A common concern with Bayesian methodology in scientific contexts is that inferences can be heavily influenced by subjective biases. As presented here, there are two types of bias for some quantity of interest: bias against and bias in favor. Based upon the principle of evidence, it is shown how to measure and control these biases for both hypothesis assessment and estimation problems. Optimality results are established for the principle of evidence as the basis of the approach to these problems. A close relationship is established between measuring bias in Bayesian inferences and frequentist properties that hold for any proper prior. This leads to a possible resolution to an apparent conflict between these approaches to statistical reasoning. Frequentism is seen as establishing figures of merit for a statistical study, while Bayes determines the inferences based upon statistical evidence. A serious concern with Bayesian methodology is that the choice of the prior could result in conclusions that to some degree are predetermined before seeing the data. In certain circumstances, this is correct. This can be seen by considering the problem associated with what is known as the Jeffreys–Lindley paradox where posterior probabilities of hypotheses, as well as associated Bayes factors, will produce increasing support for the hypothesis as the prior becomes more diffuse. Thus, while one may feel that a very diffuse prior is putting in very little information, it is in fact biasing the results in favor of the hypothesis in the sense that that there is a significant prior probability that evidence will be found in favor of the hypothesized value when it is false. It has been argued, see ,2, that While attempts have been made to avoid the Jeffreys–Lindley paradox through the choice of the prior, modifying the prior to avoid bias is contrary to the ideals of a Bayesian analysis which requires the elicitation of a prior based upon knowledge of the phenomenon under study. Why should one change such a prior because of bias? Indeed, there is bias in favor and bias against and typically choosing a prior to minimize one type of bias simply increases the other. Roughly speaking, in a hypothesis assessment problem, bias against means that there is a significant prior probability of finding evidence against a hypothesized value when it is true, and bias in favor means that there is a significant prior probability of finding evidence in favor of a hypothesized value when it is false. The real method for controlling bias of both types is through the amount of data collected. Bias can be measured post-hoc, and it then provides a way to assess the weight that should be given to the results of an analysis. For example, if a study concludes that there is evidence in favor of a hypothesis, but it can be shown that there was a high prior probability that such evidence would be obtained, then the results of such an analysis can’t be considered to be reliable.Previous discussion concerning bias for Bayesian methodology has focused on hypothesis assessment and, in many ways, this is a natural starting point. This paper is concerned with adding some aspects to those developments and to extending the approach to estimation and prediction problems as discussed in principle of evidence is adopted here: evidence in favor of a specific value of an unknown occurs when the posterior probability of the value is greater than its prior probability, evidence against occurs when the posterior probability of the value is less than its prior probability and there is no evidence either way when these are equal. The major part of what is discussed here depends only on this simple principle, but sometimes a numerical measure is needed and, for this, we use the relative belief ratio defined as the ratio of the posterior to prior probability. The relative belief ratio is related to the Bayes factor but has some nicer properties such as providing a measure of the evidence for each value of a parameter without the need to modify the prior.The measurement of bias, and thus its control, is dependent upon measuring evidence. The The inferences discussed here are based on the relative belief ratio and these inferences are invariant to any 1–1, increasing function of this quantity. For example, the logarithm of the relative belief ratio can be used instead to derive inferences. The expected value of the logarithm of the relative belief ratio under the posterior is the relative entropy, also called the Kullback–Leibler divergence, between the posterior and prior. This is an object of considerable interest in and of itself and, from the perspective of measuring evidence, can be considered as a measure of how much evidence the observed data are providing about the unknown parameter value in question. This aspect does not play a role here, however, but indicates a close association between the measurement of statistical evidence and the concept of entropy. In addition, many divergence measures involve the relative belief ratio and play a role in , which iThere is not much discussion in the Bayesian literature of the notion of bias in the sense that is meant here. There is considerable discussion, however, concerning the Jeffreys–Lindley paradox and our position is that bias plays a key role in the issues that arise. Relevant recent papers on this include ,6,7,8,9,p-value does not satisfy the principle of evidence but can still be used to characterize evidence for or against provided the significance level goes to 0 with increasing sample size or increasing diffuseness of the prior. In As already noted, the approach to inference and the measurement of bias adopted here is dependent on the principle of evidence. This principle is not well-known in the statistical community and so e to be evidence that a hypothesis h is true, it is both necessary and sufficient for e to increase h’s probability over its prior probability” which is what is called the principle of evidence here.Attempts to develop a theory of inference based upon a definition, or at least provide a characterization, of statistical evidence exist in the statistical literature. For example, see ,15,16,17One characteristic of our, and the philosophical, treatment is that evidence is a probabilistic concept and thus a proper definition only requires a single probability model as opposed to a statistical model. This explains in part why our treatment requires a proper prior as then there is a joint probability model for the model parameter and data. The following two examples illustrate the relevance of characterizing evidence in such a context. Example 1 is a simple game of chance where the probabilities in question are unambiguous. The utility aspects of the game are ignored because these are irrelevant to the discussion of evidence but surely are relevant if some action like betting was involved. This is characteristic of the treatment here where loss functions play no role in the characterization of evidence but do play a role in determining actions when required as discussed in the well-known Example 2. The examples also illustrate that characterizing evidence in favor of or against is not enough, as it is necessary to also say something about the strength of the evidence.Example 1.Card game.m cards, where Suppose that there are two players in a card game, labeled I and II, and each is dealt m in this case, grows. While There are two questions to be answered: (i) is there evidence in favor of or against m, but there can still be evidence in favor. Note that a comparison, for The measurement of the strength of evidence is not always obvious, but, in this case, effectively a binary event, the posterior probability of the event in question seems like a reasonable approach as it is measuring the belief that the event in question is true. Thus, if we get evidence in favor of Example 2.Prosecutor’s fallacy.N of which some member has committed a crime. DNA evidence has been left at the crime scene and suppose that this trait is shared by m is large. However, this probability does not reflect the evidence of guilt. If you have the trait, then clearly this is evidence in favor of guilt and indeed m is large and a conviction then does not seem appropriate. Since the posterior probability of “not guilty”is large whenever m is, it may seem obvious to conclude this. However, suppose that “guilty” corresponds to being a carrier of a highly infectious deadly disease and “has trait” corresponds to some positive, but not definitive, test for this. The same numbers should undoubtedly lead to a quarantine. Thus, the utilities determine the action taken and not just the evidence.Assume a uniform probability distribution on a population of size x and y denoting events, the following is stated.As noted, discussion concerning statistical evidence has a long history, although mainly in the philosophy of science literature, where it is sometimes referred to as confirmation theory. An introduction to confirmation theory can be found in , but they supports or corroborates a statement yincreases the probability of If we are asked to give a criterion of the fact that the evidence coefficient of influence of B upon A. In the Confirmation entry in [probabilistic relevance confirmation is what has been called here the principle of evidence. The following quote is from the third paragraph of this entry and it underlines the importance of this topic.The book  referencentry in , the defConfirmation theory has proven a rather difficult endeavour. In principle, it would aim at providing understanding and guidance for tasks such as diagnosis, prediction, and learning in virtually any area of inquiry. However, popular accounts of confirmation have often been taken to run into trouble even when faced with toy philosophical examples. Be that as it may, there is at least one real-world kind of activity that has remained a prevalent target and benchmark, i.e., scientific reasoning, and especially key episodes from the history of modern and contemporary natural science. The motivation for this is easily figured out. Mature sciences seem to have been uniquely effective in relying on observed evidence to establish extremely general, powerful, and sophisticated theories. Indeed, being capable of receiving genuine support from empirical evidence is itself a very distinctive trait of scientific hypotheses as compared to other kinds of statements. A philosophical characterization of what science is would then seem to require an understanding of the logic of confirmation. In addition, thus, traditionally, confirmation theory has come to be a central concern of philosophers of science.As far as we know, Ref.  summarizp-value cannot find evidence in favor. This one-sided aspect of a p-value is often justified by Popper’s idea that the role of science lies in falsification of hypotheses and not their confirmation. In the context of Examples 1 and 2, this seems wrong as the hypothesis in question is either true or false, so it is desirable to be able to find evidence either way. When applied to a statistical context, at least as formulated in Another aspect requiring comment is that the principle of evidence allows for finding either evidence against or evidence in favor of a hypothesis while, for example, a p-value can play a role in this aspect of a statistical argument. However, when making an inference, the model is accepted as being correct and, as such, one of the distributions in the model is true, and so it is natural to want to be able to find evidence in favor of or against a specific value of an object dependent on the true distribution. This situation is analogous to what arises in logic where a sound argument is distinguished from a valid argument. A logical argument is based upon premises and rules of inference like modus ponens. An argument is valid if the rules of logic are correctly applied to obtain the conclusions. However, an argument is sound only if the argument is valid and the premises are true. It is a basic rule of logical reasoning that one doesn’t confound the correctness of the argument with the correctness of the premises. In the statistical context, there may indeed be problems with the model or prior, but the inference step, which assumes the correctness of the model and prior, needs to be able to find evidence in favor as well as evidence against a particular value