Received: 11 May 2021 | Revised: 12 December 2021 | Accepted: 23 January 2022 DOI: 10.1111/zph.12919 ORIGINAL ARTICLE Urban landscape and infection risk in free-­roaming cats Mónica G. Candela1 | Angela Fanelli2 | João Carvalho3 | Emmanuel Serrano4 | Guillermo Domenech5 | Francisco Alonso1 | Carlos Martínez-­Carrasco1 1 Department of Animal Health, Regional Campus of International Excellence Abstract "Campus Mare Nostrum", University of Despite public concern on the role of free-­roaming cats as reservoirs of zoonotic Murcia, Murcia, Spain 2 Department of Veterinary Medicine, agents, little is known about the influence of urban and peri-­urban landscapes on the University of Bari, Valenzano, Bari, Italy exposure risk. We evaluated the seroprevalence of three zoonotic agents (Chlamydia 3 Department of Biology & Centre for felis, Coxiella burnetii and Toxoplasma gondii) in domestic cats (Felis catus). Two hundred Environmental and Marine Studies (CESAM), University of Aveiro, Aveiro, and ninety-­one free-­roaming cats were trapped in Murcia municipality (Southeast Portugal Spain), and their sera were tested for specific antibodies against T. gondii using a 4 Wildlife Ecology & Health group (WE&H) modified agglutination test (MAT), and for C. felis, C. burnetii and feline immunode- and Servei d'Ecopatologia de Fauna Salvatge (SEFaS), Universitat Autònoma de ficiency virus (FIV) antibodies with ELISA technique. Pathogen seroprevalence at Barcelona, Barcelona, Spain 5 95% CI was calculated for each sex and age category (up to and over 12 months) and Department of Food Technology, University of Murcia, Murcia, Spain compared with a chi-­squared test. The role of human population density and urban landscape characteristics on the risk of pathogen exposure in the cat population was Correspondence Mónica G. Candela, Department of Animal explored using generalized linear models. Seropositivity against a single pathogen was Health. Regional Campus of International found in 60% of the cats, while 19% was seropositive for two or three pathogens. Excellence “Campus Mare Nostrum”, University of Murcia, Murcia, Spain. Seroprevalence of C. felis was 8% (CI95%: 5–­11), 37% (CI95%: 31–­42) for C. burnetii and Email: monica@um.es 42% (CI95%: 36–­47) for T. gondii. In addition to these three pathogens, FIV seropositiv- Funding information ity was low (1%, CI95%: −0.1 to 2) and adult cats were more likely to be seropositive to Spanish Ministerio de Ciencia, Innovación C. burnetii than young individuals (OR: 2.3, CI95%: 1.2–­4.2). No sex or age class differ- y Universidades (MICINN), Grant/Award Number: RYC-­2016-­21120; Fundação ences in seroprevalence were observed for the rest of the pathogens. Seropositivity para a Ciência e a Tecnologia (FCT), Grant/ was correlated with water surface areas for C. felis, and not with crop areas. Coxiella Award Number: CEECIND/01428/2018; CESAM, Grant/Award Number: UID/ burnetii seropositivity was correlated with the percentage of urban areas (continuous AMB/50017/2019; FEDER with only buildings and discontinuous, that include buildings, parks, and pedestrian and urban green areas), human population size and peri-­urban areas with shrubs, and not correlated with other agricultural landscapes (orchards and crop areas). However, the seroprevalence of T. gondii was only associated with agricultural landscapes such as orchards. The detection of hotspot areas of high pathogen exposure risk is the basis for municipal services to implement surveillance and risk factor control campaigns in specific-­risk areas, including (a) efficient health management of urban cat colo- nies by geographical location, population census and health status monitoring of the components of each cat colony, (b) improvement of hygiene and sanitary conditions Mónica G. Candela and Angela Fanelli contributed equally to this article This is an open access article under the terms of the Creative Commons Attribution-­NonCommercial-­NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-­commercial and no modifications or adaptations are made. © 2022 The Authors. Zoonoses and Public Health published by Wiley-­VCH GmbH. Zoonoses Public Health. 2022;69:295–311.  