Parasitol Res (2014) 113:3651–3660 DOI 10.1007/s00436-014-4030-6

ORIGINAL PAPER

Development of a milk and serum ELISA test for the detection of Teladorsagia circumcincta antibodies in goats using experimentally and naturally infected animals Eleni Malama & Peggy Hoffmann-Köhler & Insa Biedermann & Regine Koopmann & Jürgen Krücken & José Manuel Molina & Alvaro Martinez Moreno & Georg von Samson-Himmelstjerna & Smaragda Sotiraki & Janina Demeler

Received: 5 March 2014 / Accepted: 4 July 2014 / Published online: 16 July 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Teladorsagia circumcincta is among the most important gastrointestinal parasites in small ruminants and the predominant species in Southern European goats. Parasite control is largely based on metaphylactic/preventative treatments, which is often seen as non-sustainable anymore. The reasons are increased consumer demand to reduce chemicals in livestock production and anthelmintic resistance against the common drugs. This study aimed at the development of a T. circumcincta-enzyme-linked immunosorbent assay (ELISA) specifically for goats. Samples were obtained from Electronic supplementary material The online version of this article (doi:10.1007/s00436-014-4030-6) contains supplementary material, which is available to authorized users. E. Malama : S. Sotiraki Laboratory of Parasitology, Hellenic Agricultural Organization Demeter, Veterinary Research Institute of Thessaloniki, Thermi Thessaloniki, Greece P. Hoffmann-Köhler : I. Biedermann : J. Krücken : G. von Samson-Himmelstjerna : J. Demeler (*) Institute for Parasitology and Tropical Veterinary Medicine, Freie Universität Berlin, Robert-von-Ostertag-Str. 7-13, 14163 Berlin, Germany e-mail: [email protected] I. Biedermann : R. Koopmann Johann Heinrich von Thuenen Institute, Federal Research Institute for Rural Areas, Forestry and Fisheries, Institute of Organic Farming, Trenthorst, Germany J. M. Molina Parasitology Unit, Department of Animal Pathology, Faculty of Veterinary Medicine, University of Las Palmas de Gran Canaria, Las Palmas, Spain A. M. Moreno Unit of Parasitology, Animal Health Department, Faculty of Veterinary Medicine, University of Cordoba, Cordoba, Spain

goats raised parasite-free or infected experimentally. Sampling continued during the following pasture season and housing period. The sensitivity for the use in bulk milk samples as an indicator of T. circumcincta infection levels in grazing goats was examined. The ELISA enables clear differentiation of negative and positive animals. With a specificity of 100 % negative cut-off values for serum and milk were 0.294 and 0.228 (sensitivity, 95 %). Positive cut-off values (sensitivity, 90 %) were 0.606 (serum) and 0.419 (milk), while a sensitivity of 95 % resulted in 0.509 and 0.363, respectively. The greyzone between negative/positive cut-offs was introduced to deal with animals in pre-patency and decreasing antibody levels after infection. There was no cross reactivity for Trichostrongylus colubriformis and Cooperia oncophora while for Haemonchus contortus and Fasciola hepatica it cannot be fully excluded currently. In bulk milk samples, 5 % of the milk had to be contributed from animals infected with T. circumcincta to be detected as positive. The results derived from experimentally and naturally infected as well as parasite naïve animals indicate the potential of the ELISA to be used in targeted anthelmintic treatment regimes in goats. Keywords Teladorsagia . ELISA . Goat . Gastrointestinal nematodes . Diagnostic . Milk . Serum

Introduction Teladorsagia circumcincta is considered one of the most important causative agents of parasitism of the gastrointestinal tract in small ruminants. It has been identified as the predominant gastrointestinal nematode (GIN) in small ruminant farms in countries of South Europe (Gallidis et al. 2009; Molina et al. 1997) as well as in sheep in the UK and in Australia/New

