Behavioural and physiological indicators of shelter dogs' welfare: reflections on the no-kill policy on free-ranging dogs in Italy revisited on the basis of 15 years of implementation.

The Italian National Law 281 of 1991 forbids the euthanatization of free-ranging dogs, unless they have an incurable illness or are proved to be dangerous. Without neglecting the undeniable benefits of the "no-kill" policy, nevertheless it has brought about a chronic overpopulation in shelters and, as a result, higher costs of management and welfare problems since some dogs remain in the shelter for life. In 2004-2008, the Istituto Zooprofilattico Sperimentale of the Lazio and Tuscany regions carried out a survey in the Lazio Region to verify the effects of the Italian National Law 281/91 on free-ranging dog management following 15 years from its implementation. One of the aims of the study was an assessment of the welfare of dogs in a shelter sample (8 shelters out of 47 censused in the Lazio Region). 97 mixed-breed dogs were selected, their behaviour was studied and a blood sample was taken for each dog in order to determine the individual blood concentration of cortisol and the amount of oxidative damage (level of dRoms), as well as the amount of antioxidants to cope with it. Moreover, the total leukocyte count (leukogram) was accomplished. We ran general backward stepwise regression models using "level of antioxidant", "level of dRoms" and "level of serum cortisol" as dependent variables respectively. The results showed that the most important variable that improved the level of welfare of dogs consisted in having the opportunity to regularly go out of the cage for a walk, whereas other variables like gender, size of the cage (small, medium, large), being alone in the cage, and being neutered/entire, had no significant effect on the physiological indicators of welfare. Dogs that enjoyed the regular walk had a higher total antioxidant capacity, and performed a lower frequency of displacing activities and stereotyped behaviour. Moreover, oxidative stress parameters seem to be indicators well matched with behavioural indicators of stress. Thus, for the first time, markers of oxidative status are utilised for the welfare evaluation in the domestic dog. Furthermore, the results of this paper give some suggestion about how small steps can help to improve shelters and, furthermore, this paper intends to solicit the debate on the no-kill policy.

Differential association between circulating testosterone and infection risk by several viruses in natural cat populations: a behavioural-mediated effect?

Testosterone is involved in the development and expression of physiological, morphological and behavioural traits. High levels are often associated with high infection risk and/or intensity, suggesting a trade-off between sexual traits and immunity. Classically invoked mechanisms are immunological or behavioural, i.e., testosterone increases susceptibility or resistance to parasites via an impact on immunity or modulates behaviours involved in parasite transmission. However, studies report contrasted patterns. Given its modes of action and the diversity of host-parasite interactions, testosterone should not act similarly on all interactions. To reduce host and context diversity, we studied 3 viruses in the same cat population: the aggressively transmitted Feline Immunodeficiency virus (FIV), and the Feline Calicivirus (FCV) and Herpesvirus (FHV) both transmitted during friendly contacts. Testosterone had a strong effect on the probability of being positive to FIV whereas its effect was significantly weaker on FCV and FHV. These findings demonstrate that testosterone can be differentially associated with parasites of the same type (viruses). The difference we observed was consistent with a behavioural-mediated effect (increased aggressiveness), supporting the idea that the testosterone effect on infection risk is at least partially driven by behavioural mechanisms in our system. Further investigations (e.g., individual immunity measures) are required to confirm this hypothesis.

Linking genetic diversity and temporal fluctuations in population abundance of the introduced feral cat (Felis silvestris catus) on the Kerguelen archipelago.

Linking temporal variations of genetic diversity, including allelic richness and heterozygosity, and spatio-temporal fluctuations in population abundance has emerged as an important tool for understanding demographic and evolutionary processes in natural populations. This so-called genetic monitoring was conducted across 12 consecutive years (1996-2007) at three sites for the feral cat, introduced onto the Kerguelen archipelago fifty years ago. Temporal changes in allelic richness and heterozygosity at 18 microsatellite DNA loci were compared with temporal changes in the adult population abundance index, obtained by typical demographic monitoring. No association was found at the island spatial scale, but we observed an association between genetic diversity and adult population indices from year to year within each study site. More particularly, the magnitude of successive increases or decreases in the adult population abundance index appeared to be the major factor linking the trajectories of genetic diversity and adult population abundance indices. Natal dispersal and/or local recruitment, both facilitated by high juvenile survival when the adult population size is small, is proposed as the major demographic processes contributing to such an observed pattern. Finally, we suggested avoiding the use of the harmonic mean as an estimator of long-term population size to study the relationships between demographic fluctuations and heterozygosity in populations characterized by strong multiannual density fluctuations.

