Enhancing bovine abortion surveillance: A learning experience.

Abortions and perinatal mortalities (APM) substantially affect cattle industry efficiency. Various infectious and noninfectious factors have been associated with bovine APM worldwide. Infections are often considered pivotal due to their abortifacient potential, leading laboratories to primarily investigate relevant infectious agents for APM cases. Some infectious causes, such as Brucella abortus, have also a zoonotic impact, necessitating monitoring for both animal and human health. However, underreporting of bovine APM is a global issue, affecting early detection of infectious and zoonotic causes. Previous studies identified factors influencing case submission, but regional characteristics may affect results. In Belgium, farmers are obliged to report cases of APM within the context of a national brucellosis monitoring program. The inclusion criteria for this monitoring program cover abortions (gestation length of 42-260 d) and perinatal mortalities of (pre)mature calves following a gestation length of more than 260 d, which were stillborn or died within 48 h after birth. The objective of the present study was to describe the evolution in submission of APM cases within a mandatory abortion monitoring program in relation to subsidized initiatives in the northern part of Belgium. Based on the proportion of APM submissions versus the proportion of bovine reproductive females, an APM proportion (APMPR) was calculated, and factors at both animal and herd level that may influence this APMPR were explored by using linear models. This evaluation revealed that the APMPR increased with the introduction of an extensive analytical panel of abortifacient agents and a free on-farm sample collection from 0.44% to 0.94%. Additionally, an increase of the APMPR was associated with an outbreak of an emerging abortifacient pathogen (Schmallenberg virus; 1.23%), and the introduction of a mandatory eradication program for bovine viral diarrhea virus (BVDv; 1.20%). The APMPR was higher in beef compared with dairy cattle, and it was higher in winter compared with fall, spring, and summer. Smaller herds categorized in the first quartile had a higher APMPR compared with larger herds. Herds that submitted an APM in the previous year had a higher APMPR in the next year compared with herds without an APM submission. Finally, herds for which there was evidence of the presence of BVDv had a higher APMPR compared with herds without evidence of the presence of BVDv. In conclusion, the number of APM submissions increased after the introduction of a free on-farm sample collection and an extensive pathogen screening panel. Production type (beef), season (winter), smaller herd size, previous APM, and presence of BVDv seemed to have a positive effect on APMPR. However, even under mandatory circumstances, APM still seems to be underreported, since the APMPR was lower than the expected minimal rate of 2%. Therefore, further research is necessary to identify the drivers that convince farmers to submit APM cases to improve submission rates and ensure an efficient monitoring program for APM and eventually associated zoonotic pathogens.

Probing hybridization parameters from microarray experiments: nearest-neighbor model and beyond.

In this article, it is shown how optimized and dedicated microarray experiments can be used to study the thermodynamics of DNA hybridization for a large number of different conformations in a highly parallel fashion. In particular, free energy penalties for mismatches are obtained in two independent ways and are shown to be correlated with values from melting experiments in solution reported in the literature. The additivity principle, which is at the basis of the nearest-neighbor model, and according to which the penalty for two isolated mismatches is equal to the sum of the independent penalties, is thoroughly tested. Additivity is shown to break down for a mismatch distance below 5 nt. The behavior of mismatches in the vicinity of the helix edges, and the behavior of tandem mismatches are also investigated. Finally, some thermodynamic outlying sequences are observed and highlighted. These sequences contain combinations of GA mismatches. The analysis of the microarray data reported in this article provides new insights on the DNA hybridization parameters and can help to increase the accuracy of hybridization-based technologies.

Machine learning modeling to predict causes of infectious abortions and perinatal mortalities in cattle.

A plethora of infectious and non-infectious causes of bovine abortions and perinatal mortalities (APM) have been reported in literature. However, due to financial limitations or a potential zoonotic impact, many laboratories only offer a standard analytical panel, limited to a preestablished number of pathogens. To improve the cost-efficiency of laboratory diagnostics, it could be beneficial to design a targeted analytical approach for APM cases, based on maternal and environmental characteristics associated with the prevalence of specific abortifacient pathogens. The objective of this retrospective observational study was to implement a machine learning pipeline (MLP) to predict maternal and environmental factors associated with infectious APM. Our MLP based on a greedy ensemble approach incorporated a standard tuning grid of four models, applied on a dataset of 1590 APM cases with a positive diagnosis that was achieved by analyzing an extensive set of abortifacient pathogens. Production type (dairy/beef), gestation length, and season were successfully predicted by the greedy ensemble, with a modest prediction capacity which ranged between 63 and 73 %. Besides the predictive accuracy of individual variables, our MLP hierarchically identified predictor importance causes of associated environmental/maternal characteristics of APM. For instance, in APM cases that happened in beef cows, season at APM (spring/summer) was the most important predictor with a relative importance of 24 %. Furthermore, at the last trimester of gestation Trueperella pyogenes and Neospora caninum were the most important predictors of APM with a relative importance of 22 and 17 %, respectively. Interestingly, herd size came out as the most relevant predictor for APM in multiparous dams, with a relative importance of 12 %. Based on these and other mix of predicted environmental/maternal and pathogenic potential causes, it could be concluded that implementing our MLP may be beneficial to design a more cost-effective, case-specific diagnostic approach for bovine APM cases at the diagnostic laboratory level.