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Zealand (Vlassoff and McKenna 1994) and is usually involved in seasonal outbreaks of mixed GIN-related gastroenteritis mainly affecting weaned kids/lambs and yearling animals (Gruner et al. 1994; Sargison et al. 2007) and also limiting milk production (Chartier et al. 2000) in lactating ewes. The economic losses due to GIN-related gastroenteritis result from the increased cost of anthelmintic treatment and the reduced nutrient conversion rates, weight gain and productive performance of the infected animals (Lawton et al. 1996; Simpson et al. 2009; Stear et al. 2003; Vlassoff and McKenna 1994). Protective immunity against T. circumcincta is manifested as cell-mediated (degranulation of sensitised mast cells and globule leucocytes) and subsequent immunoglobulin-mediated host response (McKellar 1993; Venturina et al. 2013). Although IgE production appears to play an important role in parasite expulsion, IgA are considered to be the major class of antibodies protecting against T. circumcincta (Venturina et al. 2013). An enzyme-linked immunosorbent assay (ELISA) using a recombinant protein disulphide isomerase to detect IgA in sheep has been developed (Martinez-Valladares et al. 2007). However, this antigen is more than 95 % identical to other trichostrongylids, suggesting strong cross reactivity. It has been shown that worm burden, faecal egg count (FEC) and the mean length of female parasites are negatively related to the levels of antiT. circumcincta IgA (Beraldi et al. 2008; Halliday et al. 2007; Strain et al. 2002). Age, sex and previous immunity of the host to T. circumcincta affect the level and time of antibody production against the parasite, with young ruminants and parasite-naïve animals exhibiting a slower immune response (McKellar 1993; Venturina et al. 2013). However, for diagnostic purposes IgG is generally preferable since it is present in much higher concentrations than other antibody classes (Zhao et al. 2006). Given the fact that the immunity of the host, and particularly young animals, cannot prevent the establishment of T. circumcincta infection, antiparasitic interventions are considered necessary towards the avoidance of animal disease and production losses. However, the issue of T. circumcincta parasitism in goats remains evergreen, since the extensive use of anthelmintics has led to the emergence of anthelmintic resistant parasites (Gallidis et al. 2009; Sargison et al. 2010; Silvestre and Humbert 2002; Wilson and Sargison 2007). Given the public demand on the reduction of antiparasitic drugs administered to farm animals and the increasing inefficiency of those currently used against anthelmintic-resistant nematodes, the need for targeted GIN control programmes based on the identification of individual animals or goat flocks suffering from T. circumcincta infection has emerged. For specific detection of Teladorsagia infections in sheep, a copro-antigen ELISA was published by Johnson et al. (2004). Additionally, detection of anti-T. circumcincta antibodies in individual and bulk milk samples has been

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previously described as an alternative to the gold standard of FECs to determine the parasite infections status in small ruminants (Cruz-Rojo et al. 2012; Molina et al. 2009). Molina et al. (2009) reported the preliminary use of an ELISA for the detection of anti-T. circumcincta antibodies in bulk milk samples. Remarkably, antibody levels in serum declined already within 4 weeks, while antibodies in the milk of the same animals were detectable for 16 weeks post-treatment. Levels of parasite-specific IgG in bulk milk, which have been reported to be higher than the mean value of individual milk samples, allow the detection of T. circumcincta infection as soon as in the individual milk samples (Molina et al. 2009). However, this study suffers from the fact that no defined parasitenaϊve goats were available to serve as negative controls. In addition, no calculations of specificity or sensitivity of such ELISA assays are available yet. Finally, no information has been obtained regarding the limits of sensitivity in bulk milk samples in terms of the proportion of milk from infected individuals leading to a positive bulk milk value. Overall, levels and variation pattern of antibodies in milk can potentially be affected by several factors, including milk composition, udder health status, age, number of lactation, lactation stage and health status of the animal. A recent study conducted in lactating ewes experimentally infected with T. circumcincta has shown that parasite-specific IgG levels in milk are particularly affected by the stage of lactation (Cruz-Rojo et al. 2012). Since animals between 3 and 5 years of age were used in the study reported by Molina et al. (2009), influence of these effects cannot be excluded. Therefore the present study aimed at working with as homogeneous groups as possible by using goats from the same flock, at the same age, in their first lactation and in the same stage of lactation (maximum 14 days apart). The present study aimed at the development of an ELISA specifically for goats for the detection of anti-T. circumcincta IgG using experimentally infected animals of the same age and validate its use in serum and milk samples collected from the same goats during the following pasture season. Moreover, the sensitivity for its use in bulk milk samples as an indicator of T. circumcincta infection levels in grazing goat flocks was examined. Preliminary results regarding the specificity of the ELISA were obtained using serum samples from parasite naïve sheep and goats experimentally infected with different GIN and Fasciola hepatica.

