IJP: Parasites and Wildlife 8 (2019) 221–228 Contents lists available at ScienceDirect IJP: Parasites and Wildlife journal homepage: www.elsevier.com/locate/ijppaw Exposure of yellow-legged gulls to Toxoplasma gondii along the Western T Mediterranean coasts: Tales from a sentinel Amandine Gamblea,∗, Raül Ramosb, Yaiza Parra-Torresb, Aurélien Mercierc,d, Lokman Galalc, Jessica Pearce-Duveta, Isabelle Villenae,f, Tomás Montalvog,h, Jacob González-Solísb, Abdessalem Hammoudai, Daniel Oroj,k, Slaheddine Selmii, Thierry Bouliniera a CEFE, CNRS, University of Montpellier, EPHE, University Paul Valéry Montpellier 3, IRD, Montpellier, France b Department of Evolutionary Biology, Ecology and Environmental Sciences, Faculty of Biology, Universitat de Barcelona, Barcelona, Spain c INSERM UMR_S 1094, Neuroépidémiologie Tropicale, Laboratoire de Parasitologie-Mycologie, Faculté de Médecine, Université de Limoges, Limoges 87025, France d Centre National de Référence Toxoplasmose/Toxoplasma Biological Resource Center, CHU Limoges, 87042 Limoges, France e Université de Reims Champagne-Ardenne, Laboratoire de Parasitologie - Mycologie, EA 3800, UFR Médecine, SFR CAP-SANTÉ, Reims, France f Laboratoire de Parasitologie-Mycologie, Centre National de Référence de la Toxoplasmose, Hôpital Maison Blanche, CHU Reims, Reims, France g Servei de Vigilància i Control de Plagues Urbanes, Agència de Salut Pública de Barcelona, Barcelona, Spain h CIBER Epidemiologia y salud Pública (CIBERESP), Madrid, Spain i UR "Ecologie de la Faune Terrestre" (UR17ES44), Faculté des Sciences de Gabès, Université de Gabès, Gabès, Tunisia j IMEDEA, CSIC-UIB, Esporles, Spain k CEAB, CSIC, Blanes, Spain A R T I C LE I N FO A B S T R A C T Keywords: Efficiently tracking and anticipating the dynamics of infectious agents in wild populations requires the gathering Toxoplasma gondii of large numbers of samples, if possible at several locations and points in time, which can be a challenge for some Eco-epidemiology species. Testing for the presence of specific maternal antibodies in egg yolks sampled on the colonies could Immunoassay represent an efficient way to quantify the exposure of breeding females to infectious agents, particularly when Sampling strategy using an abundant and widespread species, such as the yellow-legged gull (Larus michahellis). We used such an Stable isotope analysis Mediterranean region approach to explore spatio-temporal patterns of exposure to Toxoplasma gondii, a pathogenic protozoan re- sponsible of toxoplasmosis in humans and other warm-blooded vertebrates. First, we tested the validity of this approach by exploring the repeatability of the detection of specific antibodies at the egg level using two different immunoassays and at the clutch level using an occupancy model. Then, samples gathered in 15 colonies from France, Spain and Tunisia were analysed using an immunoassay detecting antibodies specifically directed against T. gondii. Prevalence of specific antibodies in eggs was overall high while varying significantly among colonies. These results revealed that T. gondii circulated at a large spatial scale in the western Mediterranean yellow-legged gull population, highlighting its potential role in the maintenance community of this parasite. Additionally, this study illustrates how species commensal to human populations like large gulls can be used as wildlife sentinels for the tracking of infectious agents at the human-wildlife interface, notably by sampling eggs. 1. Introduction host (Dubey, 1998a), potentially playing an important role in the parasite circulation. For instance, migratory geese are suspected to be Toxoplasma infection (toxoplasmosis) is being recognized as one of responsible for the introduction of the parasite in Arctic cat-free islands the most common parasitic zoonotic diseases in the world, notably in (Prestrud et al., 2007). Hence, more studies are needed to better un- relation to the worldwide distribution and wide range of hosts of its derstand the cycle of the parasite, notably at the interface between wild causative agent, Toxoplasma gondii (Tenter et al., 2000). However, the fauna and human populations where public health but also conserva- complex pathogenicity and life cycle of this parasite remain elusive. tion