A new approach to study exhaled proteins as potential biomarkers for asthma.

BACKGROUND: Asthma is a complex clinical disease characterized by airway inflammation. Recently, various studies reported on the analysis of exhaled breath condensate (EBC) in the search for potential biomarkers for asthma. However, in a complex disease such as asthma, one biomarker might not be enough for early diagnosis or follow-up. OBJECTIVE: The use of proteome analysis may reveal disease-specific proteolytic peptide or protein patterns, and may lead to the identification of novel proteins for the detection of asthma. METHODS: Liquid chromatography and mass spectrometry were used to separate and detect proteins (proteolytic peptides) present in EBC samples from 30 healthy children and 40 children with asthma in the age group of 6-12 years. RESULTS: Support vector machine analysis resulted in differentiating profiles based on asthma status. These proteolytic peptide patterns were not correlated to some well known (spirometry, exhaled nitric oxide) and more recently described exhaled markers (EBC pH, LTB4). The more abundant proteins in EBC were identified as cytokeratins, albumin, actin, haemoglobin, lysozyme, dermcidin, and calgranulin B. CONCLUSION: Although the exact role in the disease development or physiological state of the airways of the proteins described in the presented pattern is not clear at this moment, this is an important step in the search for exhaled biomarkers for asthma. This study shows that EBC contains proteins that are of interest for future non-invasive asthma diagnosis or follow-up.

The effects of mismatches on hybridization in DNA microarrays: determination of nearest neighbor parameters.

Quantifying interactions in DNA microarrays is of central importance for a better understanding of their functioning. Hybridization thermodynamics for nucleic acid strands in aqueous solution can be described by the so-called nearest neighbor model, which estimates the hybridization free energy of a given sequence as a sum of dinucleotide terms. Compared with its solution counterparts, hybridization in DNA microarrays may be hindered due to the presence of a solid surface and of a high density of DNA strands. We present here a study aimed at the determination of hybridization free energies in DNA microarrays. Experiments are performed on custom Agilent slides. The solution contains a single oligonucleotide. The microarray contains spots with a perfect matching (PM) complementary sequence and other spots with one or two mismatches (MM) : in total 1006 different probe spots, each replicated 15 times per microarray. The free energy parameters are directly fitted from microarray data. The experiments demonstrate a clear correlation between hybridization free energies in the microarray and in solution. The experiments are fully consistent with the Langmuir model at low intensities, but show a clear deviation at intermediate (non-saturating) intensities. These results provide new interesting insights for the quantification of molecular interactions in DNA microarrays.

Retrospective study of factors associated with bovine infectious abortion and perinatal mortality.

Abortion and perinatal mortality, leading causes of economic loss in cattle industry, are the consequence of both non-infectious and a wide range of infectious causes. However, the relative contribution of pathogens to bovine abortion and perinatal mortality is poorly documented, since available studies involved only a limited number of pathogens. Therefore, the objectives of the present monitoring study were to determine the prevalence of infectious agents associated with bovine abortion and perinatal mortality, and to identify differences in production type, gestation length, parity and seasonality by using mixed effect models (logistic regression). A pre-established sampling protocol based on the collection of the aborted fetus/calf and a corresponding maternal blood sample, involving diagnostic testing for 10 pathogens, was performed. At least one potential causal agent of the abortion or perinatal mortality was detected in 39 % of cases. In these diagnosed cases, Neospora caninum was the most detected pathogen, followed by Trueperella pyogenes, BVDv, Escherichia coli, and Aspergillus fumigatus. Neospora caninum [odds ratio (OR): 0.4; 95 % confidence interval (CI): 0.3-0.7] and Aspergillus fumigatus (OR: 0.1; 95 % CI: 0.1-0.3) were detected less in late versus early gestation. Aspergillus fumigatus was less common in dairy in comparison to beef abortion cases (OR: 0.2; 95 % CI: 0.1-0.6). Winter was associated with a lower positivity for Neospora caninum and BVDv in comparison to warmer seasons. Despite extensive diagnostic testing, an etiological diagnosis was not reached in 61 % of cases, highlighting the need for even more extensive (non-)infectious disease testing or more accurate tests.