Materials and methods Parasites An anthelmintic susceptible isolate of T. circumcincta which was initially isolated from a farm in Lower Saxony, Germany, was used for the experimental infection of animals and for

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antigen production. This isolate has been passaged in sheep at the Institute for Parasitology at the University for Veterinary Medicine Hannover, Germany, since 2005. Post-treatment FECs obtained for this isolate as well as results from in vitro assays performed using macrocyclic lactones (MLs) as well as benzimidazoles (BZs) prove the isolate to be susceptible to these anthelmintics (data not shown). Animal infection In order to exclude exposition to other GIN 16 new born male kids were kept in concrete pens without access to pasture or fresh cut grass. The parental goats had been housed 4 months prior to kidding and were dewormed using moxidectin approximately one week before parturition. The kids were born with 2 weeks and separated from their mothers at 11–12 weeks of age. To obtain blood samples for defined T. circumcincta negative animals, half of the group were left without infection while the other half received an infection dose of 35,000 infective third stage larvae (L3) orally at 12 weeks of age. Individual blood and faecal samples were taken prior to the start of the experiment, at the day of infection (d0) and every 2–3 days from d9 to d46. To be able to obtain milk from animals without previous exposure to GIN, the female kids (n=12) were only used for the second infection experiment. Until they were mated with males around 1 year of age, they were consistently kept parasite-free in concrete in-house pens without access to pasture and fresh grass. FECs were performed in regular intervals and remained negative for the whole period of time. The kids were born in late January/early February (within 14 days). Half of the female goats (n=6) were orally infected with T. circumcincta L3 (positive controls) on the 1st of April (between 8 and 9 weeks post-partum) while the other half was left uninfected (negative controls) and paired samples (milk and blood) as well as faecal samples (data not shown) were collected in similar intervals as in the first experiment. Sampling was performed prior to the experiment, at the day of infection (d0), daily from d12 to d29, every 2–4 days between d30 and d63 followed by approximately weekly sampling until the end of the experiment (d345). Serum samples were available for the whole course of the study while milk samples were only available until d210 (end of lactation period). On d64 post-infection all experimentally infected animals were treated with moxidectin (Cydectin®, 0.1 %) using 1.5× of the recommended dose for sheep (0.2 mg/kg body weight) and turned out onto pasture on d88. In order to follow the course of antibody production and decline, sampling was continued throughout the whole grazing season. FECs were monitored regularly and remained GIN positive during the grazing season. All animals were again dewormed at the time of housing (d218 of the experiment), and sampling continued in the following stabling period until d345. FECs were negative

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for GIN eggs 3 days post-treatment and remained negative until the end of the experiment. Additional to FECs larval cultures were performed once per week and larvae obtained from those cultures were subjected to DNA isolation followed by molecular species analyses using species-specific PCR as recently described (Demeler et al. 2013). All animal experiments were approved by the local authorities, the “Ministerium für Landwirtschaft, Umwelt und ländliche Räume, Schleswig-Holstein” (experimental infections and field experiments, Trenthorst) and the “Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit“(initial passaging and antigen production, Hannover). Antigen production Five male kids experimentally infected with T. circumcincta in the first experiment were euthanised (three on day 28 days post-infection and two on day 65 post-infection) using a captive bold pistol followed by cutting the jugular veins. The abomasum and first centimetres of the duodenum were collected and male and female Teladorsagia worms were collected using an active migration method as recently described by Demeler et al. (2014). Worms were intensively washed using 37 °C warm saline (0.9 %). They were subsequently washed with distilled water, transferred to a glass vial and stored at −80 °C for 24 h. 443.5 g fresh worms were lyophilised, resulting in 40.3 mg and re-stored at −80 °C. Lyophilised worms were suspended in 4 ml PBS (pH 7.2) and 24 μl 0.5 M sodium azide. Two freeze crack cycles (−80 °C vs. room temperature) were applied, followed by homogenisation using TissueRuptor® (Qiagen) and ultrasound (MSE). The obtained suspension centrifuged for 30 min at 4 °C and 10,000×g, the supernatant was discarded and protein content determined using the Bradford-reagent (Bio-Rad®). Development of the ELISA and calculation of cut-off values For the development of the ELISA the first experiments were performed using serum samples of the first experimental infection (male kids) and the commercially available Svanovir® Ostertagia ostertagi ELISA for cattle. No clear differentiation between negative and positive animals was obtained. An all ruminant antibody kindly provided by Svanovir ® as well as the use of a horseradish peroxidasecoupled rabbit anti-goat IgG secondary antibody (Dianova®) improved results slightly but results were still not satisfying concerning the discrimination of sera from infected and uninfected animals. Finally, the horseradish peroxidase-coupled rabbit antigoat IgG secondary antibody in combination with the