of the object of interest. As part of the general approach as presented in [For checking the model or checking the prior, there is one object that is being considered. Thus, it makes sense that only an indication that the entity in question is not appropriate is available, and a ented in , both mox and a proper prior probability distribution given by density For the discussion here, there is a model A, then we write A measure of the evidence that ef ratioRBΨ(ψ|x)=ef ratioRBΨ(ψ|x)=valid measure of evidence should satisfy the principle of evidence, namely, the existence of a cut-off value that determines evidence for or against as prescribed by the principle. Naturally, this cut-off is 1 for the relative belief ratio. The Bayes factor is also a valid measure of evidence and with the same cut-off. When A equals A being true with the evidence for its negation. In the continuous case, if we define the Bayes factor for Any found in  as thereSince the model and prior are subjectively chosen, the characterization and measurement of statistical evidence has a subjective component. This creates the possibility that these choices are biased, namely, they were chosen with some goal in mind other than letting the data determine the conclusions. Model checking and checking for prior-data conflict exposes these choices to criticism via the data, but these checks will not reveal inappropriate conduct like tailoring a model or prior based on the observed data. Perhaps a more important check on such behavior is to measure and control bias. As will now be shown, controlling the bias through the a priori determination of the amount of data collected can leave us with greater confidence that the data are the primary driver of whatever inferences are drawn, and this is surely the goal in scientific applications. Thus, while informed subjective choices are a good thing, there are also tools that can be used to mitigate concerns about subjectivity, as these allow an analysis to at least approach the scientific goal of an objective analysis. The lack of a precise definition of objectivity, and a clear methodology for attaining it, is not a failure since the issue can be addressed. This is a somewhat nuanced view of the objective/subjective concern and is perhaps more in line with the views on this topic as expressed in ,26.Suppose the problem of interest is to assess whether or not there is evidence in favor of or against ntly via , how strThe following example is carried along as it illustrates a number of things.Example 3.Location normal.Suppose p-value Observe that, as strength can then be measured byThe Jeffreys–Lindley paradox shows that the strength of evidence cannot be measured strictly by the size of the measure of evidence. A logical way to assess strength is to compare the evidence for sured byΠΨ.Example 3 p-value is small, this indicates that a large value of p-value A simple calculation shows that, with | fixed,  then conp-values, is a valid measure of evidence via the cut-off 0. From this, it is seen that the values of the first p-value p-value equals p-value equals n increases, these values become smaller, as, with p-value equal to p-value equals Simple algebra shows see the , howeverthose in ,28.It is very simple to elicit bias againstCertainly, default choices of priors should be avoided when possible, but even when eliciting, how can we know if the chosen prior is inducing bias? To assess this, a numerical measure is required. The principle of evidence suggests that sured byM do n do n5) dUnder general circumstances, see , both bi2. Location normal (continued).Example 3 Under /2,then  is givenn or by the diffuseness of the prior although, as subsequently shown, a diffuse prior induces bias in favor. It is also the case that ,then  equals mδ) whereM(RB(μ*|Xδ) whereM.Example 3 The value of given in  and examn recorded in To use , it is nBias in favor when estimating ly, when∫Ψ∫Ψ\{ψ*}ote that  and, in An upper bound on  is commoIt is the size and posterior content of Notice that, when 4. Location normal (continued).Example 3 It follows from  thatsupNote that, as Some elementary calculations give While the plausible region s and in  various x satisfies Consider now the bias against Suppose now that some other general rule, not necessarily the principle of evidence, is used to determine whether there is evidence in favor of or against Theorem 1.Consider When x according to some alternative evidence rule. Since C is at least as large as that of Now, consider Theorem 2.(i) The prior probability that the region C doesn’t cover a value for every Again, when The principle of evidence leads to the following satisfying properties which connect the concept of bias as discussed here with the frequentist concept.Theorem 3.(i) Using the principle of evidence, the prior probability of getting evidence in favor of The properties stated in Theorem 3 are similar to a property called unbiasedness for frequentist procedures. For example, a test is unbiased if the probability of rejecting a null is always larger when it is false than when it is true and a confidence region is unbiased if the probability of covering the true value is always greater than the probability of covering a false value. While the inferences discussed here are “unbiased” in this generalized sense, they could still be biased against or in favor in the sense of this paper, as it is the amount of data that controls this.Now, consider bias in favor and suppose there is an alternative characterization of evidence that leads to the region ). Using  to measuTheorem 4.(i) The prior probability Theorem 5.(i) The prior probability region C covers a value Thus, Theorems 4 and 5 are optimality results for the principle of evidence when considering bias in favor.Clearly, the bias against severe test is introduced in [substantively important is specified. When the test leads to the acceptance of attained powerhigh severity when the attained power is high. The motivation for adding this measure of the test is that it claimed that it is incorrect to simply accept The concept of a duced in , and thin problem” where large sample sizes will detect deviations from x leaving one wondering about their frequentist performance characteristics, see [The use of the ics, see  for moreA number of examples are now considered.Example 4.Binomial proportion.Suppose  example , so θ|nxθ, using , is giveConsider first the prior given by n or Now, consider bias in favor using . When (αAnother interesting case is when the prior is taken to be Jeffreys prior which in this case is the betaior, see , is prope, using  with δ=0The strange oscillatory nature of the plots for the binomial is difficult to understand but is a common feature with such calculations. For example, Ref.  studies Example 5.Location-scale normal quantiles.Suppose c is chosen so that the collection of intervals covers the effective support of Suppose interest is in the T given generate generate if For the bias against for estimation, the value of As n as well. The bias against as a function of The case Example 6.Normal Regression—Prediction.Prediction problems have some unique aspects when compared to inferences about parameters. To see this, consider first the location normal model of Example 3, and the problem is to make an inference about a future value y, the bias against is y will not be obtained. This baseline is dependent on both r. As y is a reasonable value from the distribution. When For a given y, namely, y correspond to values in the tails so we consider, for example, y in the tails. Again, the bias in favor has a baseline value as y satisfyingNow consider bias in favor of y, but, once again, there is a trade-off as when r increases the bias in favor goes to 1.w and, putting Prediction plays a bigger role in regression problems, but we can expect the same issues to apply as in the location problem. Suppose ussed in .For the bias calculations, it is necessary to generate values of the MSS Thus, generating For each generated p-value in Example 3 can be seen to apply quite generally, and this makes any discussion about how to characterize and measure evidence of considerable importance. The principle of evidence makes a substantial contribution in this regard as was shown in a variety of results. The major purpose of this paper, however, is to deal with a key criticism of Bayesian methodology, namely that inferences can be biased because of their dependence on the subjective beliefs of the analyst. This criticism is accepted, but we also assert that this can be dealt with in a logical and scientific fashion as has been demonstrated in this paper.There are several conclusions that can be drawn from the discussion here. First, it is necessary to take bias into account when considering Bayesian procedures and currently this is generally not being done. Depending on the purpose of the study, some values concerning both bias against and bias in favor need to be quoted as these are figures of merit for the study. The approach to Bayesian inferences via a characterization of evidence makes this relatively straight-forward conceptually. Second, frequentism can play a role in the approach to Bayesian statistical reasoning via relative belief, not through the inferences, but rather through determining the biases and then controlling these through the amount of data collected. Overall, this makes sense because, before the data are seen, it is natural to be concerned about what inferences can be reliably drawn. Once the data are observed, however, it is the evidence in this data set that matters and not the evidence in the data sets not seen. Still, if we ignore the latter, it may be that the existence of bias makes the inferences drawn of very low quality. Third, the results concerning the standard


Boipeba tayasuensis gen. et sp. nov, which extends the scolecophidian fossil record into the Mesozoic and reduces the fossil gap predicted by molecular data. The new species is estimated to have been over 1 m long, much larger than typical modern scolecophidians (<30 cm). This finding sheds light on the early evolution of blind snakes, supports the hypothesis of a Gondwanan origin for the Typhlopoidea, and indicates that early scolecophidians had large body size, and only later underwent miniaturization.Blind snakes (Scolecophidia) are minute cryptic snakes that diverged at the base of the evolutionary radiation of modern snakes. They have a scant fossil record, which dates back to the Upper Paleocene-Lower Eocene (∼56 Ma); this late appearance conflicts with molecular evidence, which suggests a much older origin for the group (during the Mesozoic: 160–125 Ma). Here we report a typhlopoid blind snake from the Late Cretaceous of Brazil,   •Boipeba tayasuensis is the oldest fossil blind snake from the Late Cretaceous of Brazil•A new phylogenetic analysis places the taxon within living typhlopoids•Boipeba is estimated to be ∼1 m in length, larger than any living blind snake•The small body size of extant blind snakes is due to subsequent miniaturization Paleontology; Animals; Systematics; Phylogenetics; Evolutionary History; Paleobiology Snakes comprise one of the most successful radiations of land vertebrates, with over 3,800 living species . With ∼6The origin of blind snakes is unclear. Their morphology includes a mixture of seemingly primitive lizard-like features and highly specialized characters , and theHere we report on a giant fossil scolecophidian found in Late Cretaceous sediments from Brazil. This finding sheds new light on the origin of blind snakes, bridging the gap between molecular and paleontological evidence . FurtherSquamata Oppel, 1811Ophidia Brongniart, 1800Scolecophidia Duméril and Bibron, 1844Boipeba tayasuensis gen. et sp. nov., and S2FGeneric epithet comes from the combination of the Brazilian native language Tupi-Guarani, “boi” meaning snake, and “peba” meaning flattened, in reference to the shape of the vertebrae. The species epithet “tayasuensis” derives from the type locality where the fossil was found, Taiaçu municipality, São Paulo, Brazil.MPMA 16-0008-08, a single precloacal vertebra with partial successive vertebra .