issues can arise (e.g., the suspicion of T. gondii introduction via While felids are considered as the only definitive hosts of T. gondii, it is feral cats Felis catus populations on remote islands; Work et al., 2002; supposed that any warm-blooded vertebrate could be an intermediate Deem et al., 2010). ∗ Corresponding author. CEFE-CNRS, 1919 route de Mende, 34293, Montpellier, France. E-mail address: amandine.gamble@gmail.com (A. Gamble). https://doi.org/10.1016/j.ijppaw.2019.01.002 Received 9 October 2018; Received in revised form 10 January 2019; Accepted 11 January 2019 2213-2244/ © 2019 Published by Elsevier Ltd on behalf of Australian Society for Parasitology. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). A. Gamble, et al. IJP: Parasites and Wildlife 8 (2019) 221–228 In this context, studies focusing on wild species commensal to to track its exposure to T. gondii at a hierarchy of spatial scales and to human populations should help to identify potential relevant hotspots explore potential relations with diet. Yellow-legged gulls are well- of circulation of T. gondii to then investigate potential risk factors. In known consumers of anthropogenic food sources, such as fishery dis- particular, predator and/or scavenger species like gulls may be espe- cards and leftovers from refuse tips (Duhem et al., 2008; Ramos et al., cially interesting to consider. Indeed, gulls could be exposed to T. gondii 2009, 2011). This feeding behaviour makes the species a potential re- via oocysts found in the environment, notably water soiled with felids’ levant sentinel to monitor disease circulation and infection risks for faeces (Dubey, 2009), but also via other sources considering their op- human and/or wild populations. The fact that in some colonies, po- portunistic feeding behaviour (Appendix A, Figure S1.1). Gulls can be pulations are controlled by egg sterilisation to limit interactions with exposed to bradyzoites by preying or scavenging on infected inter- other species and/or human populations (Oro and Martínez-Abraín, mediate hosts. Notably, large gulls can feed on smaller birds (Oro and 2007) facilitates access to eggs. In addition, because there is some Martínez-Abraín, 2007). Also, because gulls live in close proximity to confusion regarding the identity of T. gondii-like parasites in wild birds human populations, they have access to refuse tips (Ceia et al., 2014; (Dubey, 2002), we also collected tissues from dead gulls in order to Navarro et al., 2016; Ramos et al., 2009; Real et al., 2017; Steigerwald confirm the circulation of the parasite and characterize the circulating et al., 2015). Recently, it has also been suggested that marine prey, and strain. The Mediterranean area, at the crossroad between continents, is filter feeders in particular, may accumulate oocysts (Massie et al., particularly interesting to consider given the structure of T. gondii po- 2010), explaining therefore the ubiquity of this parasite in the marine pulations at the world scale and the potential relationship between environment (Dubey et al., 2005; Honnold et al., 2005; van de Velde strain and pathogenicity (Galal et al., 2018; Xiao and Yolken, 2015). et al., 2016; Verma et al., 2018). The expected results of this study may have important implications Despite the potential wide exposure of gulls to T. gondii, only a few regarding the potential role of large gulls in the maintenance commu- studies have reported information on this topic (Burridge et al., 1979; nity of T. gondii. Karakavuk et al., 2018; Prestrud et al., 2007; Uterák et al., 1992). Notably, a recent study revealed the detection of anti-T. gondii anti- 2. Material and methods bodies in yellow-legged gull (Larus michahellis) nestlings using the modified agglutination test (MAT; Cabezón et al., 2016). However, in 2.1. Study population and sampling protocols that study nestling age was not accounted for, making it difficult to interpret the results because measured antibody levels could arise from Yellow-legged gull nest in colonies that are mostly established in a mixture between maternally inherited and autogenous antibodies marshlands and on natural and man-made islets. Some are also found (Boulinier and Staszewski, 2008). Capturing adults of such species for on city roofs. The field sampling was conducted between 2009 and the collection of biological samples requires particular efforts and skills. 