When cats' ways of life interact with their viruses: a study in 15 natural populations of owned and unowned cats (Felis silvestris catus).

In natural populations, virus circulation is influenced by host behavior and physiological characteristics. Cat populations exhibit a great variability in social and spatial structure, the existence of different ways of life within a same population may also result in different epidemiological patterns. To test this hypothesis, we used a logistic regression to analyze the risk factors of Feline immunodeficiency virus (FIV), feline herpes virus (FHV), feline calicivirus (FCV), and feline parvovirus (FPV) infection in owned (fed and sheltered) and unowned (neither fed nor sheltered, unsocialized) cats living in a rural environment in the North Eastern part of France. A serological survey was carried out in 492 non-vaccinated and non-sterilized individuals from 15 populations living in the same area. The prevalence of feline leukemia virus (FeLV) was also studied, but too few were infected to analyze the risk factors of this virus. For each virus, the epidemiological pattern was different in owned and unowned cats. Unowned cats were more frequently infected by directly transmitted viruses like FIV, FHV and FCV (21.22%, 67.66%, 86.52% in unowned cats vs 9.55%, 53.88%, 77.18% in owned cats, respectively), a difference that may be explained by a more solitary and more aggressive behavior in unowned adults, and/or possibly by a higher sensitivity related to a more stressful life. On the contrary, owned cats were more frequently infected with FPV (36.41% in owned cats vs 15.61% in unowned cats), possibly as a result of their concentration around human settlements. The present study showed that owned and unowned cats living in a same area have behavioral and physiological characteristics sufficiently different to influence virus circulation. Pooling different types of cats in a single sample without taking it into account could give a wrong picture of the epidemiology of their viruses. The conclusion of this work can be extended to any epidemiological studies led in wildlife species with flexible behavior as any variations in social or spatial structure, between or within populations, could result in different virus circulation.

Estimating drop-out probabilities in forensic DNA samples: a simulation approach to evaluate different models.

Allele drop-out is a well known phenomenon that is primarily caused by the stochastic effects associated with low quantity or low quality DNA samples. Recently, new interpretation models that employ the use of logistic regression have been utilised in order to estimate the probability of drop-out. The model parameters are estimated using profiles from samples of extracted DNA diluted to low template levels in order to induce drop-out. However, we propose that this approach is over-simplistic, because several sources of variability are not taken into account in this generalised model. For example, in real-life, small (discrete) crime-stains are analysed where cells are (or were) intact. The integrity of the paired chromosomes of the diploid cell is preserved. In extracted DNA that is diluted to low template levels, we argue that the paired-chromosome integrity is lost. This directly affects the outcome of the logistic model. To date, current experimentation procedures are more akin to haploid cells and thus, different logistic models are needed for haploid and diploid cells. In order to simplify the methodology to estimate the multiple logistic regressions, we propose the use of a simulation model of the entire process associated with the analysis of STR loci, as a supplement to the purely experimental approach to support the validation of new methods. We illustrate with an evaluation of some features of the logistic model proposed by Gill et al., 2009 [12] and discuss alternative models.

The predictive value of the maximum likelihood estimator of the number of contributors to a DNA mixture.

We propose to quantify the accuracy of a likelihood-based estimator that was recently proposed for the determination of the number of contributors to a DNA mixture, when genetic data alone is considered [H. Haned, L. Pène, J.R. Lobry, A.B. Dufour, D. Pontier, Estimating the number of contributors to forensic DNA mixtures: does maximum likelihood perform better than maximum allele count? J. Forensic Sci., in press]. Using Bayes' theorem, we derive a formula for the calculation of the predictive value (PV) of the likelihood-based estimator. The PV gives the probability that a DNA stain contains the DNAs of i people given that the maximum likelihood estimator gave an estimate of i contributors for this stain. We illustrate the PV calculations for two different types of DNA evidence: traces and body fluids. The PV varied according to the number of contributors involved in the DNA stain. Setting the maximum number of possible contributors to five, the lowest predictive values were scored for five-person mixtures with a minimum value of 0.26 for traces, but values were always above 0.94 for stains comprising one, two or three contributors, for both traces and body fluids. Values remained relatively high for four-person mixtures with a minimum value of 0.69. These findings confirm that likelihood-maximization is a powerful approach for the determination of the number of contributors to forensic DNA mixtures.

Modelling the effect of temperature on transmission of dengue.