wileyonlinelibrary.com/journal/zph | 295 296 | CANDELA et al. at the feeding points of the cat colony and (c) free-­roaming cat trapping for health monitoring and, in the long term, to know the evolution of the health status of their populations. KEYWORDS Chlamydia felis, Coxiella burnetii, free-­roaming cat, geo-­epidemiology, landscape, urbanization, rural area, Toxoplasma gondii 1 | I NTRO D U C TI O N IMPACTS The domestic cat (Felis catus) is the most abundant carnivore in urban • Landscape features affect Chlamydia felis, Coxiella bur- and peri-­urban areas with high human density (Sims et al., 2008). netii and Toxoplasma gondii distribution. Feral cats include not only the offspring of domestic cats adapted • Cats in rural environments are exposed to C. felis, C. bur- to living on their own in rural and urban areas, but also un-­owned in- netii and T. gondii. dividuals that have left or lost their domestic home, surviving in the • Cats are more exposed to C. burnetii in urban than in urban environment as stray cats (Ogan & Jurek, 1997). Both types peri-­urban/rural environments. are referred to as free-­roaming cats because of their behaviour. • Free-­roaming cats are a sentinel to detect zoonotic Given that more than 75% of emerging human infectious diseases pathogens in anthropized areas. are of zoonotic origin (Jones et al., 2008), information on pathogens that may be transmitted to sympatric wildlife or humans is needed. In this context, free-­roaming cats are a major public health concern due to their large populations in contact with humans and their capac- controversy about its zoonotic potential, having been described as ity to harbour pathogens that produce disease in both humans and an etiological agent of different diseases in humans (Browning, 2004; animals (Taetzsch et al., 2018). The range of pathogens transmitted Halánová et al., 2011). In fact, pet cats seem to play an important depends primarily on the host communities, with the presence and role as sources of C. felis infection in humans (Trávnicek et al., 2002). frequency of free-­roaming pets having a significant effect on risk Although many surveys on C. felis have been carried out worldwide of disease transmission to humans (Polley, 2005). The prevalence (Sykes, 2005), limited information is available for Spain on the pres- of viruses, bacteria and parasites is usually higher in free-­roaming ence of this pathogen in cats (Millán & Rodríguez, 2009; Ravicini cats because outdoor access (Little, 2011) and roaming behaviour et al., 2016). On the contrary feline immunodeficiency virus (FIV) (Patronek, 1998) increase the risk of infection. Indeed, free-­roaming is a pathogen specific to cats that can create concomitant immu- cats have unrestricted entry to public areas such as parks and play- nosuppressive infections. This virus induces progressive lymphade- grounds where different potential hosts (e.g. rodents, birds, lizards nopathy and immunosuppression that usually ends in a fatal disease and snakes) are present (Montoya et al., 2018). Moreover, in many after several years of latency (Hofmann-­Lehmann et al., 1997). In cities in Europe and other continents, free-­roaming cat colonies fact, FIV has been associated with an increased risk of co-­infection are tolerated, deliberately fed and kept by citizens, resulting in ex- with a wide range of parasites, from viruses as feline leukaemia virus tremely high densities of cats in certain urban and peri-­urban areas (Bandecchi et al., 2006) to protozoa as T. gondii (Danner et al., 2007; (Crawford et al., 2019). Serrano & Millán, 2014). Among the zoonotic agents associated with cats that can infect Recent studies indicate that human-­induced landscape changes a wide variety of host species are Toxoplasma gondii, whose only de- are an important factor causing modifications in the transmission of finitive hosts are felids (Elmore et al., 2010), and Coxiella burnetii, the zoonoses (Bradley & Altizer, 2007; Mackenstedt et al., 2015). This is etiological agent of Q fever, of which an upsurge in urban cases in of particular importance considering that, in urban and peri-­urban both humans and animals, has been reported (Meredith et al., 2014; environments, a complex community of wild and domestic animals, Shapiro et al., 2015). In Spain, T. gondii is widespread in domestic acting as reservoirs of zoonotic pathogens, is present (Despommier and wild animals, with seroprevalence ranging between 37% and et al., 2006). Spatial distribution of diseases depends not only on 85% (Gauss et al., 2003; Millán et al., 2009; Miró et al., 2004), and land cover (e.g. vegetation cover, surface moisture, topography, soil C. burnetii has been diagnosed in animals and people in urban areas types) but also on land use (e.g. landscape uses influenced by the with varying seroprevalence (Espejo et al., 2014; van der Hoek political, social and economic context), since