2016. Each year, a set of colonies located on the Mediterranean coasts Testing for the presence of specific maternal antibodies in egg yolk of France, Spain and Tunisia was visited during the peak of the laying samples could represent an efficient way to quantify the exposure of period (late March-early April) for fresh egg collection (Appendix A, breeding females to various infectious agents, while avoiding the dif- Table S1.1). Incubation stage was assessed by a brief immersion of eggs ficult interpretation of nestling serological profiles. Indeed, in birds, in water and only one fresh egg per clutch (generally composed of three antibodies (IgY) can be transmitted from breeding females to hatchlings eggs) was collected (Hays and LeCroy, 1971); freshly laid eggs do not through the egg yolk (Boulinier and Staszewski, 2008) from which they float and non-collected eggs were put back in the nest. This method can be quantified using classical immunoassays. This approach has al- insured an efficient sampling of eggs with minimum impact on gull ready proven efficient to detect exposure of yellow-legged gulls to avian reproduction. The sampling protocol was approved by the relevant influenza viruses (Hammouda et al., 2014) and flaviviruses (Arnal local institutions (Direction régionale de l’Environnement, de l’Amén- et al., 2014), and the exposure of great cormorants (Phalacrocorax agement et du Logement Provence-Alpes-Côte d’Azur and Languedoc- carbo) to Newcastle disease virus (Alekseeev et al., 2014). Moreover, Roussillon; Syndicat Mixte des Etangs Littoraux; Parc National des Ca- eggs also contain information on the diet of breeding females. Because lanques; Conservatoire Etudes des Ecosystèmes de Provence-Alpes du food is likely to be the main source of exposure of adults to T. gondii, sud; Mairie de Marseille; Marie de Gruissan; Parc Natural de El Montgrí, one could expect that different feeding behaviours of individual gulls les Illes Medes i el Baix Ter; Parc Natural del Delta de l’Ebre; Forest can lead to different risks of exposure to the parasite. This factor is service of the Tunisian Ministry of Agriculture). particularly important to consider in species exhibiting a wide variety Spatial patterns in the prevalence of anti-T. gondii antibodies were of feeding behaviours such as gulls (Ramos et al., 2009a). For instance, first explored in the samples collected in 2009 and 2016, including 10 individuals feeding on refuse tips and sewage water could be expected and 12 colonies, respectively (Fig. 1). Second, temporal patterns were to be more at risk of exposure than individuals feeding at sea (Cabezón explored in three specific breeding sites (Frioul, Gruissan and Medes et al., 2011, 2016). Since the 1990s, stable isotopes analysis has been Islands), using samples collected along the period 2009–2016 (Fig. 2). used as a powerful approach to study animal diet (Ramos and González- Third, in the two Tunisian colonies sampled in 2009, the three eggs of Solís, 2012). This approach relies on the assumption that the isotopic every nest had been sampled (n = 201 eggs from 67 nests) in order to signature of the tissues of a consumer reflects that of its diet (Bearhop assess if the detection of antibodies from any egg provides equivalent et al., 2004), and can be used on egg components. For instance, specific information to infer the occurrence of antibodies in the mother using a isotopic ratios are known to differ among habitats: marine ecosystems patch occupancy model (Appendix A). Fourth, in order to explore the have higher isotopic ratios of carbon (13C/12C or δ13C), nitrogen pattern of exposure at a smaller scale (within a colony), we used sam- (15N/14N; δ15N), and sulphur (34S/32S; δ34S) than terrestrial and pled nest locations recorded using the global position system (GPS) in freshwater environments (Hobson, 1999; Hobson et al., 1994; Peterson colonies sampled in 2016 (Appendix A, Figure S4.1). Once at the la- and Fry, 1987). One could thus expect individuals with egg isotopic boratory, within the few days following collection, yolk and albumen signatures indicating the consumption