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Teladorsagia crude antigen obtained from adult worms (see previous section) enabled clear differentiation of positive and negative animals. Suitable antigen and antibody concentrations as well as serum dilution were evaluated using a checkerboard titration. Once milk samples from negative and positive animals became available, milk samples were used undiluted in the ELISA and optical density (OD) as well as optical density ratios (ODR) values were compared with those obtained from the paired serum samples. Milk samples were used undiluted, and serum dilution was adjusted to give similar OD values as observed for the corresponding milk sample. The final ELISA protocol as performed in the study is described in the following section. In order to test for cross reactivity, serum samples of goats experimentally infected with Haemonchus contortus were used. Since no serum samples from previously parasitenaïve goats mono-infected with other trichostrongylids were available, cross reactivity between T. circumcincta and Trichostrongylus colubriformis, Cooperia oncophora and H. contortus was analysed using serum from mono-infected (previously parasite naïve) sheep. For this purpose, the same ELISA protocol with a secondary antibody against sheep IgG was used. Additionally, serum samples from goats pre- and post-infection with F. hepatica were used. Final optimised ELISA protocol Levels of anti-T. circumcincta antibodies in blood serum and milk samples were detected and quantified as ODR values. Flat-bottomed, 96-well microplates (Nunc Maxisorp®) were coated with T. circumcincta crude antigen (32.25 mg protein/ml) at a 1:5,000 (v/v) concentration in 0.05 M carbonate-bicarbonate buffer (pH 9.6; 200 μl per well containing 1.29 μg protein), incubated at 37 °C for 1 hand then at 4 °C over night. Wells were washed three times with 200 μl 0.05 % PBS-Tween 20 solution and non-specific binding sites were blocked by adding 100 μl blocking buffer (2 % (w/v) solution of powdered milk in 0.05 % PBS-Tween 20) in each well and incubating at 37 °C for 1 h. After washing as described above, two replicates (100 μl) of undiluted milk or diluted serum samples (1:100 (v/v) with blocking buffer solution) were added. In each plate, six replicates (100 μl) of positive and six replicates (100 μl) of negative control sera were also tested. Following incubation and washing as before, 100 μl of conjugate solution (1:40,000 (v/v) secondary antibody in blocking buffer) were added into each well. The microplate was then incubated and washed as before. Substrate solution was freshly prepared by diluting one tablet ophenylenediamine dihydrochloride (P-5412 Sigma-Aldrich, Germany) in 20 ml citric acid-phosphate buffer (pH 5–5.2); 10 μl of hydrogen peroxidase were added to 20 ml of substrate solution immediately before applying to the plate. Each well was filled with 100 μl of substrate solution, and the plate was incubated at 37 °C for 30 min in the dark. At the end of the

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incubation, 50 μl of 2.5 M HCl stop-solution were added to each well. Absorbance was measured at 492 nm and expressed as OD for each sample. Levels of anti-T. circumcincta antibodies were quantified as relative ODR and calculated according to the following formula: ODR=(OD–NC)/(PC–NC), where OD is the optical density value of the sample tested and NC and PC are the mean density values computed out of the six replicates of negative and positive control, respectively. The ELISA assay was applied identically for milk and blood serum samples. Statistical analysis For the determination of cut-off ODR values true positive (day 21 post-infection or post-turnout) and true negative samples (negative control group until turnout and positive control group before infection) were used. Receiver operating characteristics (ROC) analysis was performed using GraphPad Prism 5.0. Cut-off values for the classification “negative” were defined using a specificity of 100 % and a sensitivity of 99.8 % (serum) and 98.9 % (milk). Cut-off values for the classification “positive” were defined using a specificity of 100 % and a sensitivity of 95 % (for both, serum and milk samples). As an alternative, a second positive cut-off value was calculated, using the same specificity but a sensitivity of 90 %, in order to identify fewer animals falsely positive after treatment. The area between negative and positive cut-off values is referred to as “grey zone”, including particularly animals within the pre-patency or weeks after treatment, when antibody titres are decreased. A Student’s t test with Welsh correction was applied to compare differences in ODR between different days (e.g. between two different peaks).