≤87.8 Ma) for the type stratum of Brasilestes stardusti  boundary (66 Ma)  .Medium-sized snake vertebra (∼7-mm-anteroposteriorly-long centrum) distinguished from all other ophidians in possessing the following unique combination of vertebral features: dorsoventrally compressed vertebra having oval cotyle and condyle; zygosphene with straight anterior margin; prezygapophyseal articular facets with high angle of inclination (∼25°) above the horizontal plane; presence of elongated prezygapophyseal accessory processes; undivided synapophyses  mediolaterally expanded to the level of the prezygapophyseal articular facets; synapophyses located above the ventral margin of the cotyle; low neural spine slanting posteriorly; shallowly concave posterior margin of neural arch; cylindrical centrum lacking parasagittal ridges and hemal keel ; lack of paracotylar foramina; lack of parazygantral foramina; weak precondylar constriction; and asymmetrical subcentral foramina.The holotype consists of an isolated vertebra articulated with the anterior region of a fragmentary following vertebra. It is likely to belong to the middle or posterior precloacal region due to the absence of structures such as hypapophyses, lymphapophyses, pleurapophyses, or hemapophyses. The vertebra is three-dimensionally preserved. The neural arch is mediolaterally expanded and dorsoventrally compressed. The zygantrum is deep and has a pair of foramina inside. In dorsal view the neural arch displays a shallowly concave posterior embayment. In ventral view, the centrum is cylindrical, completely smooth , with a weak precondylar constriction. On the ventral surface of the centrum there is a pair of asymmetrical subcentral foramina, where the left foramen is small but distinct, whereas on the right side only a broad shallow fossa is visible, and the foramen has not fully developed through the bone , and S2,The neural spine is low, posterodorsally inclined, and in dorsal view, extends longitudinally from the posterior region of the zygosphene roof to slightly beyond the posterior embayment of the neural arch. The zygosphene is robust and partly eroded in the three-dimensionally preserved main specimen; however, in the fragmentary successional vertebra it is well preserved and characterized by a rectilinear anterior margin . The neuThe prezygapophyseal articular facets are broad, subtriangular in shape, and inclined above the horizontal about 25° (average between left and right sides). Long prezygapophyseal accessory processes are present. The process on the left side is partially worn, but the one on the right is complete  and 2. TBoipeba retains a mix of plesiomorphic features observed in stem snakes together with apomorphic traits typical of some representatives of the crown-group (modern snakes), the clade stemming from the most recent common ancestor of all living snakes (Boipeba is most similar to blind snakes (scoleophidians) and distinct from all other snakes.Despite the apparent conservative morphology of snake vertebrae, a set of anatomical features can be used to identify them at least to some broad taxonomic level when found in isolation . Boipebag snakes ; Table 1Boipeba tayasuensis  neural spine.Among all the extant and extinct snakes known, asuensis  and 3A sis sensuB. The vecophidia  include cophidia . Additiounctatus B, exclusBoipeba also has some features in common with other basal fossil snakes. Early stem snakes, informally termed “parviraptorids” , and synapophyses subdivided into para- and diapophyseal facets , synapophyses with division between the para- and diapophyseal facets, absence of prezygapophyseal accessory processes, and the occurrence of parasagittal and arqual ridges in the neural arch  of synapophyses that are subdivided into para- and diapophyseal facets, distinct hemal keels, and smaller prezygapophyseal processes. The last feature may be due to the early ontogenetic stage of the snake, whereas the other two features cannot be so readily explained.The fossil snake marensis , albeit Seismophis septentrionalis , and the weakly developed neural spine, but is clearly distinguished from the latter in possessing a flattened hemal keel, presence of parazygantral foramina, rounded cotyle and condyle, and marked parasagittal ridges.The only unambiguous Cretaceous ophidian record from the Cenomanian of Brazil, the stem-snake rionalis  shares wTetrapodophis from the Early Cretaceous of Brazil, which was described as a stem-snake  marine Tethyan Pachyophiidae are characterized by pachyostotic vertebrae, a diagnostic feature not observed in Lunaophis aquaticus  of Venezuela, quaticus , shares Coniophis precedens  and Uropeltoidea (Anilius). On the other hand, both amerophidians and uropeltoids are distinct from Boipeba in displaying division between the articular facets of the synapophyses, distinctly trifoliate neural canal morphology, flattened hemal keel delimited by subcentral grooves, rounded cotyle and condyle, and subtriangular centrum with marked precondylar constriction. Furthermore, Boipeba differs from Cylindrophis  snakes apart from blind snakes, eltoidea F and 2G,ndrophis F due to , and a broad subtriangular centrum with prominent hemal keel and strong precondylar constriction. Boipeba shares with members of the Caenophidia  the presence of elongated prezygapophyseal processes. However, colubroids have lightly built, elongated vertebrae, synapophyses subdivided into para- and diapophyses, frequent presence of paracotylar foramina, low inclination of the pre- and postzygapophyseal facets, and retain hypapophyses throughout the vertebral column  differ from those of l column .Boipeba are fully consistent with what is found in Serpentes, and in particular modern and fossil scolecophidians , support uniting Boipeba with all scolecophidians was 72% (parsimony partitioned bootstrap) and 0.99 (Bayesian posterior probability). Within scolecophidians, Boipeba was the sister taxon to typhlopoids (here represented by Typhlops), but this relationship was not robust .All vertebral features present in phidians ; no othent study . Boipebaters see  section.ters see  and undaters see  optimaliters see . Initialakes see . For insBoipeba with Scolecophidia: the presence of a confluent synapophysis (character 116), the presence of elongated prezygapophyseal accessory processes (character 117), and a smooth ventral margin of the centrum (character 121). Furthermore, the position of the synapophyses placed dorsal to the ventral margin of the cotyle (character 249) is a synapomorphy shared between Boipeba and Typhlopoidea. Thus, despite being represented only by vertebral characters, the analyses support a scolecophidian affinity of Boipeba.There are three unambiguous morphological synapomorphies uniting Dinilysia, Najash, the paraphyletic assemblage “madtsoiids,” and the marine Pachyophiidae; the last taxon is the sister group to crown-snakes. The North American Cretaceous fossil Coniophis precedens was weakly recovered as the sister taxon to the Alethinophidia, contraTetrapodophis  and similar Bayesian support (0.99 PP)  .Boipeba tayasuensis has a centrum length (CL) of 6.8 mm , which is very large compared with the minute vertebrae typically found in extant scolecophidians , about half the length of Dinilysia (∼2 m), and is considerably shorter than most “madtsoiids,” which have an average total length of about 4.4 m (Coniophis has an estimated TL of about 0.7 m (Boipeba (1.1 m).The fossil snake phidians  , about five times the average leptotyphlopid (∼0.20 m), and nearly three and a half times the average typhlopoid (∼0.31 m). It is much closer in size to typical alethinophidians, as well as to most basal fossil snakes  of the ancestral blind snake using a dated, well-sampled (98 extant species) phylogeny of Scolecophidia , and size estimates for all ancestral nodes were obtained using parsimony/likelihood methods . The TL estimate for the ancestor of living typhlopoids is similarly affected, with an estimate of 0.40 m when the fossil is included in the analysis versus an estimate of 0.30 m when it is excluded  origin for the group  for the minimum age of the typhlopoid-leptotyphlopid divergence, in molecular divergence studies.Boipeba tayasuensis is estimated to have been slightly over 1 m in TL, a giant among scolecophidians, which are typically less than 30 cm . This scenario is even more dramatic when considering the most recent common ancestor (MRCA) of Boipeba and Typhlopoidea, where the ancestral state reconstruction produced a TL estimate of 51 cm, nearly twice the length of the average living typhlopoid , but did not robustly resolve its placement within Scolecophidia. This is likely due to the limited number of informative phylogenetic characters that could be scored from vertebral characters alone. The lack of absolute dating of the minimum age of the fossil locality hampers a more precise estimate for the age of this fossil, which impacts the estimate of the size of the ancestral scolecophidian. However, our analyses consider the most conservative minimum age (66 Ma), which means the fossil could be much older and thus closer in time to the ancestral blind snake. This means that the large size of Boipeba would exert a stronger influence on the estimated size of the ancestral blind snake. Thus, a tighter (older) constraint on the minimum age of Boipeba would potentially improve support for the body size patterns retrieved here.Our phylogenetic analyses robustly united thiagoschineiderf@usp.br)Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Thiago Schineider Fachini , and scripts for all phylogenetic and comparative analyses can be found in the https://doi.org/10.17632/4dh8fj54f6.1). Original data have been deposited to Mendeley Data: [https://doi.org/10.17632/4dh8fj54f6.1].All the relevant data for this study such as the used scripts for the phylogenetic analyses together with the unprocessed datasets, the surface reconstruction file, the supplementary figures in full resolution, and the raw CT-Scan slices are freely available at Mendeley Data repository (All methods can be found in the accompanying


Ageing of the global population represents a challenge for national healthcare systems and healthcare professionals, including medico-legal experts, who assess personal damage in an increasing number of older people. Personal damage evaluation in older people is complex, and the scarcity of evidence is hindering the development of formal guidelines on the subject. The main objectives of the first multidisciplinary Consensus Conference on Medico-Legal Assessment of Personal Damage in Older People were to increase knowledge on the subject and establish standard procedures in this field. The conference, organized according to the guidelines issued by the Italian National Institute of Health (ISS), was held in Bologna  on June 8, 2019 with the support of national scientific societies, professional organizations, and stakeholders. The Scientific Technical Committee prepared 16 questions on 4 thematic areas: (1) differences in injury outcomes in older people compared to younger people and their relevance in personal damage assessment; (2) pre-existing status reconstruction and evaluation; (3) medico-legal examination procedures; (4) multidimensional assessment and scales. The Scientific Secretariat reviewed relevant literature and documents, rated their quality, and summarized evidence. During conference plenary public sessions, 4 pairs of experts reported on each thematic area. After the last session, a multidisciplinary Jury Panel (15 members) drafted the consensus statements. The present report describes Conference methods and results, including a summary of evidence supporting each statement, and areas requiring further investigation. The methodological recommendations issued during the Conference may be useful in several contexts of damage assessment, or to other medico-legal evaluation fields. The number of people aged 65 years and over (older people) is increasing worldwide, particularly in North America and Europe . Among tThis scenario represents a challenge for national healthcare services and for healthcare professionals, including Medico-Legal experts. Indeed, the latter assess the personal damages resulting from traumatic injuries in an increasing number of older people, especially in the legal framework of third-party motor liability .The ascertainment and evaluation of personal damage are complex issues from both clinical and medico-legal standpoints . The assThe impact of the abovementioned assessment issues on the daily practice of medico-legal experts was confirmed by interviews