of items from refuse tips or other were separated and stored at −20 °C until analysed. terrestrial resources to be more likely to be anti-T. gondii antibody Complementarily, and in an attempt to isolate T. gondii from yellow- positive compared to individual with isotopic signatures associated to a legged gulls, necropsies were also conducted on three adults found marine diet (Ramos et al., 2009; Cabezón et al., 2016), on the condition freshly dead in three colonies (Frioul, Sidrière and Ebro Delta) in 2017. that dietary and immunological information are integrated on the same Brain and cardiac muscle tissues were collected and kept at 4 °C for less time scale. than 72 h. Intra-cardiac blood was also collected using a heparinized In the present study, we sampled eggs of an abundant and widely syringe and centrifuged a few hours after collection; the plasma was distributed species of the Mediterranean coasts, the yellow-legged gull, stored at −20 °C until analysed. 222 A. Gamble, et al. IJP: Parasites and Wildlife 8 (2019) 221–228 Fig. 1. Map of prevalences of anti-T. gondii anti- bodies in yellow-legged gull egg yolk samples in 2009 (a) and 2016 (b) illustrating the spatial varia- bility. RIO: Riou; FRI: Frioul; CAR: Carteau; VIC: Vic- la-Gardiole; GRU: Gruissan; HOT: Hortel; SID: Sidrière; COR: Corrège; MED: Medes; BCN: Barcelona; EBR: Ebro Delta; DRA: Dragonera; AIR: Illa de l’Aire; SSF: Sfax; HDJ: Djerba. Coloured circles highlight the colonies in which temporal variations were explored (Fig. 2).Sample sizes and confidence intervals are given in Appendix A, Table S1.1. Base map: esri©. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) frequently used for T. gondii serosurveys, notably in wild birds (Cabezón et al., 2016; Sandström et al., 2013), although, to our knowledge, it has never been used on egg yolk samples before. Details are presented in the supplementary material (Appendix A). Overall, the ELISA was proven repeatable at a much higher level than the MAT at the sample scale (i.e., repeated analyses conducted on a given sample). At the nest scale (i.e., repeated sampling of eggs from a given nest), the detection probability was suitable. In addition, the ELISA and the MAT lead to the same prevalence of specific antibodies at the colony scale. These results are in line with previous studies suggesting that ELISA are generally suitable to detect anti-T. gondii antibodies in domestic and wild animals (e.g., Dubey et al., 2005; Gamble et al., 2005). Plasma samples from necropsied individuals were analysed by MAT with positivity threshold at dilution 1/20. 2.3. Stable isotope analysis and trophic inferences δ13C, δ15N and δ34S were measured in the albumen sample as in- dicators of breeding females’ diet. δ13C and δ34S are used primarily to determine sources of primary production and are useful to trace the input of these elements into the food webs (Richards et al., 2003). In particular, the use of δ34S is recommended when the origin of the diet is heterogeneous. δ15N suffers considerable fractionation with each trophic level, making it suitable for assessing in which trophic level the Fig. 2. Limited temporal variations of the prevalence of anti-T. gondii antibody in yellow-legged gull egg yolk samples between 2009 and 2016 in three co- consumer feeds. That is, consumers at the top of a long trophic chain lonies: Frioul, Gruissan and Medes Islands. Curves correspond to cubic splined show a higher δ15N than those that feed on impoverished food chains, fitted to the yearly prevalences for visualisation purposes only. Bars indicate as might be the case for individuals feeding on refuse tips (Ramos and 95% Clopper-Pearson confidence intervals. (For interpretation of the references González-Solís, 2012). to colour in this figure legend, the reader is referred to the Web version of this Albumen samples were first dried in an oven at 60 °C to constant article.) mass, and subsequently ground to fine powder with a mortar. Subsamples of powdered materials were weighed to the nearest μg, 2.2. Anti-T. gondii antibody detection placed in tin capsules and crimped for combustion. Samples were oxi- dized at the Serveis Científico-Tècnics of the University of Barcelona Specific antibody levels were measured in yolk samples to