The main entomological parameters involved in the rate of dengue virus transmission include the longevity of female mosquitoes, the time interval between bites and the extrinsic incubation period of the virus. Field and laboratory data provide estimates for these parameters, but their interactions with other factors (e.g. host population density and environmental parameters) make their integration into a transmission model quite complex. To estimate the impact of these parameters on transmission, we developed a model of virus transmission by a vector population which predicts the number of potentially infective bites under a range of temperatures and entomological parameters, including the daily survival rate of females, the interval between bites and the extrinsic incubation period. Results show that in a stable population, an increase in mosquito longevity disproportionately enhances the number of potential transmissions (e.g. by as much as five times when the survival rate rises from 0.80 to 0.95). Halving the length of the biting interval with a 10- degrees C rise in temperature increases the transmission rate by at least 2.4 times. Accordingly, the model can predict changes in dengue transmission associated with short-term variation in seasonal temperature and also with potentially long-lasting increases in global temperatures.

Genetic markers in the playground of multivariate analysis.

Multivariate analyses such as principal component analysis were among the first statistical methods employed to extract information from genetic markers. From their early applications to current innovations, these approaches have proven to be efficient for the analysis of the genetic variability in various contexts such as human genetics, conservation and adaptation studies. However, because multivariate analysis is a wide and diversified area of statistics, choosing a method appropriate to both the data and to the question being asked can be difficult. Moreover, some particularities of genetic markers need to be taken into account when using multivariate methods. As a consequence, multivariate analyses are often used as black boxes, which results in frequent mistakes in the literature. In this review, we provide a critical analysis of the application of multivariate methods to genetic markers, using a general framework that unifies all these methods for the sake of clarity. First, we focus on some common mistakes in these applications and ways to avoid these pitfalls. We then detail the most critical particularities of allele frequencies that demand adaptations of multivariate methods, and we propose solutions to the subsequent problems. Finally, we tackle several questions of interest in which multivariate analysis has a great role to play, such as the study of the typological coherence of different genetic markers, or the investigation of spatial genetic patterns.

Revealing cryptic spatial patterns in genetic variability by a new multivariate method.

Increasing attention is being devoted to taking landscape information into account in genetic studies. Among landscape variables, space is often considered as one of the most important. To reveal spatial patterns, a statistical method should be spatially explicit, that is, it should directly take spatial information into account as a component of the adjusted model or of the optimized criterion. In this paper we propose a new spatially explicit multivariate method, spatial principal component analysis (sPCA), to investigate the spatial pattern of genetic variability using allelic frequency data of individuals or populations. This analysis does not require data to meet Hardy-Weinberg expectations or linkage equilibrium to exist between loci. The sPCA yields scores summarizing both the genetic variability and the spatial structure among individuals (or populations). Global structures (patches, clines and intermediates) are disentangled from local ones (strong genetic differences between neighbors) and from random noise. Two statistical tests are proposed to detect the existence of both types of patterns. As an illustration, the results of principal component analysis (PCA) and sPCA are compared using simulated datasets and real georeferenced microsatellite data of Scandinavian brown bear individuals (Ursus arctos). sPCA performed better than PCA to reveal spatial genetic patterns. The proposed methodology is implemented in the adegenet package of the free software R.

Spatial and temporal patterning of bank vole demography and the epidemiology of the Puumala hantavirus in northeastern France.

Epidemiological data from bank voles, Myodes glareolus, naturally infected by the hantavirus Puumala (PUUV) were collected by a capture-mark-recapture protocol from 2000 to 2002 in the French department of Ardennes. Four monitored trapping sites were established in two forests located in two cantons (Flize and Monthermé). We captured 912 bank voles corresponding to 557 different individuals during 8820 trapping nights for an overall trapping success of 10.34%. The average PUUV seroprevalence was 22.4%. Characteristics of the system reported in North European countries are confirmed in France. PUUV seroprevalence and abundance of rodents appeared weakly linked. Adult voles were more frequently antibody-positive, but no difference between sexes was established. Anti-PUUV seropositive voles were captured and high seroprevalence was observed from both forests, without human infection reported in Flize canton during the study. One site among the four exhibited peculiar infection dynamics, where vole weight and infection risk were negatively correlated.

Toxoplasmosis in prey species and consequences for prevalence in feral cats: not all prey species are equal.

