronic nature of the disease, indicate their event.7 There is still no evidence
that IgG is a good indicator of AFR in dogs.2, 9 Although IgG-ELISA tests are already performed
in human,10, 11 it is questionable whether IgG is the suitable antibody for testing AFR in dogs, as
the pathogenesis of AFR is not fully elucidated.1, 2, 5, 7
Diagnosis
There are several ways to diagnose cutaneous adverse food reactions, but the eliminationchallenge diet trial is the mostly used method and with the most reliable results.1, 2, 4, 6 This
food trial is seen as the golden standard.1 For this diagnostic method the owner’s education and
compliance is very important.4 An elimination diet is a home cooked or commercial diet,
consisting of novel proteins and carbohydrates.1, 2, 4 The elimination diet is first fed, during 8 10 weeks.1 Mostly, the reduction in symptoms is seen within 4 weeks, but in some dogs
improvement is seen after 10 – 12 weeks.4 With this elimination diet the clinical symptoms,
primary caused by an allergic food reaction, disappear. Secondary infections might remain and
will need medical therapy. When the dogs are fully recovered, dogs are provoked with their
original diet. When the clinical symptoms reappear during provocation, the diagnosis AFR is
confirmed.1, 2, 4 After diagnosis a suitable maintenance feed has to be found by ‘trial and error’.
2
One (commercial) feed after another has to be tried, until a feed is found on which the dog
does not respond allergic.
Aim of the study
Because of the high effort needed to make a reliable diagnosis and to find a suitable
maintenance feed nowadays, an easier diagnostic method is desired. This method preferably is
as reliable as the elimination challenge and gives information about which ingredients are
suitable. The aim of this study is to determine the efficacy of IgG-ELISA/IEA as a new
diagnostic method in dogs with AFR.
Materials and Methods
Experimental design
40 Dogs were included in this study; 24 dogs diagnosed, using the elimination-challenge diet
trial, with cutaneous adverse food reactions (test group) and 16 dogs without clinical signs of
adverse reactions to food (control group). Blood was taken by jugular venipuncture and
sampled in two serum tubes. Blood was stored in the refrigerator before sending it to the
laboratory for an IgG-ELISA/IEA. The results of the lab were compared with the ingredients of
the regular diet of the dogs.
Animals and inclusion criteria
All 40 dogs included in this study belonged to the customer base of the dermatology
departments of the clinic MCD Amsterdam or Veterinaire Specialisten Oisterwijk. Dogs
selected for this study where over one year of age. All dogs were owned privately. The dogs
were free of systemic or topical corticosteroid therapy or other pharmaceuticals which
influence the immune system of the skin. All dogs were free of ectoparasites. The 24 dogs in
the test group were included in the study after the diagnosis ‘food allergy’ was confirmed by
the dermatologists. This diagnosis was made with the elimination-challenge diet trial. The 16
dogs of the control group did not suffer from skin problems or from intestinal problems caused
by food which was concluded after a physical exam by the dermatologists and after
questionnaire for the owner’s about their dogs health.
Diet
Background information about food consumption, functioning of the gastro-intestinal tract
and the dermatologic health of the dogs over the last half year, was collected using specific
questionnaires and interviews with the owners. Food composition was first determined by
consulting the package labels and internet website of the specific brands. In case of a closed or
inconclusive declaration on the packaging, manufacturers were contacted by phone or via email to obtain further compositional information of the diet. All ingredients fed were
compared with the ELISA outcome.
Blood analyses
After the blood was collected, the serum tubes were stored in the refrigerator, at 7°C, before
sending to the laboratory once every two weeks. A minimum of 3 patients (6 tubes) was sent at
a time for ELISA analysis. All tubes had a code matched to the dogs, the laboratory staff was
not aware of the coding-system, so the ELISA IgG/IEA tests was performed single-blind.