infer the (SCT-UB; Spain) by means of elemental analysis-isotope ratio mass past exposure of breeding females to T. gondii using a commercially spectrometry, using a Thermo Finnigan Flash 1112 (CE Elantech, available indirect enzyme-linked immunosorbent assay (ELISA) de- Lakewood, NJ, USA) elemental analyser coupled to a Delta-C isotope signed to detect chicken IgY directed against the T. gondii p30 antigen ratio mass spectrometer via a CONFLOIII interface (Thermo Finnigan (ID Screen® Avian Toxoplasmosis Indirect, IDvet, France). This study is MAT, Bremen, Germany). Stable isotope ratios were expressed in the the first to investigate the detection of anti-T. gondii antibodies in gull standard δ-notation (in ‰) relative to Vienna Pee Dee Belemnite egg yolk samples, and one of the few to investigate their detection in (VPDB; δ13C), atmospheric N2 (VAIR; δ15N) and Vienna Cañon Diablo seabirds in general (Dubey, 2002 for a review). We thus included sev- Troilite (VCDT; δ34S). Isotopic ratio mass spectrometry facility at the eral validation procedures on a subset of samples in our laboratory SCT-UB applies international inorganic standards (IAEA CH7, IAEA analyses. Notably, as no gold standard is available for the detection of CH6 and USGS 24 for C, IAEA N1, IAEA N2 and IAEA NO3 for N and anti-T. gondii antibodies in wild birds, a second assay was run on a IAEA-S1, IAEA-S2, IAEA-S3, NBS-127 and YCEM for S) as well as in- subset of samples (Enøe et al., 2000). The MAT was chosen as it is ternal organic standards (Human Hair CRM 397, Community Bureau of Reference, Commission of the European Community; δ13C: 223 A. Gamble, et al. IJP: Parasites and Wildlife 8 (2019) 221–228 −19.5 ± 0.2, δ15N: 10.0 ± 0.2, δ34S: 4.5 ± 0.2) inserted every 12 18, 1986) and approved by the Regional Ethics Committee Limousin samples to calibrate the system and compensate for any drift over time. (Registration Number: CREEAL 3-07-2012). Replicate assays of standard materials indicated measurement errors of ± 0.1, ± 0.2 and ± 0.2‰, for C, N and S respectively but these are 3. Results likely underestimates of true measurement errors for complex organics like albumen samples. 3.1. Spatio-temporal variation in antibody prevalence 2.4. Statistical analyses In total, 1122 egg yolk samples from 988 nests were screened for anti-T. gondii antibodies by ELISA (Appendix A, Table S1.1). Anti-T. We calculated the prevalence of anti-T. gondii antibodies in a sam- gondii antibodies were detected in 233 screened nests, corresponding to pled colony as the proportion of nests containing at least one positive an overall antibody prevalence [95% confidence interval] of 0.19 [0.14; egg over the total number of nests sampled. We then assessed variation 0.24] in 2009 and 0.33 [0.29; 0.39] in 2016 (note that the set of in antibody prevalence among years and colonies by means of logistic sampled colonies is different between the two years). Prevalence ranged regression models. Two separate regressions were carried out: (1) one from 0.03 to 0.52 among years and colonies (Fig. 1). In 2009 and 2016, focusing on 2009 and 2016 to explore spatial patterns and (2) one fo- year and colony had significant effects on the occurrence probability of cusing on the temporal series collected on Frioul, Gruissan and Medes anti-T. gondii antibodies (year: χ2 = 5.8, d.f. = 1, p = 0.02; site: to explore temporal patterns. Best models were selected based on χ2 = 46.5, d.f. = 6, p < 0.01; n = 414 samples; Appendix A, Table Akaike information criteria (AIC) with a threshold of ΔAIC = 2 S3.1). When focusing on Frioul, Gruissan and Medes (for which tem- (Burnham and Anderson, 2002). poral series where analysed), year did not provide a significant pre- Spatial auto-correlation of antibody prevalence (as a continuous dictor of antibody occurrence probability, as it was not included or not variable) among colonies was assessed using 2009 and 2016 data, by significant in the selected models (Appendix A, Table S3.2). However, calculating the Moran's I (Moran, 1950). Within colonies, spatial auto- there was a significant difference between colonies (χ2 = 23.1, d.f. = 2, correlation of nest antibody status (anti-T. gondii antibody-negative or p < 0.01; n = 525 samples). Overall, these results suggest some