with experts in personal damage assessment carried out in December 2017, and represents the starting point of the Consensus Conference on Medico-Legal Assessment of Personal Damage in Older People.The aim of a consensus conference is to produce recommendations through “the analysis of the available evidence on specific controversial subjects for which there is no shared opinion, and often leading to non-standard practices in the clinical, organisational, and managemental aspects of healthcare” . In fielImprove knowledge on the subjectDefine standard medico-legal assessment proceduresAgree upon a common terminologyIdentify qualitative indices, andSuggest areas requiring further investigationThe Consensus Conference on Medico-Legal Assessment of Personal Damage in Older People focuses on the medico-legal assessment of personal damage (both as unbiased ascertainment and rating) in the elderly person. The consensus conference aims to:The consensus conference target audience includes Legal Medicine experts and Geriatricians, and insurance and law professionals in the field of personal damage, as well as the lay public. Also, the consensus conference’s recommendations can help all physicians  who take care of older people.The consensus conference was held in Bologna  on June 8, 2019. This report describes the Conference methods, presents the summary of evidence, and the statements/recommendations formulated by the Jury Panel, which also pointed out limitations and areas requiring further investigations.The consensus conference, promoted and endorsed by several Italian Scientific Societies and Institutions (see Appendix The organization of the consensus conference was a 2-year long process (May 2017–May 2019), preceded by a 5-month evaluation period during which the feasibility of the project was assessed (December 2016–April 2017). The early phases of conference organization involved the constitution of (1) the Promotion Committee, which promoted the conference, appointed the Scientific Technical Committee (STC) members, identified the field experts, and solicited questions concerning selected topics; (2) the STC, which selected the members of the Scientific Secretariat, prepared the questions, and drafted the guidelines for the literature review and the preparation of conference presentations; and (3) the Scientific Secretariat (including a literature review expert) was in charge of reviewing the scientific literature and preparing summary documents containing the information collected during the review.Consensus Conference Promoters identified 8 field experts , responsible for providing the Jury Panel with the summary of evidence and presenting it during the Conference. To constitute a multidisciplinary and multiprofessional Jury Panel, multiple stakeholders  were asked to designate a representative who would sit on the Jury Panel. The Jury Panel also included a nursing professor with an interest in geriatric research, a methodologist specialized in searching for and assessing evidence, and a representative of elderly people’s associations. The Conference organogram is presented in Appendix Evidence that traumatic injury outcomes are different between older and younger people and its relevance for the assessment of personal damage Pre-existing status reconstruction and evaluationMedico-legal examination proceduresMultidimensional assessment and scalesThe 16 consensus conference questions addressed the following 4 thematic areas:All questions focused on personal damage assessment in older people. Starting from questions, a systematic literature review for each thematic area/topic of the conference was carried out. To deal with the great variability of possible clinical conditions, the necessity of also including theoretical/doctrinal documents, the existence of a considerable amount of literature, and the difficulty of identifying documents inherent to the medico-legal practice, the systematic review followed some general criteria. First, a hierarchical search strategy favouring the selection of the most recently published systematic reviews was applied. Non-systematic reviews were included when systematic reviews were not available. Primary studies were included if no reviews were available on a specific topic, or if primary studies were more recent than systematic reviews on the same topic. Then, for questions concerning clinical practices and medico-legal technical aspects, the search focused on available guidelines, narrative review, and grey literature.In particular, for the first thematic area, only reviews discussing trauma outcomes in general or major trauma outcomes were included, excluding those that did not analyze associations between outcomes and age ≥ 65 years. For questions on pre-existing status, only reviews investigating the role of the pre-injury status on trauma outcomes in older people and theoretical/doctrinal documents were included. For questions concerning examination procedures, when reviews were not available, observational studies were included. For questions concerning the multidimensional assessment, guidelines, evidence-based synthesis, health technology documents, documents by scientific societies, and primary studies on the functional status assessment tools validated in Italian were included. Also, experts were asked to recommend additional documents, which were included only if they matched the above-mentioned criteria.https://dejure.it) database. This search led to a list of 10 relevant documents. The CISMeF (www.chu-rouen.fr/cismef), Cochrane Library (www.cochranelibrary.com), and NHS Evidence (www.evidence.nhs.uk) databases were also used for the literature search. For grey literature retrieval, webpages of scientific societies, point-of-care evidence-based medicine websites (UpToDate), as well as Google and Google Scholar search engines were used. This search produced 10 relevant documents, to which 1 document recommended by an expert was added.A first round of literature search was performed between July and August 2018, using the PubMed and EMBASE databases ; the search led to the identification of 3924 documents, most of which were of little or no relevance to the Conference questions. Therefore, at the beginning of 2019, a second round of literature search, that included documents published until the end of 2018, was performed. The new search led to the identification of 5437 articles, 29 of which were included in the review. Theoretical/doctrinal documents and court rulings were identified from the list of non-indexed journals of Legal Medicine that publish articles on the topic, and the DeJure , main measured outcomes, follow-up duration, and main findings with reference to the questions. Similarly, the Scientific Secretariat prepared summary tables for the theoretical/doctrinal material and other included documents. The experts and Jury Panel members were provided with the reviewed documents, together with the summary tables of the results of the quality assessment (see below), ahead of consensus conference. The experts used a further 20 non-reviewed documents during their presentations of summary of evidence.AGREE II  [AMSTAR 2 [Standard Quality Assessment Criteria for Evaluating Primary Research Papers from a Variety of Fields [The following tools were used for the evaluation of documents included in the revision: version) , for guiAMSTAR 2  for systf Fields  for primOverall, the quality of the observational studies was good, while that of systematic and non-systematic reviews was poor ; one of the guidelines was of high quality, while the other was of average quality Table . TheoretThe consensus conference was held on June 8, 2019, in Bologna . The invited audience included field experts and citizens.During the morning sessions, the experts presented a summary of the literature before the Jury Panel and the audience. For each session, the speakers were a medico-legal expert and a geriatrician giving a 40-min-long oral presentation, followed by 30 min of discussion. After the last session, the Jury Panel gathered in a secluded place to discuss the evidence presented and to draft a preliminary consensus document including the answer to each question  that was publicly presented by the Jury Panel’s Chair at the end of the Conference.The final version of the consensus document was drafted during the months following the conference, and finally approved on February 29, 2020. The agreement on the Jury Panel statements was reached through a two-round Delphi-like method . AccordiTwo reviews suggest that after traumatic injuries older people, regardless of injury type, have a lower quality of life, longer recovery times, less probability to regain independence in activities of daily living, and a reduced functional status , 30. GooSeveral reviews found a direct association between ageing and mortality after major traumas –27, 30. In conclusion, several studies show that the elderly population experience worse outcomes with regard to mortality, complications, functional status, and quality of life compared to a younger population. Different factors, including the pre-existing functional status, contribute to an increased risk of post-traumatic unfavourable outcomes and mortality in older people. However, further studies are required to better understand the role of these factors and their interactions with age.an  and the quantum (methods for damage ascertainment and rating criteria). Concerning the causation, these lines of evidence provide an empirical unbiased basis to establish that, on average, injury outcomes are more severe in a specific group of older people compared to the reference group composed of younger people . In addition, these lines of evidence provide objective support in explaining the particular severity of the outcomes in that specific older person undergoing a medico-legal assessment . In this assessment, the evidence of the harmful mechanism has a key role, and should always be assessed in the ascertainment of causation [evidence-based medicine [quantum, the criteria used in current rating methods depend upon decisions not stemming from empirical evidence, but available evidence might support these decisions in a concrete manner.According to experts’ opinions, available evidence is relevant to the evaluation of personal damage with regard to the ausation . The undmedicine . With reQuestions and Jury Panel statements/recommendations are reported in Table The available literature on this topic consists mainly of theoretical/doctrinal contributions.According to the doctrine, the reconstruction of the pre-existing status of the injured party is essential , 56, 65.In order to reconstruct the pre-existing status, several studies recommend the need to investigate, besides pre-existing diseases and therapies, the following aspects: what the person was able to do beforehand ; clinicaSome authors believe that the reconstruction of pre-existing status should only rely on objective information: ongoing treatments, General Practitioner’s opinion, laboratory and diagnostic tests, family context, and personal autonomy  . Others According to some authors, the evaluation should not only rely on standard ratings but should consider the actual negative impact of the injury on the general homeostatic balance of the person , includiQuestions and Jury Panel statements/recommendations are reported in Table A search of the literature did not provide results on the topic, partly because an unambiguous definition of clinical stability is still lacking . Some stSeveral observational studies found that the time needed for a medical examination increases with ageing and in the case of elderly patients , 47, 51.Several observational studies showed that accompanying family members and/or caregivers play a key role in the examination process of elderly patients, especially with regard to female patients ; patientFamily members and caregivers usually have an encouraging attitude during the medical examination , 42, thuAccording to the only available review , the firQuestions and Jury Panel statements/recommendations are reported in Table The multidimensional assessment (MDA)  can identify factors influencing the health and functional status of the older person even when the patient has poor self-perceived health conditions . AlthougMultidimensional assessments have long been used for the epidemiological study of disability , 77 and Clinical status (comorbidity): Cumulative Illness Rating Scale; Charlson IndexFunctional status: Basic Activities of Daily Living-BADL  and Instrumental Activities of Daily Living-IADL Cognitive status: Short Portable Mental Status Questionnaire, Mini Mental Status Examination, Clock Drawing testAffective status: Geriatric Depression Scale (15- or 5-item version)Social interactions: Oslo-3 Social Support ScaleIndependently of the specific purpose of an MDA, there is international consensus on the minimum set of domains that should be assessed , and on Some authors suggest using a smaller set of domains  .When specific alterations are found, a II level assessment should be performed .Four-Meter Gait Speed test Get-Up and Go test , 20Short Physical Performance Battery , 13, 14Grip Strength In some cases, a functional assessment with physical performance measurements, which better define the functional status, may be useful ; the mosIn literature, several frailty assessment methods are reported; the “physical frailty” and the “cumulative deficit frailty” are the most cited conceptual frameworks . The fraThe abovementioned tools have been routinely used for decades in geriatric practice. They provide a quantitative assessment of the patient status resulting in a discrete score that allows an accurate comparison between different examinations, when available.Additional tools for the evaluation of older patients that are validated in Italian are available , 41, 46,Questions and Jury Panel statements/recommendations are reported in Table unicum, without taking into account the heterogeneity of the elderly population with regard to several critical factors: global health conditions, frailty, odds of recovery, and social relationships and social capital. Even the most used assessment tools are generally not adequate for the older population. Further studies that consider these factors and investigate the relations between injury outcomes and pre-injury conditions are required. Also, additional studies are needed to validate, in the medico-legal context, the evaluation tools used to assess functioning of older people and the usefulness, for the purposes of personal damage assessment, of the frailty scales and the bio-psycho-social model issued by the World Health Organization [Most of the available studies on traumatic injury outcomes consider the older person as an nization . The latFinally, some members of the Jury Panel required an in-depth discussion of the most appropriate criteria for the monetary quantification and settlement of personal damage in older people, considering, in addition, the possible settlement by an annuity. On the contrary, other Jury Panel members expressed their disagreement on the inclusion of compensation settlement issues, or, in any case, for the mention of possible annuity compensation, in the present report.The consensus conference referred to personal damage assessment, with a focus on third-party liability. Nevertheless, for the medico-legal discipline unity, the methodological recommendations issued during the Conference may be useful to other contexts of damage assessment, or to other medico-legal evaluation fields.


Mendelian disorders. But the quality of services differs widely across laboratories in China, particularly in variant classification, even with the adoption of the ACMG guidelines. As an effort of quality control and improvement for better clinical utilization of exome sequencing, we assessed the exome data analysis and clinical reporting among Chinese laboratories. Five raw datasets of real clinical samples with associated phenotypes were sent to 53 laboratories. The participants independently performed secondary analysis, variant classification, and reporting. The first round of results was used for identifying problems associated with these aspects. Subsequently, we implemented several corrective actions and a training program was designed based on the identified issues. A second round of five datasets were sent to the same participants. We compared the performances in variant interpretation and reporting. A total of 85.7% (42/49) of participants correctly identified all the variants related with phenotype. Many lines of evidence using the ACMG guidelines were incorrectly utilized, which resulted in a large inter-laboratory discrepancy. After training, the evidence usage problems significantly improved, leading to a more consistent outcome. Participants improved their exome data analysis and clinical reporting capability. Targeted training and a deeper understanding of the ACMG guidelines helped to improve the clinical exome sequencing service in terms of consistency and accuracy in variant classification in China.Exome sequencing has become an effective diagnostic method for  Exome sequencing has proven to be an effective strategy for gene discovery and clinical diagnosis for patients with Mendelian disorders, which herald a new era of genomic medicine . HoweverIn terms of variant classification, previous reports revealed extensive discordance between laboratories . A totalThe initial survey involved three probands and two trios. The clinical manifestation and/or family history of these test cases suggested a possible genetic disease.Genomic DNA was isolated from peripheral blood samples using the Gentra Puregene Blood Kit  following the manufacturer’s protocol. The target regions for proband-only samples were captured by the ClearSeq Inherited Disease panel kit . The whole exome for trio samples were captured by the SureSelect Human All Exon V6 kit . Sequencing was performed on Hiseq X Ten  using paired-end 150-bp reads according to the manufacturer’s protocol. Raw data (FASTQ files) were generated via the on-board Hiseq control software and reporter software (Illumina).Chinese laboratories that offer exome sequencing for routine clinical molecular testing were invited to participate in the survey. Participation was voluntary and free of charge. Participating laboratories downloaded the FASTQ datasets from a central portal. Laboratories were asked to process the data using their validated pipelines for alignment, variant calling, annotation and filtration, and variant interpretation and reporting as they would normally do for a clinical case. Participants were asked to provide a detailed description of the bioinformatic pipelines and quality metrics. In addition to the results of the variant classification, the laboratories were also asked to provide individual lines of evidence following the ACMG/AMP guidelines. The NCCLs collected all the results. The participants were asked to provide an explanation for missing identifying the causative variants.After identifying the problems based on the initial assessment, a training program was designed and conducted, focusing on training the laboratories on how to apply the ACMG-AMP criteria correctly. The training included a one time in-person training course with workshops plus months of online educational lectures. After training, another five cases with putative disease-causing variants were sent to the same laboratories. The criteria applied by each laboratory were tracked to identify to what extent training improved the consistency and accuracy of variant interpretation.The initial set (labeled as -1) and the second set (labeled as -2) of clinical cases are shown in FBP1(NM_000507.3) c.720_729delTTATGGGGCC in sample 1815. Two institutions (lab4 and lab6) reported wrong variants by using the wrong phenotype. One participating laboratory (lab7) called a wrong indel variant (FBP1 c.718_727delCCCCATAAGG).A total of 53 clinical laboratories from hospitals or commercial entities providing genetic testing participated in this survey. The necessary experience with WES data analysis was at least one year for these participates. At the deadline of the survey, 49 laboratories returned the results meeting the requirements. Each laboratory provided the variants associated with a phenotype, clinical diagnostic reports for each sample, and a brief description of their bioinformatics pipeline. Most participants  were from a commercial entity. As a result, 85.7% (42/49) of participating laboratories correctly identified all the variants related with a phenotype. A total of 42 laboratories reported the intended variant nomenclature as shown in Generally, a bioinformatics pipeline consists of the following major steps: alignment, pre-variant call, variant calling, variant annotation, and filtration. The bioinformatics pipeline and its components used by participating laboratories for each step were highly variable as shown in The most used population frequency database was the 1000 Genomes project (94%), followed by ExAC (88%), gnomAD (63%), dbSNP (80%), ESP6500 (51%), and an in-house database (51%). A variety of knowledgebases and databases were used, including but not limited to: ClinVar (84%), OMIM (94%), Human Gene Mutation Database (HGMD) (84%), HPO (20%), CHPO (6%), COSMIC (16%), Orphanet (8%), an in-house annotation (22%), dbNSFP (69%), LOVD (14%), and DECIPHER (10%). A diverse range of pathogenicity prediction tools were utilized often in combinations of more than two.Forty-five laboratories submitted clinical reports for each case in the first survey. The elements of the report are summarized in A training meeting was subsequently conducted in order to standardize the content and format of clinical reports. The reports submitted for the second round of surveys had a much improved consistency as shown in FBP1:c.490G > A, SLC26A4:c.281C > T, and c.720_729del) were quite discordant, the classification crossed clinical categories . For 3.3% (11/335) of the variant assessments, the ACMG evidence listed by the laboratories did not support the classification chosen.All participating laboratories claimed that they assessed the pathogenicity of variants following the ACMG/AMP guidelines , but thein silico evidence), and PVS1 (variant predicted null where loss of function is a mechanism of disease) were the most frequently used lines of evidence in the first survey. Lines of evidence that should have been used but were not are plotted in (category 1 error) and those that should not have been used but were are plotted in (category 2 error). Category 1 error occurred in over 30% of instances for some lines of evidence. PP1 (segregation data) (47.52%), PM3  (32.07%), and PS4 (case-control difference) (34.11%) were among the most significantly misused lines of evidence as part of category 1 error. PP4 (using phenotype to support variant claims) (42.86%) was also the most inconsistently used evidence among the laboratories. Category 2 error occurred in over 15% of instances for some lines of evidence. PP5 (reputable source) was still used for variant classifications (32.94%), though ACMG-AMP had proposed that laboratories discontinue the use of this criteria. PS1 (same amino acid change) (19.24%), PP3  (19.24%), PM1  (19.24%) were among the most significantly misused lines of evidence as part of category 2 error. The misusage of PP3 was due to ‘double counting’ when PVS1 had already been used. Some laboratories used PS1 when the same missense variant had been reported before. Some used PS1 instead of the PM5 (novel missense at the same position) criteria for the MYH7 c.2167C > G (p. Arg723Gly) variant. The misusage of PM1 was due to a misunderstanding of the well-established functional domain. Only three laboratories correctly up- and downgraded lines of evidence .A total of 16 different lines of evidence were invoked for variant classifications in the survey. PM2 (absent from controls), PP3  metrics. The summary of quality metrics provided by participating institutions are provided in 1. It specified that the in vitro enzyme activity < 50% compared to wild-type control or RT-PCR evidence of mis-splicing could be used as PS3 . However, PS3 was not used by 13.6% (6/44) of laboratories in variant classifications for the PAH gene in the second survey. Also, there was a lot of uncertainty associated with the use of PS3. It was not used in variant classifications for 14.57% of variants in the second survey, illustrating that the evidence is still challenging for many laboratories; 2) up and downgrading evidence became the main cause of discordance for lines such as PP1, PM3, and PS2/PM6. Many laboratories had not implemented the upgraded guidelines or had difficulty in