Toxoplasma gondii is largely transmitted to definitive felid hosts through predation. Not all prey species represent identical risks of infection for cats because of differences in prey susceptibility, exposure and/or lifespan. Previously published studies have shown that prevalence in rodent and lagomorph species is positively correlated with body mass. We tested the hypothesis that different prey species have different infection risks by comparing infection dynamics of feral cats at 4 sites in the sub-Antarctic Kerguelen archipelago which differed in prey availability. Cats were trapped from 1994 to 2004 and anti-T. gondii IgG antibodies were detected using the modified agglutination test (> or =1:40). Overall seroprevalence was 51.09%. Antibody prevalence differed between sites, depending on diet and also on sex, after taking into account the effect of age. Males were more often infected than females and the difference between the sexes tended to be more pronounced in the site where more prey species were available. A difference in predation efficiency between male and female cats may explain this result. Overall, our results suggest that the composition of prey items in cat diet influences the risk of T. gondii infection. Prey compositon should therefore be considered important in any understanding of infection dynamics of T. gondii.

Detecting population structure using STRUCTURE software: effect of background linkage disequilibrium.

STRUCTURE is the most widely used clustering software to detect population genetic structure. The last version of this software (STRUCTURE 2.1) has been enhanced recently to take into account the occurrence of linkage disequilibrium (LD) caused by admixture between populations. This last version, however, still does not consider the effects of strong background LD caused by genetic drift, and which may cause spurious results. STRUCTURE authors have, therefore, suggested a rough threshold value of the distance (1.0 cM) between two loci below which the pair of loci should not be used. Because of the sensitiveness of LD to demographic events, the distance between loci is not always a good indicator of the strength of LD. In this study, we examine the link between genomic distance and the strength of the correlation between loci (r(LD)) in a free-ranging population of mouflon (Ovis aries), and we present an empirical test of effect of r(LD) on the clustering results provided by the linkage model in STRUCTURE. We showed that a high r(LD) value increases the probability of detecting spurious clustering. We propose to use r(LD) as an index to base a decision on whether or not to use a pair of loci in a clustering analysis.

Predicting the emergence of human hantavirus disease using a combination of viral dynamics and rodent demographic patterns.

The paper proposes a model explaining the spatial variation in incidence of nephropathia epidemica in Europe. We take into account the rodent dynamic features and the replicative dynamics of the virus in animals, high in the acute phase of newly infected animals and low in the subsequent chronic phase. The model revealed that only vole populations with multi-annual fluctuations allow for simultaneously high numbers of infected rodents and high proportions of those rodents in the acute excretion phase during the culminating phase of population build-up. This leads to a brief peak in exceptionally high concentrations of virus in the environment, and thereby, to human exposure. Such a mechanism suggests that a slight ecological disturbance in animal-parasite systems could result in the emergence of human diseases. Thus, the potential risk for public health due to several zoonotic diseases may be greater than previously believed, based solely on the distribution of human cases.

Waning of maternal immunity and the impact of diseases: the example of myxomatosis in natural rabbit populations.

Myxomatosis is a leporipoxvirus that infects the european rabbit, inducing a high mortality rate. Observations lead us to hypothesize that a rabbit carrying maternal antibodies (or having recovered) can be infected (or re-infected) upon being exposed (or re-exposed) to the virus. Infection will lead to mild disease, boosting host immune protection. Using a modelling approach we show that this phenomenon may lead to a difference of impact of myxomatosis according to its transmission rate. Young are exposed when they still carry maternal antibodies and develop a mild disease in high transmission populations. Our results show that the impact of myxomatosis is generally higher in epidemic situations compared to populations where the virus circulates all the year. As a consequence, waning of acquired immunity and the continuous supply of newborn along the year may reduce the impact of the disease.

Incidence and persistence of classical swine fever in free-ranging wild boar (Sus scrofa).

Although veterinary authorities aim to limit persistence of classical swine fever (CSF) in wild boar (Sus scrofa), to avoid potential transmission to pigs, factors influencing CSF transmission and persistence are not clearly understood. Here we analyse incidence and persistence in a CSF epidemic that occurred in the French Vosges Forest. Higher incidence was found in large forests compared to smaller isolated ones, being highest near the starting point of the epidemic, but poorly related to the local density. We hypothesize that the spatial and social structure of wild boar populations may be responsible for this variability of incidence over space. Persistence was highest near the starting point of the epidemic and where initial density was highest. We hypothesize that persistence was favoured by the abundance of young wild boar, itself encouraged by CSF. Our results allow us to propose management measures aimed at limiting CSF persistence.

Measuring diversity from dissimilarities with Rao's quadratic entropy: are any dissimilarities suitable?