Establishing the spread of bluetongue virus at the end of the 2006 epidemic in Belgium.

Bluetongue (BT) was notified for the first time in several Northern European countries in August 2006. The first reported outbreaks of BT were confirmed in herds located near the place where Belgium, The Netherlands and Germany share borders. The disease was rapidly and widely disseminated throughout Belgium in both sheep and cattle herds. During the epidemic, case reporting by the Veterinary Authorities relied almost exclusively on the identification of herds with confirmed clinical infected ruminants. A cross-sectional serological survey targeting all Belgian ruminants was then undertaken during the vector-free season. The first objective of this study was to provide unbiased estimates of BT-seroprevalence for different regions of Belgium. Since under-reporting was suspected during the epidemic, a second goal was to compare the final dispersion of the virus based on the seroprevalence estimates to the dispersion of the confirmed clinical cases which were notified in Belgium, in order to estimate the accuracy of the case detection based on clinical suspicion. True within-herd seroprevalence was estimated based on a logistic-normal regression model with prior specification on the diagnostic test's sensitivity and specificity. The model was fitted in a Bayesian framework. Herd seroprevalence was estimated using a logistic regression model. To study the linear correlation between the BT winter screening data and the case-herds data, the linear predicted values for the herd prevalence were compared and the Pearson correlation coefficient was estimated. The overall herd and true within-herd seroprevalences were estimated at 83.3 (79.2-87.0) and 23.8 (20.1-28.1)%, respectively. BT seropositivity was shown to be widely but unevenly distributed throughout Belgium, with a gradient decreasing towards the south and the west of the country. The analysis has shown there was a strong correlation between the outbreak data and the data from the survey (r=0.73, p<0.0001). The case detection system based on clinical suspicion underestimated the real impact of the epidemic, but indicated an accurate spatial distribution of the virus at the end of the epidemic.

Gene expression profiling of in vitro cultured macrophages after exposure to the respiratory sensitizer hexamethylene diisocyanate.

Occupational exposure to chemicals is one of the main causes of respiratory allergy and asthma. Identification of chemicals that trigger allergic asthma is difficult as underlying processes and specific markers have not yet been clearly defined. Moreover, adequate classification of the respiratory toxicity of chemicals is hampered due to the lack of validated in vivo and in vitro test methods. The study of differential gene expression profiles in appropriate human in vitro cell systems is a promising approach to identify selective markers for respiratory allergy. As alveolar macrophages display important immunological and inflammatory properties in response to foreign substances in the lung, we aimed at gaining more insight in changes of human macrophages transcriptome and to identify selective genetic markers for respiratory sensitization in response to hexamethylene diisocyanate (HDI). In vitro cultures of human THP-1 cells were differentiated into macrophages and exposed to 55 microg/ml HDI for 6 and 10h. Using human oligonucleotide microarrays, changes were observed in the expression of genes that are involved in diverse biological and molecular processes, including detoxification, oxidative stress, cytokine signaling, and apoptosis, which can lead to the development of asthma. These genes are possible markers for respiratory sensitization caused by isocyanates.

Dynamic range extension of hybridization sensors.

In hybridization based nucleic acid sensors the stringency of hybridization poses a challenge to design and experiment. For a given set of experimental parameters the affinity window of probe-target interaction is always limited and vice versa for a given probe set design, changes in experimental conditions can easily bring some measurements out of detection range. In this paper we introduce and apply a strategy to extend this dynamic range for affinity sensors, sensors which measure the amount of hybridized molecules after equilibrium is reached. The method relies on concepts of additivity of nucleic acids hybridization free energies and on equilibrium isotherms. It consists in combining the measurements from probes with different lengths, by appropriately rescaling the measured signals. We test the validity of the approach on experiments and show that by combining probes with hybridizing regions of length 21, 23 and 25 nucleotides we manage to extend the dynamic range of the intensity signals by a factor of 25. The presented concept is easy to extend, platform free and applies to any hybridization based affinity sensor.

Serological diagnosis of bovine neosporosis: a Bayesian evaluation of two antibody ELISA tests for in vivo diagnosis in purchased and abortion cattle.