judging when it was appropriate to up or downgrade the evidence, or literature searching was incomplete so some raw evidence was missed. On the other hand, we trained laboratories on how to interpret the loss of function PVS1 in the ACMG/AMP variant criterion (CHD7:NM_017780.3, c.1666-2A > G, because they thought the provided information was the “whole observable phenotype”, and assumed that the patient did not have any other abnormalities. The lack of personnel with clinical experience will be a significant problem for genetic testing in China if a certification program is not implemented. It should be noted that the number and type of variants in the surveys were limited and not all relevant issues had been identified by this initial assessment. But improving the understanding of the guidelines can help with certain issues right away. Our near-future efforts will focus on further training for personnel and the development of additional practice guidelines to assist with the application of the ACMG-AMP guidelines in Chinese laboratories. During the training we will illustrate the importance of data and experience sharing, NCCLs will organize a clinical consortium to facilitate fair data sharing and resolve inter-laboratory discrepancies, as was already implemented by ClinGen (It has been a major challenge to accurately and consistently classify variant pathogenicity. The ACMG/AMP guidelines provided a great framework for variant clinical interpretation and helped to significantly reduce discrepancies . But theriterion . In a fu ClinGen . In the Although specific international guidelines  and ChinThe overall purpose of this initial survey in China was to investigate the performance of exome variant analysis and clinical reporting and to identify the shortcomings of the process and its product. We have used training as one of the solutions to amend the identified problems. We also implemented several corrective actions based on the specific issues exposed during the surveys including the adjustment and improvement of the secondary analysis pipeline, the implementation of appropriate validation, and a more vigorous process of variant interpretation and reporting. In conclusion, the practice of NGS-based exome sequencing for genetic disease in China is still in the early stages of its development. The initial survey revealed a significant number of issues associated with but not unique to the clinical exome test in Chinese laboratories. We also illustrated the approaches that can help to achieve optimal exome testing quality in Chinese diagnostic laboratories.All datasets presented in this study are included in the article/KZ drafted the manuscript. YS and JL revised the manuscript. JW, DH, YH, and GL contributed to the acquisition and interpretation of data. KZ, YS, and JL designed the work. All authors contributed to the manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Numerous groups, such as the tobacco industry, have deliberately altered and misrepresented knowable facts and empirical evidence to promote an agenda, often for monetary benefit, with consequences for environmental and public health. Previous research has explored cases individually, but none have conducted an in-depth comparison between cases. The purpose of this study was to compile a comprehensive list of tactics used by disparate groups and provide a framework for identifying further instances of manufactured doubt.We examined scholarly books, peer-reviewed articles, well-researched journalism pieces, and legal evidence related to five disparate industries and organizations selected for their destructive impacts on environmental and public health . These documents provided evidence for a list of tactics used to generate pro-industry spin and manufacture doubt about conferred harm. We then identified trends among sets of strategies that could explain their differential use or efficacy.We recognized 28 unique tactics used to manufacture doubt. Five of these tactics were used by all five organizations, suggesting that they are key features of manufactured doubt. The intended audience influences the strategy used to misinform, and logical fallacies contribute to their efficacy.This list of tactics can be used by others to build a case that an industry or group is deliberately manipulating information associated with their actions or products. Improved scientific and rhetorical literacy could be used to render them less effective, depending on the audience targeted, and ultimately allow for the protection of both environmental health and public health more generally.The online version contains supplementary material available at 10.1186/s12940-021-00723-0. The term ‘manufactured doubt’ refers to actions that deliberately alter and misrepresent knowable facts and empirical evidence to promote an agenda –4, oftenThere are multiple examples of organizations that have manufactured doubt, obscuring the scientific link between their work/actions and harmful effects. These include, but are not limited to, the NFL and chronic traumatic encephalopathy (CTE), manufacturers of the insecticide DDT and wildlife destruction, pharmaceutical companies and the addictive nature of opioids, and asbestos companies and mesothelioma , 4, 7, 8tobacco industry has managed to maintain its clientele for many decades in part due to manufactured scientific controversy about the health effects of active and secondhand smoking [In a recent review, we described the deceptive actions of five different industries or organizations, chosen for their unique and varied contributions to the list of methods used to manufacture doubt among diverse audiences with ultimate impacts on environmental or public health . The fir smoking , 10–13.the coal industry, whose employees often suffer from black lung disease [the sugar industry, which distracted from its role contributing to metabolic and cardiovascular diseases [the agrochemical business, Syngenta, manufacturer of the herbicide atrazine [the Marshall Institute, a conservative think tank comprised of Cold War physicists eager to maintain their proximity to government, and associated scientists who deliberately misrepresented information to the government to both minimize and normalize the effects of fossil fuels on global temperatures [The other industries we examined include  disease , yet the disease –19; the diseases  by deflediseases –25; the atrazine –28, whicatrazine , 30; anderatures , 31.The stories of these five industries and organizations reveal an extensive variety of tactics used to manufacture doubt within numerous organizations with impacts on environmental health and public health. A great deal of literature has examined these examples on a case-by-case basis, but to our knowledge, no evaluation has compared and contrasted these campaigns to identify common elements or strategies used across organizations to create pro-industry spin and manufacture doubt. In addition, to our knowledge, no prior work has provided a formal definition of manufactured doubt, though a close relative of the term is manufactured controversy, e.g., a controversy that is “motivated by profit or extreme ideology to intentionally create public confusion about an issue that is not in dispute” .Here, we identify a list of tactics that were used by one or more groups to manufacture doubt. We describe each of these approaches and identify common trends that inform their use and efficacy. By distinguishing the breadth of tactics used by industries that impact public health, a definition of manufactured doubt was produced along with a framework that can be used to identify and characterize other instances in historical or emerging industries.We previously conducted a thorough review of literature concerning five doubt manufacturers: Big Tobacco, the coal industry, the sugar industry, Syngenta, and the Marshall Institute and associated scientists addressing climate change in the 1980s [Starting with documents that are attributed to the Tobacco Industry , anAfter fully investigating the five examples, we identified the strategies that were found in documents related to all five industries/organizations. We consider these common tactics to be the most effective tools for manufacturing doubt. We also identified the intended audience associated with each strategy. This information was organized into Venn diagrams to clearly demonstrate the shared or separate use of various techniques across audiences and industries.Given the extensive literature relevant to these five case studies, we do not assume that our analysis is absolute; we may have overlooked evidence linking one or more strategies to one of the five organizations. There are no established criteria for identifying strategies so our decisions regarding which strategies to include, how to define them, and which were documented for each case study were dependent on our choice of literature and the breadth of literature available. In addition, determining the intent behind an organization’s actions is not always possible, and thus inferring whether an action involves willful deceit is dependent on our interpretation of the authors’ phrasing. We also did not conduct a systematic literature review, which was beyond the scope of this study.We identified 28 unique strategies Table  used by Many other tactics were used by four of the five organizations that we evaluated, suggesting that they are individually effective though not consistently used to manufacture doubt; their presence adds to the weight of evidence that a group is manufacturing doubt. This set of tactics also suggests that there may be aspects of the industry, product, or intended audience, that dictate the actions taken or not taken by a specific industry.Only three strategies were used by a single industry each: the tobacco industry altered their product to make it appear safer #20); the tobacco industry inappropriately questioned causality (#24); and prominent climate denialist scientists from the 80’s created straw man arguments to combat action against climate change (#25). Of note, several strategies utilized by one or more industry or organization rely on logical fallacies information.We identified five strategies that can be used regardless of the intended audience Fig. : gain suMany other strategies target only three of the audiences, but due to their nature are unlikely to be effective at altering perceptions in the fourth audience. For example, taking advantage of scientific illiteracy and posing as a defender of health or truth likely will not be useful in the scientific community specifically because literacy and objectivity are core tenets of scientific inquiry. Similarly, attacking opponents and abusing credentials are unlikely to hold weight in the legal community because the legal system prevents personal attacks  and expert witnesses can only serve after passing evaluation as an expert in their field. A few strategies are singular to an audience. For example, appealing to mass media (#16) is aimed toward a general lay audience. Scientists, government officials, and the court system rely on other sources of information for decision making. Attacking study design (#1) is aimed at the scientific community, where a basic understanding of study design and bias are needed to understand both a paper and its criticism.We have identified a list of 28 unique tactics used in five diverse case-studies to manufacture doubt. Five of these strategies were used by all five organizations we examined, suggesting that they are sufficient for the protection of products, to the detriment of public health. Based on all information gathered herein, we have created a working definition for manufactured doubt: the deliberate alteration and misrepresentation of knowable facts and empirical evidence to promote an agenda, often for financial benefit.Many of these strategies require funds for implementation. For example, tactics such as the recruitment of reputable individuals require monetary resources as these in-demand professionals typically expect to be compensated for their time and/or efforts; contributing to the published literature can be costly if ghostwriters or professional scientists are hired; hosting conferences and seminars can require funds to pay for travel costs, honoraria for reputable speakers, and venue fees; and the use of professional firms to develop PR