3
IgG-ELISA/IEA
The microtitre plates for the IgG-ELISA were coated with the antigen extracts of European
hake, herring, salmon, tuna, calf liver, calf, turkey, chicken, lamb, beef, beef liver, tripe, pork,
potato, corn, beetroot, butter bean, sugar beet, tomato, carrot, garden pea, soy pulp, garlic,
cress, casein, cow’s milk, linseed oil, barley, oats, wheat, buckwheat, cassava manioc, millet,
rice, wild rice, bakingsoda, brewer’s yeast, candida albicans, gliadin, gluten, chicken egg yolk
and chicken egg white. Diluted serum (1:401) was placed in duplicate into the wells, after which
the plates were incubated at 5°C for 48 hours. After washing the plates, anti-dog peroxidase
conjugate was added, followed by a 1-h incubation period at 37°C. After a second washing, a
substrate solution of tetramethylbenzin (TMB) was pipetted into the wells. The plates were
incubated at 22°C for 20 minutes in a dark space. The blue coloring that arose during the last
incubation was stopped by adding an inhibitor into the wells. Within 30 minutes after adding
the inhibitor the extinction was measured using spectrophotometry (at 450 nm). The ELISA
was performed split sample. An outcome of ≥0.4U was considered positive, and a 10%
difference between both samples was considered acceptable.
Statistics
Mann Whitney U-test
IgG titers of individual ingredients were compared between control dogs and patient with an
independent samples Mann Whitney U-test using SPSS version 19. The level of significance
was set at p<0.05. An outcome of ≥0.4U/ml from the IgG-ELISA was considered positive. The
following hypotheses were tested:
H0: “The IgG-ELISA/EIA is not able to differentiate between healthy dogs (the control group)
and dogs with an adverse food reaction (the test group)”
H1: “The IgG-ELISA/EIA is able to differentiate between healthy dogs (the control group) and
dogs with an adverse food reaction (the test group)”
Pearson Correlation-test
IgG titers of all 39 ingredients were compared, using the two tailed Pearson Correlation-test
using SPSS version 19. p<0.05 was considered significant. The possibility that the IgGELISA/IEA could not differentiate between the different ingredient(group)s was hereby tested.
Results
Mann-Whitney U-test
In Table 1 the mean IgG-titer for the test group and control group per tested ingredient is
shown. The mean IgG-titer for 89,74% ingredients of the test group and for 94,87% of the
control group was positive (>0.4U/ml). The mean (±SEM) number of ingredients that showed a
positive titer in individual dogs was 17 (±2,0) for the control group and 16 (±1,3) for the test
group, which was not significantly different (p=0,59). The p-value in Table 1 shows whether
there is any significance (p<0.05) between the groups, per ingredient. With the p-values
between 0.090 and 0.989, no significance is shown.
4
Pearson Correlation-test
Significant correlations (p<0.05) has been seen between different antigens, but mostly between
specific groups of ingredients, which are pointed out in appendix 1 – 4.
Remarkable is the vegetable group (potato, corn, beetroot, butter bean, sugar beet, tomato,
carrot and garden pea) 64.29% has a significant correlation (p<0.05), appendix 1. The cereals
also show correlation within the group. 25.00% of this group shows a significant correlation
(p<0.05), appendix 2. Remarkable is the fact that rice shows a significant correlation (p<0.05)
with 31.71% of all antigens tested in the ELISA.
Another remarkable group is the meat and dairy group (calf liver, calf, turkey, chicken, lamb,
beef, beef liver, tripe, pork, casein and cow’s milk). 38.18% of the ingredients in this group
show a significant correlation (p<0.05), appendix 3. Remarkable in the meatgroup is tripe, it
has a significant correlation (p<0.05) with 41.46% of all antigens tested. The fishgroup (hake,
herring, tuna and salmon) also show correlation, 33.33% has a significant correlation (p<0.05),
see appendix 4. Tuna has a significant correlation (p<0.05) with 31.70% of all antigens tested.
Correlation between the different groups is also seen. Between the vegetable- and cerealgroup
44.64% has a significant correlation (p<0.05). Between meat and fish a correlation is seen
between 37.50% of the included ingredients.
Ingredient – ELISA outcome comparison
In Figure 1 and 2 show a graphic overview of the ingredient intake per dog, compared with the
ELISA titers. All dogs, both in the control and test group, had one or more positive titers
(>0,4U/ml). The dark blue colour indicates a positive titer and also showing that the ingredient
is fed. Except for 2 dogs in the test group, all dogs are exposed to one or multiple food
ingredients in their current diet for which they have a positive IgG titer. However, all of these
dogs were free of clinical signs on their current diet.
5
Table 1: Mean IgG-titers per ingredient, inclusive the Standard Error of Mean (SEM) and the p-value which shows whether there was
a significant difference between the test and control group.