Currently, there are no perfect reference tests for the in vivo detection of Neospora caninum infection. Two commercial N caninum ELISA tests are currently used in Belgium for bovine sera (TEST A and TEST B). The goal of this study is to evaluate these tests used at their current cut-offs, with a no gold standard approach, for the test purpose of (1) demonstration of freedom of infection at purchase and (2) diagnosis in aborting cattle. Sera of two study populations, Abortion population (n=196) and Purchase population (n=514), were selected and tested with both ELISA's. Test results were entered in a Bayesian model with informative priors on population prevalences only (Scenario 1). As sensitivity analysis, two more models were used: one with informative priors on test diagnostic accuracy (Scenario 2) and one with all priors uninformative (Scenario 3). The accuracy parameters were estimated from the first model: diagnostic sensitivity (Test A: 93.54 per cent-Test B: 86.99 per cent) and specificity (Test A: 90.22 per cent-Test B: 90.15 per cent) were high and comparable (Bayesian P values >0.05). Based on predictive values in the two study populations, both tests were fit for purpose, despite an expected false negative fraction of ±0.5 per cent in the Purchase population and ±5 per cent in the Abortion population. In addition, a false positive fraction of ±3 per cent in the overall Purchase population and ±4 per cent in the overall Abortion population was found.

Follow-up of the Schmallenberg Virus Seroprevalence in Belgian Cattle.

Schmallenberg virus (SBV), which emerged in Northwestern Europe in 2011, is an arthropod-borne virus affecting primarily ruminants. Based on the results of two cross-sectional studies conducted in the Belgian ruminant population during winter 2011-2012, we concluded that at the end of 2011, almost the whole population had already been infected by SBV. A second cross-sectional serological study was conducted in the Belgian cattle population during winter 2012-2013 to examine the situation after the 2012 transmission period and to analyse the change in immunity after 1 year. A total of 7130 blood samples collected between 1st January and 28 February 2013 in 188 herds were tested for the presence of SBV-specific antibodies. All sampled herds tested positive and within-herd seroprevalence was estimated at 65.66% (95% CI: 62.28-69.04). A statistically significant decrease was observed between the beginning and the end of 2012. On the other hand, age-cohort-specific seroprevalence stayed stable from 1 year to the other. During winter 2012-2013, calves between 6 and 12 months had a seroprevalence of 20.59% (95% CI: 15.34-25.83), which seems to be an indication that SBV was still circulating at least in some parts of Belgium during summer-early autumn 2012. Results showed that the level of immunity against SBV of the animals infected has not decreased and remained high after 1 year and that the spread of the virus has slowed down considerably during 2012. This study also indicated that in the coming years, there are likely to be age cohorts of unprotected animals.

A competitive inhibition ELISA for the differentiation of serum antibodies from pigs infected with transmissible gastroenteritis virus (TGEV) or with the TGEV-related porcine respiratory coronavirus.

A competitive inhibition ELISA was developed to detect non-neutralizing antibodies to the peplomer protein of transmissible gastroenteritis virus (TGEV) in porcine sera using a monoclonal antibody as an indicator. It was demonstrated that field strains of the TGEV-related porcine respiratory coronavirus (PRCV) did not induce this antibody, whereas the Miller strain and field strains of TGEV did. The sensitivity of the competitive inhibition ELISA appeared to be similar to that of the virus neutralization (VN) test. The test enables differentiation of pigs which were previously infected with TGEV or PRCV and which cannot be distinguished by the classical anti-TGEV neutralization test. The present test is useful for selective serodiagnosis.

One-dimensional contact process: duality and renormalization.

We study the one-dimensional contact process in its quantum version using a recently proposed real-space renormalization technique for stochastic many-particle systems. Exploiting the duality and other properties of the model, we can apply the method for cells with up to 37 sites. After suitable extrapolation, we obtain exponent estimates that are comparable in accuracy with the best known in the literature.

Generalized contact process with n absorbing states.

We investigate the critical properties of a one-dimensional stochastic lattice model with n (permutation symmetric) absorbing states. We analyze the cases with n/=3 with exponents z=nu( ||)/nu( perpendicular)=2, nu( perpendicular)=1, and beta=1. These exponents coincide with those of the multispecies (bosonic) branching annihilating random walks. For n=3 we also show that, upon breaking the symmetry to a lower one (Z2), one gets a transition either in the directed percolation, or in the parity conserving class, depending on the choice of parameters.