campaigns can require significant funds depending on the nature of the work. Other strategies take advantage of a victim’s lack of money or influence, which necessarily requires vast amounts of resources to maintain a power imbalance. For example, the coal industry’s victims - its own employees - often have limited funds, scientific and medical knowledge, or political and social capital.The industries and organizations involved in all five of the case studies we selected had both money and influence at their disposal. The coal, sugar, and tobacco industries are composed of multiple large companies that possess the funds needed to purchase support and attack opponents. Syngenta is one of the largest agrochemical companies operating worldwide. The Marshall Institute received private funding, often from fossil fuel shell companies or think tanks, to continue its work and held sway over governmental opinions and policies, thus it did not need to purchase access . FundingThe true reasons for the common use of five strategies  among all five case-studies is unclear, though our analysis of the intended audience provides some insight. Strategies #2, #3, and #8 target all audiences  is a form of appealing to a false authority; individuals with expertise in one field are routinely recruited to support industry actions in unrelated fields, and their reputations as experts are exploited to promote industry’s viewpoint to the detriment of truth and health. An illustrative example of this logical fallacy comes from disgraced physician Andrew Wakefield. Wakefield was a gastroenterologist who claimed to have found an association between use of the vaccine for measles, mumps and rubella and autism in children . Not onlIn an increasingly interdisciplinary world, identifying logical fallacies may become an essential part of scientific training. Learning to identify their use requires “training in critical thinking and avoiding illogical thought patterns that often come naturally to humans” . Even wewho can manufacture doubt. Our approach provides evidence that there are two related but distinct parties in the doubt manufacturing process: manufacturers and perpetuators. All five of the groups we examined worked with others such as PR firms, scientists, researchers and physicians, as well as hired “ringers” posing as lay-people, who served as active doubt manufacturers   .In contrast, doubt perpetuators knowingly or unknowingly spread false information given to them by an original doubt manufacturer. These can include, but are not limited to, journalists, bloggers, citizen scientists, and lay-people that disseminate information and spread pro-industry spin. For example, one member of the Marshall Institute found that the Wall Street Journal  served aThe digital age has provided additional opportunities to spread misinformation. Doubt manufacturers have taken advantage of new media platforms, such as blogs and social media, to unite journalists, industry representatives and ‘citizen scientists’ with the aim of recruiting these individuals to perpetuate manipulated information ,The average individual can be easily swayed to denial, perhaps due to confirmation bias stemming from ideological, political, or religious beliefs or a lack of subject-specific knowledge for critically examining scientific evidence. Thus, the general public may participate in “muddying the waters” by acting as unknowing recipients and perpetuators of manufactured doubt. When manufacturing doubt, industries capitalize on grassroots support, encouraging citizens to take part in actions that impede public health-promoting activities and spread disinformation among family, friends, and relevant parties .Of note, in the case studies we examined, governmental bodies were not doubt manufacturers; instead, outside industries influenced federal government agencies to become unknowing doubt perpetuators. Affiliated groups  or lobbyists working for industries provided information to federal agencies, and, unfortunately, members of those agencies could not always sort truth from lies. For example, the Marshall Institute presented a report to members of the federal government that blamed the sun for increasing global temperatures; in this report, they selectively displayed data visualizations that supported their claims and omitted those that ran counter . The repExamples where government employees and elected officials have perpetuated doubt suggest that even these individuals with great power can fall victim to the same scientific illiteracy and logical fallacies that influence the general public. Further, employees in many federal agencies benefit from the “revolving door” between industries and the agencies tasked with their regulation, leading these employees to favor decisions that benefit industries  . Thus, tOf note, evidence from other cases not studied here demonstrates that government can also be a manufacturer, not just a perpetrator, of doubt. For example, the US federal government  contaminated drinking water with perchlorate, a chemical used in missiles and rockets that interferes with iodine uptake . StudiesWe propose that our list of 28 tactics can be used as a tool for both identifying and countering new cases of manufactured doubt as they arise in emerging industries with obvious implications for environmental health, and public health more broadly  –61. SimiWe note an important caveat regarding this type of evaluation. Some of the 28 tactics we identified can be used for legitimate purposes, so these tactics must be evaluated in a broader context. For example, public health defendants with strong evidence may err but nonetheless choose to use hyperbole. Advocates for environmental health issues may host one-sided conferences without the intention to misinform; of course, there is no need for legitimate scientific organizations to invite speakers representing a non-scientific viewpoint to their conferences in an attempt to avoid appearing biased. Additionally, anyone is capable of unintentionally employing a logical fallacy, and as noted above, the use of logical fallacies alone is not evidence that a group is manufacturing doubt. Thus, expert knowledge or informed judgment must be used when applying this analysis to other cases.Furthermore, the use of each of these strategies alone does not necessarily indicate deception; we suggest that both the strength and weight of evidence must be considered. The use of a variety of strategies provides stronger evidence of an agenda to manufacture doubt. Similarly, the use of a weightier strategy, such as suppressing information, compared to one that may be used for non-manipulative purposes, such as hosting conferences, also provides more compelling evidence for manufactured doubt. For this reason, the 28 strategies should not be considered to have equal weight. As new cases are evaluated, we would expect that the five strategies that were common to the case studies we examined here  would similarly be documented; such observation is likely to provide the most convincing evidence for manufactured doubt.lay public can verify sources and investigate who is making claims by checking for conflicts of interest. Though public health may not motivate all individuals, and not everyone is interested in overcoming confirmation bias, knowledge of potential threats to personal health through intentional deception may inspire more thorough personal investigation. Enhanced understanding of logical fallacies would also increase the public’s recognition of their use. Media must also avoid presenting evidence and industry-generated manufactured doubt or “spin” as if these two views are equal. As shown in these cases, this kind of “balance” does not reflect truth.The Scientists, who have a greater understanding of the evidence-based literature, must also identify conflicts of interest such as funding, consider the credentials of “experts”, and be wary of meta-analyses or attacks on scientific articles by consulting original studies. Peer-reviewed journals have an obligation to require conflict of interest disclosure and should refuse papers that are designed to mislead. Lack of disclosure should be followed by the retraction of studies. Research funding from big businesses may not necessarily indicate bias, but reviewers must be trained to consider bias when a relationship is disclosed. Scientists also benefit from positions of power within organizations . They should utilize their roles to advocate for change within these institutions, which will help to improve the public’s access to scientific information [ormation .legal system, there is much room for improvement regarding equity. Policies can be enacted that protect the rights of individuals affected by industry’s actions  to effectively defend their cases, without the influence of a power or financial disparity. Expert testimony must also be held to a very high standard of impartiality.In the political system has demonstrated that funding influences regulations and policy. Though we cannot expect all government officials to be experts in scientific literacy, government agencies must actively consult and act upon information from truly impartial individuals that can provide support and information regarding scientific and non-scientific evidence not subject to manufactured doubt. We can ask that the executive branch nominate qualified individuals to lead scientific agencies and advisory boards; that federal officials face punishment for tampering with scientific reports; and that administrations, regardless of their political party, avoid creating hostile work environments for scientific staff [The current ic staff . RegulatUltimately the responsibility for the production of disinformation, and its effects on individuals, public health, and the loss of public trust lies with the doubt manufacturers. Until principles of scientific integrity are incorporated into the mission statements of all industries, organizations, and government agencies, recipients of information must be better prepared to identify falsehoods. Policies must also be specifically designed by government agencies, academic institutions, scientific and medical societies, and other stakeholders to stop its spread. Furthermore, the ramifications of regulatory “reform” legislation intended to stem the creation of doubt, or the spread of doubt, should be made clear to constituents who may be unaware of its impact on evidence-based policy . DefininIndividuals and organizations, especially those tasked with protecting the public’s health, must arm themselves with knowledge of these tactics and develop or hone protective strategies to recognize and prevent cases of manufactured doubt. This investigation will enable people from all spheres to assess the vast amount of information they are exposed to in the most objective way possible and discern truth from falsehood.Our evaluation provides a thorough, but by no means exhaustive, list of tactics used by industries and organizations to manufacture doubt. Our list is limited by the scope of the works cited, the documentation available, and the current understanding of stakeholders involved in these cases; there may have been strategies that were used but not mentioned in the covered texts, and information regarding the covert actions taken by manufacturers may not yet be available to the public. Null findings may indicate a true absence of use or the limitations of publicly available documentation. We also make no assessments of the scientific literature involved in these cases ; rather we have focused this research on efforts made by organizations to misrepresent evidence by any means and the possible repercussions of those falsified narratives.The conclusions and definitions reached in this analysis may be specific to the groups we investigated. Our list is also defined by the scope of the case studies selected (i.e. a specific company vs. an industry), and a more inclusive case-study may contribute a wider range of tactics. With more cases, an even more refined definition of manufactured doubt can be produced. Future analyses could include instances of government-sanctioned manufactured doubt or more recent cases. The methods described herein, however, are integral to understanding industry ‘spin’ and warn against the implications of as-of-yet undiscovered tactics and doubt mongering.Additional file 1.