Patients
Controls
Patients
Controls
Ingredients
Mean
SEM
Mean
SEM
p-value
Hake
Herring
Salmon
Tuna
Calf liver
Calf
Beef
Beef liver
Tripe
Lamb
Pork
Turkey
Chicken
Potato
Corn
Beetroot
Butter bean
Sugar beet
Tomato
Carrot
7,39
0,00
0,13
9,10
13,59
23,33
20,74
24,23
33,80
0,54
4,63
4,73
0,40
7,42
4,86
1,99
1,60
4,41
21,26
0,46
4,32
0,00
0,09
5,72
6,83
8,23
6,82
7,77
8,12
0,20
4,15
2,03
0,18
4,39
4,15
0,83
0,69
4,16
7,84
0,19
1,50
0,05
0,22
9,38
9,97
21,91
37,10
19,92
36,06
7,83
0,77
12,45
0,81
11,27
2,84
8,77
3,16
17,08
33,18
1,18
0,68
0,03
0,10
6,21
6,68
9,65
11,20
9,44
10,45
6,21
0,43
6,57
0,43
6,85
1,30
5,72
1,29
8,85
11,35
1,18
0,613
0,521
0,404
0,389
0,304
0,713
0,539
0,420
0,633
0,420
0,733
0,652
0,452
0,795
0,967
0,774
0,345
0,174
0,613
0,989
Ingredients
Mean
SEM
Mean
SEM
p-value
Garden pea
Soy pulp
Garlic
Cress
Casein
Cow's milk
Linseed oil
Barley
Oats
Wheat
Buckwheat
Cassava manioc
Millet
Rice
Wild rice
Bakingsoda
Brewer's yeast
Chicken egg yolk
Chicken egg white
2,38
0,60
2,91
2,46
15,94
5,42
14,69
0,32
0,24
0,89
0,16
2,18
3,45
0,44
0,52
36,73
32,79
4,53
1,96
0,85
0,21
0,93
0,80
6,75
4,13
6,25
0,13
0,06
0,37
0,05
1,67
1,41
0,24
0,19
8,80
9,06
4,15
1,24
10,71
9,52
16,23
11,73
10,47
8,66
1,55
1,77
1,75
7,17
3,25
1,11
3,63
1,81
1,55
21,30
27,07
1,58
1,55
6,19
6,20
7,19
6,90
6,52
6,21
0,66
0,93
0,98
6,21
2,75
0,55
0,92
0,98
0,60
9,84
10,92
0,84
0,89
0,292
0,090
0,183
0,946
0,733
0,774
0,967
0,157
0,452
0,557
0,304
0,613
0,613
0,345
0,436
0,107
0,486
0,420
0,389
6
Figure 1: Food ingredient exposure versus IgG ELISA results in healthy control dogs.
7
Figure 2: Food ingredient exposure versus IgG ELISA results in patient dogs.
Discussions
In this study the efficacy of IgG-ELISA/IEA as a diagnostic tool in case of cutaneous adverse
food reaction was tested. Based on the available results of other studies the expectation was
that the ELISA would not be suitable as a new diagnostic tool.6, 9, 11, 12 The results of this study
confirms that the IgG-ELISA was not able to differentiate between the control- and test group,
nor between the different ingredients tested.
In 2003, 2004 and 2012, 3 similar studies were published. Foster, published in 2003, determined
the serum IgG and IgE responses to food antigens in 3 groups of dogs; healthy dogs, dogs with
gastrointestinal disease and atopic dogs. A higher total mean titer level for healthy dogs than
for dogs with gastrointestinal disease or atopic dogs was shown.13 This is in contrast to the
results of this study and the results of Halliwell, 2004 and Bethlehem 2012. Halliwell studied
the IgE and IgG antibodies to food in sera from dogs with AFR, dogs with atopic dermatitis and
healthy control dogs. This study showed that the dogs with AFR had the highest titers of these
8
3 groups.6 The study of Bethlehem was designed to determine whether patch tasting, ELISA
IgE and IgG are suitable as a diagnostic tool in case of AFR. Hereby they tested healthy control
dogs and dogs with proven AFR. The results show no titer level difference between the testand control dogs,12 the results of this study show the same. Bethlehem also found that there
were many high serum levels in dogs without clinical symptoms12; this is also seen in the
present study. As stated before, a significant correlation is seen between different groups of
ingredients. Foster et al. also mentioned an association between different
ingredients.13Similarly to this study, between pork and beef and between chicken and beef.