these environmental et al., 2010; Ruiz-­Seco et al., 2011). In addition to these pathogens, features determine the probability of contact between human and cats can also be infected with Chlamydia felis, an obligate intracel- animal hosts (Lambin et al., 2010). In spite of the large number of lular bacterium with a predilection for conjunctival epithelial cells, methods to investigate spatiotemporal patterns of infectious dis- the most common cause of feline chlamydiosis. Moreover, there is eases (Blasdell et al., 2016; Chowell & Rothemberg, 2018; Lewis CANDELA et al. | 297 et al., 2017; Ortega et al., 2020), these have rarely been applied to made up of two large tertiary basins occupied by areas of high study pathogen spread in free-­roaming cats from urban and peri-­ agricultural activity, separated by the Alpine massif of the Sierra urban areas. de Carrascoy, which is part of the Penibetic System of the Baetic In this work, we explore the role of urban landscape features Mountain Ranges (Estrella et al., 2018) and divides the municipal- and human population density on exposure risk to three zoonotic ity into Southern/Western (predominantly unirrigated landscape) pathogens (C. felis, C. burnetii and T. gondii) in cats from Murcia mu- and Northern (predominantly irrigated and urbanized landscape) nicipality (Southeast Spain). We assume that there are high densi- regions. The city of Murcia, settled in the basin to the north of ties of wild and synanthropic hosts species increasing the risk of the Sierra de Carrascoy, is drained by the Segura River, an area tick infestations and amplifying the sources of C. burnetii (Barbu that has been traditionally cultivated giving rise to the ‘Huerta et al., 2013; Meredith et al., 2014; Pfäffle et al., 2013; Rizzoli de Murcia’, one of the six orchard landscapes that exist in Europe et al., 2014), T. gondii (Aronson et al., 2017; Barros et al., 2018; (Martí-­C iriquián & Moreno-­V icente, 2014). In the southern basin, Hill & Dubey, 2002) and C. felis (Halánová et al., 2019). The high the traditional predominant use was unirrigated agriculture, but environmental stability of C. burnetii, which allows it to survive in in recent years, this has changed to intensive irrigated agriculture, soil for long periods (Eldin et al., 2017), the route of infection via especially in an extensive area known as Campo de Cartagena. inhalation (Angelakis & Raoult, 2010) and the possibility of wind The study area has a hot semi-­arid climate, with mild winters and currents spreading from manure accumulates (King et al., 2011; very hot summers, sometimes with absolute maximum tempera- Tissot-­D upont et al., 2004) influence the prevalence of infection tures above 45ºC. The average annual rainfall is less than 300 mm, in agricultural landscapes with small ruminant farms (De Lange showing irregular but intense rains, which can cause large floods et al., 2014; Eldin et al., 2017; van der Hoek et al., 2010). In the (Espín Sánchez et al., 2018). From a social point of view, the mu- same sense, humidity linked to green areas (agricultural and nicipality of Murcia has a population of 459,403 inhabitants urban parks) favours the survival of infective T. gondii oocysts (INE, 2020), unevenly distributed due to the fact that the city (Afonso et al., 2008; Shapiro et al., 2019; Yilmaz & Hopkins, 1972). of Murcia and its surroundings comprise more than 90% of the Chlamydia felis spreads through direct contact, and transmission study area, with a human population density of 525 inhabitants/ between cats is due to contact with infectious eye secretions km2 . Administratively, the study area is divided into 55 districts (Halánová et al., 2011). Moreover, free-­roaming cats usually show (called ‘pedanías’, Figure 2) formed from small population centres higher C. felis seroprevalence than cats kept strictly indoors (Helps scattered throughout the territory. The land covers and uses are et al., 2005), probably linked to malnutrition and a possibly weak- very heterogeneous, showing an unequal distribution throughout ened immune system, and increased exposure in overpopulated the study area. The municipality covers 88,473 hectares, of which urban cat colonies (Halánová et al., 2019). 