Rao has developed quadratic entropy to measure diversity in a set of entities divided up among a fixed set of categories. This index depends on a chosen matrix of dissimilarities among categories and a frequency distribution of these categories. With certain choices of dissimilarities, this index could be maximized over all frequency distributions by eliminating several categories. This unexpected result is radically opposite to those obtained with usual diversity indices. We demonstrate that the elimination of categories to maximize the quadratic entropy depends on mathematical properties of the chosen dissimilarities. In particular, when quadratic entropy is applied to ultrametric dissimilarities, all categories are retained in order to reach its maximal value. Three examples, varying from simple one-dimensional to ultrametric dissimilarity matrices, are provided. We conclude that, as far as diversity measurement is concerned, quadratic entropy is most relevant when applied to ultrametric dissimilarities.

Is there geographical variation in human handedness?

Right- and left-handed individuals are present in all cultures. However, while it is known that handedness is a heritable trait, little is known about how handedness varies between populations-and without this knowledge, the significance of the left/right polymorphism is hard to interpret. We reviewed the literature to assess the extent of geographical variation of throwing or hammering handedness. These two tasks were chosen because they are present in all known cultures (unlike, for example, writing), and make sense within the context of several adaptive theories on the origin of laterality, or maintenance of handedness polymorphism, which state that tool or weapon manipulation are pivotal. A total of 81 samples were found with primary data on throwing or hammering handedness, spanning 14 countries and concerning more than 1,214,000 individuals studied between 1922 and 1998. A global logistic regression was performed to assess the significance of the country of the study, controlling for several potentially confounding variables (date of the study, sex and age of individuals). Country always had a significant effect, consistent with substantial geographical variation of throwing and hammering handedness. Curiously, left-handedness frequency estimates for a given country were not always consistent across datasets, perhaps due to missing variables, such as educational level or socio-economic status. Results are discussed in the context of the evolution of handedness and the significance of the current polymorphism.

Effective size of two feral domestic cat populations (Felis catus L): effect of the mating system.

A variety of behavioural traits have substantial effects on the gene dynamics and genetic structure of local populations. The mating system is a plastic trait that varies with environmental conditions in the domestic cat (Felis catus) allowing an intraspecific comparison of the impact of this feature on genetic characteristics of the population. To assess the potential effect of the heterogenity of males' contribution to the next generation on variance effective size, we applied the ecological approach of Nunney & Elam (1994) based upon a demographic and behavioural study, and the genetic 'temporal methods' of Waples (1989) and Berthier et al. (2002) using microsatellite markers. The two cat populations studied were nearly closed, similar in size and survival parameters, but differed in their mating system. Immigration appeared extremely restricted in both cases due to environmental and social constraints. As expected, the ratio of effective size to census number (Ne/N) was higher in the promiscuous cat population (harmonic mean = 42%) than in the polygynous one (33%), when Ne was calculated from the ecological method. Only the genetic results based on Waples' estimator were consistent with the ecological results, but failed to evidence an effect of the mating system. Results based on the estimation of Berthier et al. (2002) were extremely variable, with Ne sometimes exceeding census size. Such low reliability in the genetic results should retain attention for conservation purposes.

Puumala hantavirus infection in humans and in the reservoir host, Ardennes region, France.

We compared the occurrence of nephropathia epidemica cases, over a multi-annual population cycle, in northeastern France with the hantavirus serology for bank voles captured in the same area. We discuss hypotheses to explain the pattern of infection in both humans and rodents and their synchrony.

Trans-dominant suppression of plant TGA factors reveals their negative and positive roles in plant defense responses.

Salicylic acid (SA) is a key regulator for the induction of systemic acquired resistance (SAR), and NPR1 is a critical mediator for the biological effects of SA. Physical interactions between NPR1 and TGA factors, a conserved family of basic-leucine-zipper (bZip) proteins in plants, have suggested a role for these transcription factors in mediating SAR induction via the regulation of defense genes. To elucidate this function, we constructed a trans-dominant mutant that specifically eliminates DNA-binding activities of this class of bZip proteins in transgenic tobacco plants. Our results demonstrate that the loss of TGA DNA-binding activities is correlated with suppression of two xenobiotic-responsive genes, GNT35 and STR246, and enhanced induction of pathogenesis-related (PR) genes by SA. In addition, these TGA-suppressed plants exhibited higher levels of PR gene induction by pathogen challenge and an enhanced SAR. These results suggest that TGA transcription factors serve both negative and positive regulatory roles in mediating plant defense responses.