Results Calculation of cut-off values The individual ODR results obtained during the course of the experiment are plotted in Figs. 1 (serum) and 2 (milk). The calculation of the negative cut-off value for serum samples resulted in 0.294, for milk samples a value of 0.228 was obtained. The positive cut-off value using a sensitivity of 90 % resulted in 0.606 (serum) and 0.419 (milk). If a sensitivity of 95 % is used, the cut-off values are 0.509 and 0.363, respectively. All cut-off values and statistical details are given in Table 1. Development of the ELISA using experimentally infected animals Different antigen and antibody concentrations as well as blood dilution ranges were tested (results not shown). Milk was used undiluted and blood samples were diluted 1:100.

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Fig. 1 Time course of optical density ratio (ODR) values of serum samples derived from negative control animals (a) and animals initially experimentally infected with Teladorsagia circumcincta (b). Dots (black) represent individual measurements; the squares (blue) are values derived from a single animal where the experimental infection produced only a very low or no epg (not included in the calculation of cut-off values); the solid line is the calculated negative cut-off; the dotted line represent the calculated positive cut-off (sensitivity, 95 %); and the dashed line is the calculated positive cut-off (sensitivity, 90 %). T turnout, P start of patency, D deworming, H housing

All samples from non-infected animals of the first experiment remained negative throughout the course of the experiment. In experimentally infected animals, an increase in ODR values due to antibody production was obtained already on day 12–14 post-infection (pre-patency) and remained high until the end of the experiment. These serum samples were only used for initial determination of ELISA parameters (results not shown). Blood and milk samples from the goats of the second experiment followed similar patterns as blood samples from the kids, with all non-infected goats remaining negative throughout the infection part of the experiment. Antibody responses for serum samples All animals of the infected group showed an increase in antibody titre between day 14 and 19 post-infection. The exception was one technical replicate series, which gave lower results compared with the corresponding four other replicate series of the same animal. Though ODR values obtained from positive animals were slightly different, all animals were detected clearly positive at day 23 or 27 using the 95 or 90 % sensitivity-based cut-off, respectively. ODR values

decreased slightly after anthelmintic treatment at day 64 but turnout onto pasture was (accidently) too early (d88). After turnout the ODR values of the previously infected group immediately increased and reached values higher (peak at d178) than during the peak of the first infection (d33), but this was not statistically significant (p=0.195). This was different for the previously negative group. Here ODR values were also elevated at day 17 after turnout but increase was much slower. The peak of antibody titres was reached at day 168 (80 days post-turnout vs. 33 days post-experimental infection) with ODR values only slightly lower to those of the experimental infection group during primary (experimental) infection (p=0.204). For all animals, a decrease in ODR values was obtained following deworming prior to housing (d210) and the subsequent housing period without exposure to parasites with ODR values for all animals below (or approximately at) the negative cut-off value of 0.281. Comparison of the ODR values before infection and at the last day of the experiment (d345) revealed no differences for the negative control group (p=0.490). For the experimental infection group, the ODR values did steadily decrease but were still slightly higher compared with day 1 (p=0.017). The

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Additionally, third stage larvae obtained from faecal cultures subjected to species specific PCRs revealed the presence of T. circumcincta in all experimentally infected as well as all pastured animals. The negative group remained negative until turnout. Besides, T. circumcincta also T. circumcincta and H. contortus were detected in the majority of but not all individuals.

Antibody responses for milk samples

Fig. 2 Time course of optical density ratio (ODR) values of milk samples derived from negative control animals (a) and animals initially experimentally infected with Teladorsagia circumcincta (b). Dots (black) represent individual measurements; the squares (blue) are values derived from a single animal where the experimental infection produced only a very low or no epg (not included in the calculation of cut-off values). The solid line shows the calculated negative cut-off; the dotted line is the calculated positive cut-off (sensitivity, 95 %), and the dashed line is the calculated positive cut-off (sensitivity, 90 %). T turnout, P start of patency, D deworming, H housing

course of antibody levels (mean of all animals) in serum is provided in Fig. 3.