10% is dedicated to anthropic uses (half of which is urban area), Our hypothesis is that the landscape influences the exposure with agricultural activity accounting for the largest part at 65%, risk to cat pathogens, especially in those urban and peri-­urban and natural surfaces cover the remaining 25% (Corine Land Cover areas where land cover and land use favour the persistence of in- (CLC), 2018). fective forms in the environment. Also, areas of high landscape biodiversity would influence free-­roaming cats' interaction (di- rect/indirect contact or predation) with infected and spreader 2.2 | Cat sampling hosts, either conspecifics or other animal species. Finally, urban landscapes are likely to be areas with higher cat pathogens sero- In the period 2005–­2007, 291 free-­roaming cats were trapped in prevalences due to the presence of urban colonies of this felid. the study area (Figure 2), as a part of an annual programme run by The objectives of this study were (a) to determine the seroprev- the Department of Public Health of Murcia. Animals were trapped alence of zoonotic pathogens (C. burnetii, T. gondii and C. felis) in during October, November and December in different urban and free-­roaming cats from Murcia municipality (SE Spain) and (b) to peri-­urban areas and euthanized for sanitary reasons (according to evaluate the host and environmental risk factors associated with European Union protocols regarding animal welfare and bioethics) exposure to these zoonotic pathogens. by official Veterinary Services at Centro Municipal de Control de Zoonosis de la ciudad de Murcia (CMCZ). Cats were grouped by sex, age and provenance (district where they were trapped). Sex was de- 2 | M ATE R I A L A N D M E TH O DS termined by visual inspection during the necropsy, and the age of the animals assigned by observing tooth replacement (The Humane 2.1 | Study area Society of United States, 2006). Two classes were considered for age: young (up to 12 months old) and adult (more than 12 months The study area covers the Municipality of Murcia, located in the old). The sample population included 128 males, 162 females, 69 Southeast of Spain (37º59′10″N, 1º7′49″W, Figure 1), which is young and 221 adult cats. The age and sex of one of the animals a complex geomorphologic territory. Broadly, the study area is could not be determined. 298 | CANDELA et al. F I G U R E 1 Geographic location of the municipality of Murcia. Demarcation and description of the landscape of the municipal districts CANDELA et al. | 299 F I G U R E 2 Number of cats sampled per district in the municipality of Murcia 2.3 | Serological analysis of samples to detect an inactivated mixture of C. burnetii phase I and II anFIVtigens (Nine Mile strain). Both tests were modified in house Immediately after death, a blood sample was taken from each cat, with a monoclonal goat anti-­c at IgM antibody HRP conjugate which was then allowed to coagulate at laboratory temperature (Bethyl A20-­100P©, Bionova Científica S.L. Madrid, Spain), ac- for 24 hr to obtain the serum samples. The serum obtained was cording to the recommendations of Candela et al. (2017). The kept in aliquots at −20ºC until analysis. Sera were tested for C. fe- samples were also tested for FIV antibody detection using lis-­ and C. burnetii-­s pecific antibodies using a commercially avail- INGEZIM FIV-­R .16.FIV.K.1© (Ingenasa, Inmunología y Genética able ELISA kit according to the manufacturer protocols (CHEKIT© Aplicada, S.A., Madrid, Spain), assuming that the presence of Chlamydia, Bonmelli Diagnost 2356789 ics. IDEXX Laboratories virus-­s pecific antibodies in serum is indicative of FIV infection B.V. Netherlands; CHEKIT© Q-­f ever C. burnetii Antibody Test Kit, (Fish & Altman, 1989). IDEXX Switzerland AG, Bern, Switzerland). The test showed a A modified agglutination test (MAT) was used to detect T. gondii sensitivity of 90.9% and specificity of 85.9% to detect Chlamydia antibodies using a commercial test kit; the antigen (Antigène ToxoAD, LPS antigen, and a sensitivity of 99% and specificity of 88.57% Catalogue No. 7-­542-­2, Lot No. 640558-­A) and control serum 300 | CANDELA et al. (Toxotrol A, Catalogue No. 7-­543-­1, Lot No. 613896-­A) were obtained between landscape characteristics using variance inflation factors from bioMerièux (Marcy l’Etoile, 69260-­Charbonnieres-­les-­Bains, (VIF) in the ‘HH’ package Version 3.1-­43 (Heiberger, 2018). Highly France). correlated variables were removed based on their multicollinearity (VIF > 5) (Kim, 2019) (Table 1). Information on human population size was used as an additional covariate. Details on the covariates 2.4 | Statistical analysis are provided in Table 1. Some districts were excluded because of the low sample size (e.g. less than one cat sampled). Pathogen seroprevalence (i.e. number of seropositive animals over Quantitative analysis