Dirofilaria immitis, Leishmania infantum, Anaplasma spp., and Ehrlichia canis) in Spain. Between September 2018 and February 2020, blood was sampled from 4643 client-owned dogs from 111 veterinary clinics from the 17 autonomous communities of Spain. All samples were tested for the detection of D. immitis antigens, and for antibodies against L. infantum, Anaplasma spp. and E. canis. Of the studied dogs, 22.14% were positive for one or several diseases while the prevalence was 6.25% (CI: 5.59–6.98) for D. immitis, and the seroprevalences were 10.36% (CI: 9.52–11.27) for L. infantum, 5.06% (CI: 4.47–5.73) for Anaplasma spp., and 4.26% (CI: 3.72–4.88) for E. canis. Co-infections by two and three vector-borne diseases were reported in 13% and 2% of the infected dogs, respectively. The studied vector-borne diseases are widely distributed throughout the Spanish geography, being observed and expanding northward in the case of D. immitis and L. infantum. The results point to an insufficiency of preventive measures to avoid the infection, and the need of the implementation of awareness campaigns among veterinarians and owners. Furthermore, a close collaboration between veterinarians, physicians and health authorities would be necessary for such zoonotic vector-borne diseases.Currently, climate change, modifications of landscapes and habitats due to human activities, as well as an increase in the movement of reservoirs and new species of competent vectors, have contributed to the spread of canine vector-borne diseases. These are mostly emerging and neglected diseases, some of them with zoonotic potential. Therefore, the objective of this study was to assess the prevalence and distribution of four major canine vector-borne diseases ( It is described that climatic factors  influence the distribution of animal diseases, such as vector-borne diseases, causing dynamic changes in their geographical distribution, epidemiology, pathogenicity, and control. In addition, anthropogenic factors such as globalization with the increasing circulation of people and goods, the transport of infected animals from endemic areas, environmental changes related to human activities and climatic change, favor the spread of vector-borne diseases –3.Canine vector-borne diseases include a variety of diseases which are mainly transmitted by culicid mosquitoes, phlebotomine sand flies, fleas, and ticks. This research focuses on four canine vector-borne diseases that are among the most clinically relevant and most prevalent in the Spanish geography , 3, 4.Dirofilaria immitis, is a chronic and potentially lethal disease transmitted by culicid mosquitoes , granulocytic anaplasmosis (A. phagocytophilum), and monocytic ehrlichiosis (E. canis). These mostly affect dogs and are mainly transmitted by Rhipicephalus sanguineus and Ixodes ricinus  is the type of climate which covers most of the Iberian Peninsula and the Balearics, occupying ~40% of its surface. This climate is mainly found in Catalonia, Valencian Community, the Balearic Islands, Andalusia, Castilla La Mancha, Madrid, and Extremadura. This climate is characterized by having hot summers with the average temperature in the warmest month >22°C. The temperate with dry or temperate summer climate (Csb) is the second most common climate in Spain, occurring in ~22% of the territory and is mainly present in Galicia and Castile and León. Like the Csa climate, it has a minimum of rainfall in the summer, but the summer is mild as it does not exceed 22°C on average in the warmest month. The third most common climate in Spain is the temperate with a dry season and temperate summer climate (Cfb). It is located in the northern region and mainly presented in Asturias, Cantabria, Basque Country, La Rioja, Navarra, and Aragón. Murcia is mainly represented by the cold steppe climate (Bsk); this correspond to a dry climate which is characterized as such because the evaporation exceeds precipitation on average, but is less than potential evaporation; average temperature is <18°C. Finally, the Canary Islands present different climates but, overall, the climate is subtropical and desert, moderated by the sea and in summer by the trade winds (Subtr).The study included a total of 4,643 blood samples from domestic dogs, collected between September 2018 and February 2020. Samples were collected from dogs presented for routine health examination to 111 veterinary clinics and hospitals located in the 17 autonomous communities present in Spain ; practicD. immitis antigens and for the detection of antibodies against L. infantum, E. canis, and Anaplasma spp. following immunochromatography techniques by using Uranotest Quattro  following manufacturer's instructions. Briefly, one drop of serum or plasma was added along with two drops of reagent to each of the test strips. The sensitivity of the tests was: 97% for L. infantum (vs. IFI), 95% for Ehrlichia (vs. IFI), 94% for D. immitis (vs. necropsy) and 96% for Anaplasma (vs. IFI). The specificity was: 99% for L. infantum (vs. IFI), 94.6% for Ehrlichia (vs. IFI), 100% for D. immitis (vs. necropsy), and 99% for Anaplasma (vs. IFI).Blood samples were collected from the cephalic or jugular vein, placed in 3 ml serum tubes and centrifuged. Serum samples were kept at −20°C until tests were performed. All samples were tested for the detection of Dirofilaria, Leishmania, Anaplasma, and Ehrlichia. Each variable was analyzed individually by obtaining crude odds ratios (ORs) and their 95% confidence intervals. Subsequently, the OR values adjusted for the rest of the factors, using logistic regressions, and their corresponding 95% confidence intervals were estimated as well.Data were analyzed using SPSS Base 20.0 software . Descriptive analysis of the qualitative variables was carried out considering the number of cases and percentages. Univariate and multivariate logistic regression models were carried out to establish the degree of association between the variables gender, age, presence of co-infection, climate, habitat, region and the presence of Of the studied dogs, 54.3%  were male and 45.7%  were female. The age ranged from 2 months to 19 years old, and dogs were further divided into four age groups: dogs <1 year , from 1 to 5 years , from 5 to 10 years , from 10 to 15 years , and dogs >15 years . Regarding habitat, 30%  of the dogs were indoor , 37.2%  were outdoor  and 32.8%  for dogs that spent at least 1–50% of their time outdoors (indoor/outdoor).D. immitis was 6.25%  (95% CI: 5.59–6.98) and the seroprevalences of L. infantum, Anaplasma spp., and E. canis were 10.36%  (95% CI: 9.52–11.27), 5.06%  (95% CI: 4.47–5.73) and 4.26%  (95% CI: 3.72–4.88), respectively. Results by autonomous communities are shown in The results showed that 22.14%  were positive for one or several diseases. The overall prevalence of D. immitis), 10.78% (L. infantum), 5.32% (Anaplasma spp.), and 4.69% (E. canis).Taking into account the sensitivity and specificity of the tests used, true prevalence could be estimated as 6.65% (L. infantum + E. canis (n=41), L. infantum + Anaplasma spp. (n = 32), L. infantum + D. immitis (n = 19), D. immitis + Anaplasma spp. (n = 19), E. canis + Anaplasma spp. (n = 17), D. immitis + E. canis (n = 6). Furthermore, 2% of them were co-infected by three vector-borne infections .Co-infections were observed in 15% of the infected dogs. Of them, 13% were infected by two vector-borne infections, being D. immitis and L. infantum trended to show lower prevalences in the autonomous communities dominated by the Cfb climate; while higher prevalences were observed in the Canary Islands (D. immitis), and Csa and Bsk climates (L. infantum). Anaplasma spp. and E. canis were quite homogeneously distributed in the country.By climates, D. immitis, being females 43% more likely to present dirofilariosis. No significant association was observed between gender and the presence of Leishmania, Anaplasma and Ehrlichia , southern Spain , and the Balearic Islands, which have been traditionally considered the endemic areas of the disease in Spain  is present in Spain   . However Islands , 23, 69; Islands . The resDirofilaria infections; although other epidemiological studies found no significant differences . This highlights a lack of preventive measures to avoid the interaction with the vectors, and the need of the implementation of awareness campaigns among veterinarians and owners. The efficacy of the preventive measures against D. immitis has been observed in Gran Canaria with a constant decrease of the prevalences across 15 years (Co-infections were observed in 15% of the dogs; it has been previously reported that co-infections of vector-borne diseases are common, and studies have reported that up to half of all patients with positive test results for vector-borne diseases may have evidence of co-infection or co-exposure –74. Furt15 years , 70, 75.D. immitis, the detection of parasitic antigens allows to determine current infections. However, the detection of antibodies that occur in the case of Leishmania, Ehrlichia and Anaplasma indicates exposure to disease-causing organisms, but the presence of antibodies does not always indicate disease, which, in some cases, may be confirmed by molecular techniques (In the case of chniques , 12. In chniques . Furtherchniques .This study has a number of limitations. First, even though the samples were chosen randomly by veterinary clinicians, there is a possibility that they have inadvertently selected the samples out of some sort of personal preference. Likewise, it must be taken into account that the sample collected is not representative of each Autonomous Community, especially in those of great extension, despite the fact that efforts have been made to recruit the maximum number of veterinary clinics from different points of geography.The results show a wide distribution of the vector-borne diseases studied; therefore, awareness of these diseases should be raised among the veterinary clinicians and dog owners, and prophylactic plans and measures should be implemented routinely. This is especially important in the vector-borne diseases studied in the present research, because all they are characterized by a wide range of symptoms, highlighting the presence of non-specific clinical signs or clinical-pathological anomalies that can lead to misdiagnosis. Also, many of these animals remain asymptomatic, being uncontrolled reservoirs of the infection; moreover, it has been described that animals with subclinical infections present an increased risk of disease transmission . In addiThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.This animal study was reviewed and approved by ethical committee of Veterinary Medicine Service of the University of Las Palmas de Gran Canaria. Written informed consent was obtained from the owners for the participation of their animals in this study.EC, RM and JM-A designed the study and wrote the manuscript. RM, NC-R, JM, and YF-C performed the fieldwork and collected the data. All authors participated in the discussion of the results, corrected, and read and approved the final manuscript.Although the article has been partially financed by Boehringer Ingelheim Spain, none of the authors maintains a contractual relationship with the company. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.