large –positive as a binary variable) was assessed by calculating the BB join- scale spatial variation but limited local temporal variation in gull ex- count statistic (Cliff and Ord, 1981). These statistics were calculated posure to T. gondii. using the ‘spdep’ R package and p-values were corrected for multiple There was no significant spatial auto-correlation of the prevalence tests using the Bonferonni correction. Maps were drawn with QGIS among colonies (2009: I = 0.46, p = 0.12, n = 10; 2016: I = −0.30, 2.18.21 and the ‘QuickMapServices’ plugin. p = 1.00, n = 12). Similarly, there was no significant spatial auto- The relationship between nest status (antibody-negative or -posi- correlation of nest status within colonies (all p > 0.10 for the five tive) and mother's diet was explored by comparing the distribution of concerned colonies; Appendix A, Table S4.1; Fig. 3). the isotopic ratios (δ13C, δ15N, and δ34S) between antibody-negative and -positive nests. To confirm observations, a logistic regression with 3.2. Relationship between exposure to T. gondii and diet the three isotopic signatures as fixed effects, the year and site as random effects and nest status as the response variable was run on the subset of Complete antibody and isotopic data were available for 129 egg samples for which all measures were available. yolk samples (Appendix A, Table S1.2). Distributions of the isotopic More details regarding the run models are given in Appendix A. All ratios (δ13C, δ15N and δ34S) largely overlapped between negative and statistical analyses were conducted in R 3.3.3 and codes are available in positive nests (Fig. 4), suggesting no relationship between nest antibody Appendix B. status and inferred breeding females' diet. This observation was con- firmed by the fact that none of the isotopic signature had a significant 2.5. T. gondii isolation and genotyping effect on the nest status in the selected models (Appendix A, Table S3.3; n = 129 samples). Tissues for parasite isolation were collected from the necropsied gulls that tested positive for anti-T. gondii antibodies. Brains and cardiac 3.3. T. gondii isolation and genotyping muscles were prepared and inoculated intraperitoneally to three female Swiss mice (Mus musculus) following the procedure described in Mercier The three necropsied gulls were seropositive for anti-T. gondii an- et al. (2010). Given the low parasitic burden found in the tissues of tibodies. Two individuals showed positive real-time PCR (heart and most T. gondii hosts, mouse bioassay was necessary for an upstream brain) with threshold values (CT) of 30.50 and 34.74 respectively for amplification of parasitic burden to reach the sensitivity thresholds of individuals 170329Lmic01 ES (from Ebro Delta, Spain) and most genotyping techniques. Considering the risk of bacterial pro- 170402Lmic0111 (from Sidrière, France). One live strain was isolated liferation due to the sampling conditions and the decomposition state of from the individual originating from Ebro Delta, Spain, as indicated by the gull samples, those were treated with an antibiotic solution (1000 the observation of T. gondii tissue-cysts under the microscope in brain U/ml penicillin and 100 μg streptomycin/ml in saline solution) before samples from inoculated mice. This isolate was genotyped as type II inoculation. A 200 μl aliquot of the gull brain and heart preparations (Appendix A, Table S5.1). was also used for DNA extraction using the QIAamp® DNA MiniKit (Qiagen, France) and quantification of T. gondii by real-time PCR tar- 4. Discussion geting the T. gondii 529 bp repetitive element as previously described (Homan et al., 2000). The mice were monitored daily and all surviving Using egg sampling and an easily available immunoassay, we ex- mice were euthanized at four weeks post-inoculation. Brain from the plored patterns of yellow-legged gull exposure to T. gondii at different MAT-positive mice were screened for cysts by microscopic examination. spatial and temporal scales. The results revealed a wide exposure of this Live parasites were cryopreserved until analysed. DNA from 200 μl of species to the parasite, which highlights its broad presence at the mouse brain tissue was extracted and T. gondii isolates were genotyped wildlife-human