The results of this study also show that even the dogs in the control group or test group that
are not eating particular ingredients can have high IgG serum levels. Notwithstanding, all dogs
were symptom free on their current feed. The appearance of positive titers could be explained
by the fact that IgG might be an indicator for food exposure.9, 12 For this study a feed history of
the dogs of six months is known. It is possible that the dogs have had exposure to some of the
ingredients before these six months. The fact that in this study all dogs in the control group
and 22 out of 24 dogs in the test group had positive titers and also had these ingredients fed
(see figure 1 and 2) but were free of clinical symptoms, makes the ELISA outcome less reliable.
Another difficulty is that not all the feed ingredients are known. Contact with manufactures of
cheaper feed brands revealed that some of the feeds are produced according to a least-cost
formulation model. This means that the composition of the feeds may vary with varying
ingredient prizes. It is known that the amount of feed/allergen intake may influence the
immune response.10
Beef, wheat, dairy products, lamb,1, 2 chicken, soybean, eggs and corn are most commonly
reported as causing AFR.21 But also rabbit, venison, fish, rice and potato are reported.2 The
results show that chicken, lamb, cow’s milk and egg are not the most important allergens
according to this ELISA test. A possible reason for this remarkable outcome is the fact that the
antigen extracts on the ELISA plates could have been manufactured incorrectly.11 The method
used to manufacture the plates for this test is unknown. According to Miller11, a possible risk of
homemade plates is the fact that all food is coated with microorganisms. Also the presence of
pesticides or other organic solvents in the wells cannot be excluded.11 These possible
disturbances could explain the fact that all dogs (n=40) showed high IgG titers for tripe. It is
not sure that the antibodies really bound to the tripe-antigens.
Furthermore, the exact allergenic molecules have not been identified.1 Since the exact
molecules are unknown, it is possible that the ELISA plates used in this test are not coated
with the right antigen extracts. The typical result seen for herring, the only antigen with no
positive titers at all, shows that there could have been nothing in the ELISA-wells for the antiherring-antibodies to bond on.
Remarkable is the fact that the ELISA tests for calf, calf liver, beef, beef liver and tripe antigens
separately. Since a calf is a baby cow, it is not unthinkable that these antigens could have cross
reactivity and so create a similar output of the ELISA. It is known that in dogs with cow’s milk
and beef related AFR the major allergen is bovine IgG.1, 2, 4 The correlation between cow’s milk
and calf-/beef liver and tripe, seen in this study between these antigens supports this theory. In
this study no correlation between beef- and calf meat and cow’s milk is seen, this is not fitting
9
in the cross reactivity theory; the same results were mentioned in the articles of Verlinden et
al. and Roudebush et al2, 7 Although the results of the ELISA show that all 40 dogs suffer from a
multi antigen AFR, adverse food reactions to multiple food ingredients are unusual in dogs.1, 4
It is possible that a dog is multisensitive,6 it had been identified in 35% to 48% of food allergic
dogs.4, 7 Cross reactivity, as mentioned before, is better known.4, 11 The amount of positive titers,
in this test, was almost 50/50 in both groups. The study of Bethlehem in 2012 also showed that
the amount of positive titers was about 50/50, respectively it is hard to tell that there is a
positive correlation between a positive titer and AFR because even 57% of the healthy dogs in
the control group of this study showed a positive titer.
In this study the mean titer per ingredient for the control group was not significantly different
from the test group. This is in contrast to what is mentioned by Roudebush et al2, they
mentioned that the mean titer for healthy dogs where higher than for test dogs, this is in
contrast to what is seen in human.2, 10 A study of Stapel et al showed that it is more likely that
the presence of IgG is an indicator for prolonged exposure to food components rather than
being an indicator for AFR in human9, 12 This way the IgG food antibodies seen by healthy
human can be explained. Knowing that the presence of IgG might be an indicator for exposure
to the feed in the past, it might be helpful to use IgG-ELISA-IEA to determine which food is, or
is not, safe for the elimination diet. This usage of the IgG-ELISA is also suggested in the study
of Bethlehem.12
To support the outcome of this study, a food challenge based on the ELISA results is
recommended. This way the sensitivity and the specificity of the IgG-ELISA/EIA can be
determined. Because of the prevalence of cutaneous adverse food reactions a minimal
specificity and sensitivity of 80% is appropriate to call the IgG-ELISA/EIA a good diagnostic
method.