Sites of replication of a porcine respiratory coronavirus in 5-week-old pigs with or without maternal antibodies.

On farms, where the porcine respiratory coronavirus (PRCV) is enzootic, pigs usually become infected between 5 and 10 weeks of age while losing their maternal antibodies. It was examined whether PRCV replicates in the small intestine in such pigs. This point is important since intestinal replication with PRCV might induce immunity against TGEV not only by stimulating mucosal intestinal immunity, but also by the induction of a lactogenic IgA response at later age via the gut-mammary link. Five week old pigs with and without maternal antibodies were inoculated by aerosol or directly into the intestinal lumen. In aerosol inoculated pigs, virus replication was observed to high titres in the respiratory tract. Replication occurred in epithelial cells of nasal mucosa, trachea, bronchi bronchioli and alveoli and in alveolar macrophages. Small amounts of virus produced in the respiratory tract were ingested, but no intestinal replication of PRCV was demonstrated. Differences were not observed in virus titre and sites of replication in seronegative pigs compared to those in pigs with maternal antibodies. Upon inoculation of 10(5) or 10(7) TCID50 directly into the lumen of the cranial jejunum, no intestinal replication could be demonstrated.

Sites of replication of a porcine respiratory coronavirus related to transmissible gastroenteritis virus.

A porcine respiratory coronavirus (PRCV) was inoculated by aerosol into nine hysterectomy-derived and colostrum-deprived pigs at the age of one week. They were killed at different times after inoculation and tissues were sampled for virus isolation and immunofluorescence. Results indicate that virus replicated to high titres in the respiratory tract. Replication mainly occurred in alveolar cells but also in epithelial cells of nasal mucosa, trachea, bronchi, bronchioli, in alveolar macrophages and in tonsils. After primary replication in the respiratory tract, viraemia occurred. Virus also reached the gastrointestinal tract after swallowing. Subsequently, PRCV was observed to replicate in the ileum. The infection spread within a few days from the ileum to the duodenum. Replication in the small intestine remained limited to a few cells located in or underneath the epithelial layer of villi and, or, crypts. The cell type could not be identified. Virus was isolated from mesenteric lymph nodes in all pigs, but immunofluorescence was not observed. Results show that small changes in molecular structure between transmissible gastroenteritis virus and PRCV resulted in important changes in host cell tropism.

Large-scale cross-sectional serological survey of Schmallenberg virus in Belgian cattle at the end of the first vector season.

A cross-sectional survey was conducted in the Belgian cattle population after the first period of infection of the emerging Schmallenberg virus. A total number of 11 635 cattle from 422 herds sampled between 2 January and 7 March 2012 were tested for the presence of Schmallenberg-specific antibodies using an ELISA kit. Between-herd seroprevalence in cattle was estimated at 99.76% (95% CI: 98.34-99.97) and within-herd seroprevalence at 86.3% (95% CI: 84.75-87.71). An Intraclass Correlation Coefficient of 0.3 (P < 0.001) was found, indicating that the correlation between two animals within a herd with respect to their serological status was high. Those results corroborate the conclusion that the Schmallenberg virus was widespread in Belgium during winter 2011. Seroprevalence was shown to be statistically associated to the animal's age (P < 0.0001): with 64.9% (95% CI: 61.34-68.3) estimated for the 6-12 months of age, 86.79% (95% CI: 84.43-88.85) for the 12-24 months of age and 94.4% (95% CI: 93.14-95.44) for the animals older than 24 months. Based on the results of the described serological survey, we can conclude that after the first Schmallenberg virus episode, almost every Belgian cattle has already been in contact with the virus. In consequence, the vast majority of the host animals should have developed post infection protective immunity against the virus.

Bovine tuberculosis surveillance alternatives in Belgium.