of risk factors was performed using gener- the total sampled) was calculated for each sex and age class and com- alized linear models (GLM) with binomial family and logit link. These pared with a chi-­squared test (χ2 test). Differences were considered were fit using the function glm (R Core Team, 2018). Pathogen oc- significant at p-­values <.05. Odds ratio (OR) was used as a measure currence (0 = absence and 1 = presence) was considered as a re- of the strength of association between variables. Confidence inter- sponse variable and landscape characteristics at the district level as vals (CI) at 95% were also calculated using Epi InfoTM 7.0 version the explanatory variables (Table 1). (Dean et al., 2000). Model selection was done using a backward approach based on In regard to the spatial analysis, the landscape information cov- the Akaike information criterion (Akaike, 1973) (Table S2). The mod- ered land use and land cover characteristics potentially linked to els with the lowest AIC were used. Goodness of fit was evaluated zoonosis risk at the district level (Barros et al., 2018; McFarlane using diagnostic plots of the residuals. Overdispersion, however, was et al., 2013) and cat ranging behaviour (Hanmer et al., 2017; Metsers assessed using the ‘testOverdispersion’ function implemented in the et al., 2010). Land cover is related to landscape attributes such as DHARMa package (Harting, 2019). All statistical analyses were con- vegetation cover, surface moisture, topography and soil types, ducted in the R software 3.5.2 version (R Core Team, 2018). The final among others. Land uses are related to anthropogenic landscape model for each pathogen was used to build a risk map using QGIS uses influenced by the political, social and economic context (Lambin 3.6.0 version (QGIS Development Team, 2017). et al., 2010). All landscape variables used were representative of the study area, selected to respond to the hypotheses proposed on the risk of potential contact of free-­roaming cats with the pathogens 3 | R E S U LT S selected and, consequently, to achieve the objectives of our study. Some similar or spatially under-­represented landscape categories The overall seroprevalence was 8% (CI95%: 5–­11) for C. felis, 37% were grouped into a new spatial variable. We have used nine land- (CI95%: 31–­42) for C. burnetii and 42% (CI95%: 36–­47) for T. gon- scape categories, which group specific landscape variables and char- dii. There were only three FIV positive cats (P: 1%, CI 95%: −0.1 acteristics and are detailed in Table 1. The proportion of land cover to 2); they were all adults, specifically one female and two males. in each district was calculated intersecting a shapefile containing the Seroprevalence for all the different groups is presented in Table 2. urban districts in SIOSE (Sistema de Información sobre Ocupación del Statistically significant differences were observed between age and Suelo de España, downloaded from Instituto Geográfico Nacional, the seroprevalence of C. burnetii, with the adult cats at higher risk https://www.ign.es/web/ign/portal). We checked for collinearity (OR: 2.3, CI 95%: 1.2–­4.2, χ2 = 6.4, p-­value = .01). All the information TA B L E 1 Overall listing of variables considered in the study (* variable with VIF>5 and removed because of multicollinearity) Variable Description Source Human population size Number of resident human population at district level Instituto Nacional de Estadística (INE) https://www.ine.es/ % of crops area The category includes herbaceous crops different from rice SIOSE (Sistema de Información sobre % of unproductive area The category includes rocky outcrops, nude soil, ground Ocupación del Suelo de España) without construction downloaded from Instituto Geográfico Nacional (IGN) % of orchards and other farming The category includes citrus fruit plants, non-­citrus fruit plants, https://www.ign.es/web/ign/portal area olive grove, vineyard and other woody plants. % of pasture area* The category includes pasture % of urban area The category includes buildings (as continuous urban areas), and buildings mixed with parks, pedestrian and urban green areas (as discontinuous urban areas) % of water surface area The category includes water courses and artificial water surfaces % of wood area The category includes coniferous trees and leafy trees % of shrubs and small bush areas The category includes scrubs and small brushes CANDELA et al. | 301 related to odds ratio and chi-­squared values performed with sex and whereas for C. burnetii, it ranged from 0 to 