Table 1 Results of the receiver operating characteristics analysis for negative and positive cut-off values including optical density ratio (ODR), sensitivity and specificity with confidence intervals (CI) in percent

Serum Negative cut-off 1. Positive cut-off 2. Positive cut-off Milk Negative cut-off 1. Positive cut-off 2. Positive cut-off

As observed for the serum samples, for all infected animals an increase in antibody level in the milk was detected and at day 15 all values were above the negative cut-off value of 0.228. Again ODR values obtained from individuals showed little variation but all animals were detected clearly positive at day 21 or 23 using the 95 or 90 % sensitivity-based cut-off, respectively. Generally, the same time course of antibody increase/decrease as for serum samples was observed. One exception is one animal of the infected group, which generally displayed low antibody titres throughout the course of infection. This animal also had much lower egg counts than the rest of the group throughout the period of patency (data not shown) and “lost” the infection that early, that the experimental infection was considered as not successful. Due to the beginning of the dry period milk samples were only available until d210, so the decrease in specific antibody levels could not be followed as long as in serum samples. The course of antibody levels (mean of all animals) in milk is provided in Fig. 3. Additionally, the sensitivity of the ELISA using artificially composed bulk milk samples was assessed. For this purpose, one positive sample was diluted 1:20–1:200 using the corresponding amount of negative samples. The 1:20 up to the 1:80 dilutions resulted in an ODR value above the negative cut-off, and only for the 1:20 dilution an ODR value above the positive cut-off (90 % sensitivity) was obtained. Nevertheless, that means, that any bulk milk sample is detected as positive when 5 % of the sample is derived from T. circumcinctainfected animals.

ODR

Specificity

95 % CI

Sensitivity

95 % CI

0.294 0.509 0.606

100 100 100

99.53–100 99.53–100 99.53–100

99.91 95.07 90.01

99.50–100 93.62–96.24 88.11–91.69

0.228 0.363 0.419

100 100 100

98.88–100 98.88–100 98.88–100

98.88 94.94 90.17

97.15–99.69 92.13–96.98 86.59–93.06

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Fig. 3 Time course of mean optical density ratio (ODR) values of serum and milk samples derived from negative control animals and animals initially experimentally infected with Teladorsagia circumcincta. Whiskers represent the SD, either the SD above or below the mean value. T turnout, P start of patency, D deworming, H housing

Cross reactivity with other GIN using serum samples from goat and sheep Cross reactivity of the ELISA was assessed using samples from animals mono-infected with other GINs (Trichostrongylus, Cooperia and Haemonchus) and with liver fluke. Since serum samples from goats infected with other GIN were not available, the cross reactivity to T. colubriformis, C. oncophora and H. contortus was tested using serum samples from previously parasite naive, experimentally monoinfected sheep and the respective corresponding secondary antibody. Additionally, serum samples from seven goats before and after the infection with F. hepatica were tested. However, these animals were not raised parasite naïve and some probably had contact with T. circumcincta before the experimental infection. Three out of seven samples showed no clear cross reactivity, however, the ODR values for all three were slightly increased after infection but not exceeding the negative cut-off. For the other four animals, higher ODRs (0.37–0.53) were already obtained before infection with F. hepatica and after infection sample ODRs were between 0.9 and 1.1. Goats infected with H. contortus showed moderately increased ODR values (0.35–0.64). Though not reaching considerably high values, at least partial cross reactivity for this parasite species in goats cannot be fully excluded. Unfortunately, it was not known, if these animals had previously been exposed to T. circumcincta, so possible persistence of respective antibodies cannot be excluded. Performance of the ELISA with sera from sheep revealed no cross reactivity for H. contortus and C. oncophora. ODR values for T. colubriformis were slightly higher but still did not exceed the negative cut-off value. All animals infected with T. circumcincta appeared clearly positive while negative control animals remained negative. Additionally, serum samples

from new born lambs (truly naïve animals) were tested. The ODR values of this group were much lower than of noninfected animals after uptake of colostrum (Fig. 4). The ODR values of the individual animals are presented in Table S1 (Electronic supplementary material). Statistical analysis (ANOVA with Bartlett’s test for equal variances) revealed highly significant (p