Belgium obtained the bovine tuberculosis (bTB) officially free status in 2003 (EC Decision 2003/467/EC). This study was carried out to evaluate the components of the current bTB surveillance program in Belgium and to determine the sensitivity of this program. Secondly, alternatives to optimize the bTB surveillance in accordance with European legislation (Council Directive 64/432/EEC) were evaluated. Separate scenario trees were designed for each active surveillance component of the bTB surveillance program. Data from 2005 to 2009 regarding cattle population, movement and surveillance were collected to feed the stochastic scenario tree simulation model. A total of 7,403,826 cattle movement history records were obtained for the 2,678,020 cattle from 36,059 cattle herds still active in 2009. The current surveillance program sensitivity as well as the impact of alternative surveillance protocols was simulated in a stochastic model using 10,000 iterations per simulation. The median (50% percentile) of the component sensitivities across 10,000 iterations was 0.83, 0.85, 0.99, 0.99, respectively, for (i) testing the cattle only during the winter screening, (ii) testing only imported cattle, (iii) testing only purchased cattle and (iv) testing only all slaughtered cattle. The sensitivity analysis showed that the most influential input parameter explaining the variability around the output came from the uncertainty distribution around the sensitivity of the diagnostic tests used within the bTB surveillance. Providing all animals are inspected and post mortem inspection is highly sensitive, slaughterhouse surveillance was the most effective surveillance component. If these conditions were not met, the uncertainty around the mean sensitivity of this component was important. Using an antibody ELISA at purchase and an interferon gamma test during winter screening and at import would increase greatly the sensitivity and the confidence level of Belgium's freedom from bTB infection status.

Bluetongue sentinel surveillance program and cross-sectional serological survey in cattle in Belgium in 2010-2011.

Bluetongue virus serotype 8 (BTV-8) emerged in Central Western Europe in 2006 causing a large scale epidemic in 2007 that involved several European Union (EU) countries including Belgium. As in several other EU member states, vaccination against BTV-8 with inactivated vaccines was initiated in Belgium in spring 2008 and appeared to be successful. Since 2009, no clinical cases of Bluetongue (BT) have been reported in Belgium and BTV-8 circulation seemed to have completely disappeared by spring 2010. Therefore, a series of repeated cross-sectional surveys, the BT sentinel surveillance program, based on virus detection in blood samples by means of real-time RT-PCR (RT-qPCR) were carried out in dairy cattle from the end of 2010 onwards with the aim to demonstrate the absence of BTV circulation in Belgium. This paper describes the results of the first two sampling rounds of this BT sentinel surveillance program carried out in October-November 2010 and January-February 2011. In addition, the level of BTV-specific maternal antibodies in young non-vaccinated animals was monitored and the level of herd immunity against BTV-8 after 3 consecutive years of compulsory BTV-8 vaccination was measured by ELISA. During the 1st sampling round of the BT sentinel surveillance program, 15 animals tested positive and 2 animals tested doubtful for BTV RNA by RT-qPCR. During the 2nd round, 17 animals tested positive and 5 animals tested doubtful. The positive/doubtful animals in both rounds were re-sampled 2-4 weeks after the original sampling and then all tested negative by RT-qPCR. These results demonstrate the absence of BTV circulation in Belgium in 2010 at a minimum expected prevalence of 2% and 95% confidence level. The study of the maternal antibodies in non-vaccinated animals showed that by the age of 7 months maternal antibodies against BTV had disappeared in most animals. The BTV seroprevalence at herd level after 3 years of compulsory BTV-8 vaccination was very high (97.4% [95% CI: 96.2-98.2]). The overall true within-herd BTV seroprevalence in 6-24 month old Belgian cattle in early 2011 was estimated at 73.4% (95% CI: 71.3-75.4).

Nonequilibrium effects in DNA microarrays: a multiplatform study.

It has recently been shown that in some DNA microarrays the time needed to reach thermal equilibrium may largely exceed the typical experimental time, which is about 15 h in standard protocols (Hooyberghs et al. Phys. Rev. E2010, 81, 012901). In this paper we discuss how this breakdown of thermodynamic equilibrium could be detected in microarray experiments without resorting to real time hybridization data, which are difficult to implement in standard experimental conditions. The method is based on the analysis of the distribution of fluorescence intensities I from different spots for probes carrying base mismatches. In thermal equilibrium and at sufficiently low concentrations, log I is expected to be linearly related to the hybridization free energy ΔG with a slope equal to 1/RT(exp), where T(exp) is the experimental temperature and R is the gas constant. The breakdown of equilibrium results in the deviation from this law. A model for hybridization kinetics explaining the observed experimental behavior is discussed, the so-called 3-state model. It predicts that deviations from equilibrium yield a proportionality of log I to ΔG/RT(eff). Here, T(eff) is an "effective" temperature, higher than the experimental one. This behavior is indeed observed in some experiments on Agilent arrays [Hooyberghs et al. Phys. Rev. E2010, 81, 012901 and Hooyberghs et al. Nucleic Acids Res. 2009, 37, e53]. We analyze experimental data from two other microarray platforms and discuss, on the basis of the results, the attainment of equilibrium in these cases. Interestingly, the same 3-state model predicts a (dynamical) saturation of the signal at values below the expected one at equilibrium.