0.70 (mean: 0.38) with age variables is detailed in Table S1. some districts at higher risk (probability: 0.68–­1, Figure 4). Contact Seropositivity against more than two pathogens was found in risk for C. burnetii in urban landscapes was higher than in peri-­urban 19% of the cats studied (56/291). Specifically, 16% (47/291) were areas (OR: 6.86). On the contrary, cats from peri-­urban areas are positive to two pathogens (47/291) and 3% (9/291) to three. Among more prone to contact with T. gondii (probability from 0.34 to 0.67) the cats that tested positive to three pathogens, two were positive than their counterparts living in more urbanized areas (0.21–­0.22, to FIV. Figure 5). The statistically significant factors selected in the final GLMs were (a) for C. felis, the percentage of water surface areas (OR: 1.74e−08, CI 95%: 1.3e−18–­0.065) with a p-­value equal to the cut-­off 4 | DISCUSSION alpha; (b) for C. burnetii, the percentage of shrub area (OR:4.15e−09, CI 95%:3.7e-­16–­0.01, p-­value: .01), the percentage of urban area This work aimed to explore the exposure of free-­roaming cats to (OR: 5.06, CI 95%:1.3–­20, p-­value: .02) and the human population three selected zoonotic pathogens by understanding the landscape size (OR:0.99, CI 95% 0.993–­0.99, p-­value: .02); and (c) for T. gondii, variables that influence pathogen contact rates with cats. This ap- the percentage of orchards (OR: 0.13, CI 95%: 0.02–­0.7, p-­value: .02) proach was based on the use of complementary research tools to in peri-­urban areas. determine risk factors associated with the environmental charac- Final models for each pathogen species are shown in Table 3. teristics of the study area. This provides more precise knowledge Neither residual patterns nor overdispersion was observed in our of the risk factors and, consequently, allows for more effective and GLM. All models with their AIC values and weights are available in localized implementation of control plans for these zoonoses in the Table S2. urban and peri-­urban environments. Here, we provide a comprehen- A map of infection risk for each pathogen at the district scale sive assessment of the effect of landscape on the exposure of cats is shown in Figures 3–­5. The probability of C. felis occurrence is to C. felis, C. burnetii and T. gondii in a highly urbanized landscape. low in all districts ranging from 0.09 to 0.18 (mean: 0.11; Figure 3), One of the limitations of our study has been that the sampled cats TA B L E 2 Seroprevalences (%) Sex Age and CI95% of Chlamydia felis, Coxiella burnetii, Toxoplasma gondii and feline Agent Male Female Young Adult immunodeficiency virus (FIV) according to C. felis 9 (4–­10) 9 (4–­12) 3 (0–­6) 10 (6–­14) host sex and age C. burnetii 34 (26–­42) 40 (30–­46) 23 (5–­33) 40 (34–­47) T. gondii 40 (30–­46) 45 (37–­52) 32 (12–­42) 45 (39–­51) FIV 1.5 (0–­3) 0.6 (0–­1) 0 1.3 (0–­2) TA B L E 3 GLM results for Chlamydia Predictors OR (CI95%) p-­value felis, Coxiella burnetii and Toxoplasma gondii Reduced GLM of C. felis Percentage of crop area 0.04 (0.0003–­1) .14 Percentage of water surface area 1.74e−08 (1.3e−18–­0.065) .05 AIC:69 quasi R2: .23 Reduced GLM of C. burnetii % of crop area 0.12 (0.006–­1) .15 % of orchard and other farming areas 6.86 (0.89–­56) .067 % of shrub and small bush areas 4.15e−09 (3.7e−16–­0.01) .01 % of urban area 5.06 (1.3–­20) .02 Human population size 0.99 (0.9999873–­0.99999885) .02 AIC:93 quasi R2: .45 Reduced GLM of T. gondii % of orchard and other farming areas 0.13 (0.02–­0.7) .02 AIC: 87 quasi R2: .16 302 | CANDELA et al. F I G U R E 3 Probability of occurrence and intervals of Chlamydia felis prevalence in the municipality of Murcia CANDELA et al. | 303 F I G U R E 4 Probability of occurrence and intervals of Coxiella burnetii prevalence in the municipality of Murcia 304 | CANDELA et al. F I G U R E 5 Probability of occurrence and intervals of Toxoplasma gondii prevalence in the municipality of Murcia CANDELA et al. | 305 could not be accurately geo-­referenced. Another limitation has been Although the transmission of C. burnetii by ticks is still debated, the number of samples per district, which in some cases was less the rise of tick populations in urban and peri-­urban areas is of par- than 5. Finally, although serological studies based on the detection ticular importance considering that this arthropod is able to amplify of antibodies should be interpreted with caution, since they do not sources of C. burnetii infection by vertical transmission to their prog- unequivocally indicate that the