interface of the western Mediterranean Sea, in line with using the length polymorphism of 15 multilocus microsatellite markers results obtained from other wild avian species (e.g., Cabezón et al., located on 11 different chromosomes in a multiplex PCR assays as de- 2011). Additionally, this study suggests that large gulls could poten- tailed in Ajzenberg et al. (2010). All experimental procedures in mice tially play a role in the reservoir community of this zoonotic parasite, were conducted according to European guidelines for animal care notably through predation or scavenging by other intermediate hosts (‘‘Journal Officiel des Communautés Européennes’’, L358, December (e.g., other gulls or rodents) or by felids, although this would be 224 A. Gamble, et al. IJP: Parasites and Wildlife 8 (2019) 221–228 Fig. 3. Location and status of yellow-legged gull nests sampled in 2016 and screened for anti-T.gondii antibodies. Base map: Google©. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) difficult to quantify and may vary depending on the characteristics of humans in Europe and is also the predominant lineage among both the colonies (e.g., predators are likely to be absent from most of the domestic and wild fauna on this continent (Shwab et al., 2014; colonies that are on small islets). It also stresses that gulls can be used as Ajzenberg et al., 2015). The genotype described in this study using 15 general wildlife sentinels for tracking certain infectious agents at the MS genotyping exhibited an unreported combination of alleles within wildlife-human interface, especially through egg sampling. the diversity of type II lineage which supports the substantial genetic In gulls, reports of T. gondii isolation are scarce (Karakavuk et al., diversity within the Type II lineage (Shwab et al., 2018). Future work 2018; Literák et al., 1992). Isolating T. gondii from its intermediate using molecular epidemiology approaches should thus aim at exploring hosts is difficult, notably because it relies on the availability of fresh the phylogenetic relationship between the strains circulating among internal tissues. For detection and exposure quantification purposes, gulls and the strains infecting other T. gondii hosts, including humans. serology offers a useful tool (Dubey et al., 2005; Elmore et al., 2016, This approach may be useful to quantify parasitic exchanges between 2014; Opsteegh et al., 2011) but does not allow genetic characteriza- these species and thus the potential involvement of yellow-legged gulls tion. In the present study, identification of a type II T. gondii isolated in the reservoir community of this parasite. from a yellow-legged gull using mouse bioassay confirmed that gulls As mentioned above, yellow-legged gulls could be exposed to T. can be infected by T. gondii. Type II is the main lineage infecting gondii through various sources of exposures notably in relation to their 225 A. Gamble, et al. IJP: Parasites and Wildlife 8 (2019) 221–228 could limit the environmental transmission of T. gondii, leading to lower prevalences (Brouat et al., 2018; Dubey, 1998b). If the impact of cli- mate on T. gondii transmission was confirmed, it would suggest that environmental transmission of oocysts may represent a relatively im- portant source of exposure for gulls, in line with a previous study conducted on yellow-legged gulls (Cabezón et al., 2016). The diversity of sources of exposure could explain the absence of an evident re- lationship between the past exposure to the parasite and diet pre- ferences of the individuals and colonies (inferred through δ13C, δ15N and δ34S), highlighting the complexity of the epidemiology of T. gondii. In addition, immunological information contained in egg yolk and diet information contained in albumen may represent processes happening at different time scales, blurring potential relationships. Yellow-legged gulls exhibit individual specialization, leading to some consistency of individual diets, and thus of individual isotopic signatures, at the inter- annual scale (Ceia et al., 2014). However, individual diets and isotopic signatures are expected to vary among and within years in relation to food availability. Gulls have been described as income breeders, i.e., females rely on local