Conclusion
The results show that H0 is the right hypothesis and that H1 can be rejected. This means that
the tested IgG-ELISA/IEA is not able to differentiate between healthy dogs and dogs with
adverse food reactions. The fact that correlations were found between the different
antigen(groups) and the fact that dogs showing a positive titer to some ingredients, while
having these specific ingredients in the diet without clinical symptoms of allergy, suggests that
this test is not sensitive enough. It may thus be concluded that the IgG-ELISA/IEA tested in
this study is not efficient as a diagnostic tool in case of AFR.
Acknowledgement
I would like to thank the owners of the dogs for being so cooperative during this study. Also,
the dermatologist for helping to select the dogs and collecting the blood needed for the ELISA
tests. I might not forget to thank Esther Plantinga for her support and sharing her experiences
writing a good article. And last but not least I would like to thank Koen Bleeker, for living with
me in such turbulent moods and giving pep talks.
10
Appendix 1, correlation matrix of vegetables
Tomato
Tomato
Carrot
Gardenpea
Sugarbeet
Beetroot
Corn
Potato
Butterbean
0,007
0,006
0,039
0,006
0,113
0
0,007
Carrot
0,007
0,367
0,058
0,304
0,229
0
0,012
Gardenpea Sugarbeet Beetroot
0,006
0,039
0,006
0,367
0,058
0,304
0
0
0
0
0
0
0,136
0
0,131
0,009
0,024
0,003
0,003
0,056
0
Corn
0,113
0,229
0,136
0
0,131
0,429
0,457
Potato
0
0
0,009
0,024
0,003
0,426
Butterbean
0,007
0,012
0,003
0,056
0
0,457
0
0
Appendix 2, correlation matrix of cereals
Barley
Barley
Oats
Wheat
Cassava
Buckwheat
Millet
Rice
Wildrice
0,976
0,585
0,961
0,794
0,08
0,444
0,011
Oats
0,976
0,734
0,805
0
0,338
0,004
0,632
Wheat
0,585
0,734
0,791
0,847
0,65
0,795
0,048
Cassava Buckwheat
0,961
0,794
0,805
0
0,791
0,847
0,53
0,53
0,852
0,205
0,279
0
0,839
0,161
Millet
0,08
0,338
0,65
0,852
0,205
0,037
0,003
Rice
0,444
0,004
0,795
0,279
0
0,037
Wildrice
0,011
0,632
0,048
0,839
0,161
0,003
0,225
0,225
11
Appendix 3, correlation matrix of meat
Casein
Cow's
milk
Calf liver
Calf
Beef
Beef liver
Tripe
Turkey
Chicken
Lamb
Pork
Casein Cow's milk Calf liver Calf
0,047
0,55
0,123
Beef
0,074
Beef liver
0,812
Tripe
0,07
Turkey Chicken
0,093
0,03
Lamb
0,755
Pork
0,003
0,047
0,55
0,123
0,074
0,812
0,07
0,093
0,03
0,755
0,003
0,188
0,265
0,006
0,001
0
0,098
0,013
0,006
0,705
0,71
0,028
0,007
0,652
0,396
0,616
0,004
0,496
0,273
0,818
0,903
0,014
0,016
0,77
0,432
0,83
0,991
0,845
0,783
0,04
0,043
0,665
0,648
0,017
0,574
0,973
0
0
0,2
0,188
0,001
0,006
0,652
0,113
0,818
0,845
0,2
0,015
0,015
0,265
0
0,705
0,396
0,176
0,903
0,783
0,006
0,098
0,71
0,616
0,123
0,014
0,04
0,013
0,028
0,004
0
0,016
0,043
0,007
0,496
0,101
0,77
0,665
0,273
0,014
0,432
0,648
0
0,83
0,017
0,113
0,176
0,123
0
0,101
0,014
0
0,991
0,574
0,973
Appendix 4, correlation matrix of fish
Hake
Hake
Herring
Tuna
Salmon
Herring
Tuna
Salmon
0,867
0,672
0,508
0,867
0,013
0,053
0,672
0,013
0,001
0,508
0,053
0,001
12
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