nutrient sources for egg formation during the clutch production period (Ramírez et al., 2011; Ruiz et al., 2000; Saino et al., 2010). Therefore, the isotopic composition of egg albumens should reflect the females’ diet consumed during the egg laying period. In contrast, high anti-T. gondii antibody levels are considered to be maintained over the years following exposure and could thus reflect an exposure event that happened several years before laying. This meth- odological constraint may mask potential links between inferred diet and immunological status. Knowledge on the dynamics of the specific immune response against T. gondii however remains elusive. Although protective antibody levels are considered to be maintained life-long in humans, some evidence suggest high seroreversion rates in wild mammals and birds (Opsteegh et al., 2011; Sandström et al., 2013). The lack of knowledge on the specific antibody kinetics after exposure to T. gondii makes it difficult to interpret temporal patterns of ser- oprevalences, which would require the longitudinal monitoring of marked individuals (e.g., Sandström et al., 2013). In addition, clinical cases of toxoplasmosis have been reported from wild and domestic avian species (Dubey, 2002), but the impact of the parasite on wild populations has never been quantified. In particular, because vertical transmission is frequent in humans, leading to abortions and congenital malformations (Tenter et al., 2000), the presence of T. gondii in eggs and its impact on breeding should be further investigated. A first step in the exploration of the possibility of vertical transmission in wild birds could be to screen egg yolk samples for parasitic DNA (e.g., Rahaus et al., 2008). Beyond T. gondii epidemiology, understanding the ecology and evolution of infectious diseases in wildlife has been acknowledged as critical for public health and biodiversity conservation (Daszak et al., 2000). However, wildlife investigations have been often hampered by the difficulties in collecting enough data at broad spatial and temporal scales, which has limited our current knowledge on eco-epidemiolo- gical dynamics. In this epidemiological context, tracking infectious agents in some wild species may be more rewarding than in others, depending on their epidemiological and ecological characteristics (Halliday et al., 2007). Here we illustrate how gulls could be used as Fig. 4. Biplots of δ13C and δ15N (a) and δ34S and δ15N (b) representing the sentinel to report spatial patterns of exposure to a zoonotic infectious isotopic variability of yellow-legged gull albumen samples as a response of their agent, notably by sampling eggs. This approach could be applied to immunological status against T. gondii. Isotopic signatures (as a proxy of fe- males' diet) and egg/nest immunological status (as a proxy of females' exposure quantify the circulation of a wide range of other infectious agents of to the parasite) does not appear to be related. (For interpretation of the refer- interest for human or animal populations (e.g., avian influenza viruses: ences to colour in this figure legend, the reader is referred to the Web version of Hammouda et al., 2011; tick-borne viruses: Arnal et al., 2014). In ad- this article.) dition, eggs can be used to quantify exposure to various mineral and organic pollutants (Ackerman et al., 2016; Bertolero et al., 2015; Morales et al., 2016, 2012; Ramos et al., 2013; Vicente et al., 2012) and opportunistic feeding behaviour. The spatial variations of the pre- to study food availability around colonies in interaction with density- valence of antibodies against T. gondii could be linked to differences in dependence strength (Real et al., 2017). Because large gulls are abun- environmental conditions among the considered colonies. Notably, the dant and widely distributed (e.g., Hebert, 2015; Navarro et al., 2016), dryer and hotter climate in Tunisia compared to the European coasts this approach could be applied at larger spatial scales to track exposure 226 A. Gamble, et al. IJP: Parasites and Wildlife 8 (2019) 221–228 to various infectious agents susceptible to circulate at the human- Bertolero, A., Vicente, J., Meyer, J., Lacorte, S., 2015. 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