animal is a carrier of the pathogens eny (i.e. transtadially and transovarially) (Walker & Fishbein, 1991). investigated, they are useful to know, indirectly, the spatial distribu- In urban and peri-­urban habitats in Europe, rodents, hedgehogs, tion of pathogens, since contact with them generates a serological shrews, birds, lizards and pets (dogs and cats), as well as red foxes response in the host. (Vulpes vulpes), roe deer (Capreolus capreolus) and wild boar (Sus Several surveys have been carried out on domestic rumi- scrofa) in peri-­urban areas, play a major role as hosts and reservoirs nants, highlighting the relevant role of C. burnetii as a zoonotic of tick-­borne pathogens (Pfäffle et al., 2013). Some authors have also agent (Astobiza et al., 2012; García-­Pérez et al., 2009; Ruiz-­Fons claimed that ticks may play an important role in the maintenance of et al., 2010), although the risk of transmission from cats to hu- C. burnetii infection in rodents and lagomorphs (Marrie et al., 1993), mans has not been previously investigated in the Iberian Peninsula. and thus, a very complex network of transmission could be pres- Candela et al. (2017) reported C. burnetii infection in free-­living ent in an urban area. In fact, increasing urbanization may alter the European wildcats (Felis sylvestris sylvestris) in Central Spain, sug- biology and population density of ticks and hosts and, accordingly, gesting that this wild feline should be considered as part of the ep- could lead to an increased risk of vector-­borne disease emergence in idemiological cycle, just like domestic and feral cats. Other authors peri-­urban and urban areas (Barbu et al., 2013; Rizzoli et al., 2014). have reported high seroprevalence of C. burnetii in cats; specifically, It is interesting to note that the percentage of shrub and small Meredith et al. (2014) found a prevalence of 61.5% in rural cats in the bush areas in peri-­urban zones in the selected model shows a pro- United Kingdom, and Komiya et al. (2003) detected a prevalence of tective effect, which contradicts our initial hypothesis that hedge- 41.6% in stray cats in Japan. Our study revealed a high probability rows and shrubs, which harbour greater biodiversity and therefore of C. burnetii exposure (seroprevalence close to 40%) in adult free-­ more ticks, may favour exposure to C. burnetii. Interpreting this re- roaming cats from urban and peri-­urban areas. Together with the sult is difficult, but it is known that in semi-­arid landscapes (such above-­mentioned studies, our findings provide a valuable overview as Murcia), shrubs and small bushes are important determinants of of high seroprevalence of C. burnetii in cats and, consequently, the biodiversity (Chu et al., 2015; Giladi et al., 2007) and ecosystem en- potential zoonotic risk to humans should be of wide interest, con- richment (Eldridge et al., 2011). Thus, these landscapes favour the sidering the public health threat posed by this pathogen (Espejo presence of predators (medium-­sized birds of prey, lizards, snakes, et al., 2014; Murcia et al., 2002; Ruiz-­Seco et al., 2011). etc.) that reduce rodent populations in urban and peri-­urban areas The GLM analysis highlighted the link between urban and peri-­ (Aronson et al., 2017; Lepczyk et al., 2017). No correlation was de- urban landscapes and the risk of pathogen infection at the district tected between seroprevalence and unirrigated agricultural land- level. We detected a significant relationship between C. burnetii scapes, frequent in the south of Murcia, and with a greater presence exposure, not only with more urbanized and densely populated of sheep and goat farms. areas, but also with the more populated traditional orchard areas Cats play a central role in the epidemiology of T. gondii (Robert-­ surrounding the metropolitan area of Murcia. C. burnetti has a Gangneux & Dardé, 2012) because felids are the only definitive very wide host range that may serve to amplify hosts, favouring hosts that can excrete oocysts in the environment. Although cats transmission to humans in densely populated urban areas (Comer shed oocysts for a short period after primoinfection (Dubey, 2010), et al., 2001; Meredith et al., 2014). These hosts include domestic some individuals may have new episodes of oocyst shedding during animals (cats, dogs and cattle), commensal rodents (Rattus nor- their lifetime (Zulpo et al., 2018). In this study, the seroprevalence vegicus and Mus musculus), wild birds and lagomorphs. Important of T. gondii was the highest among the pathogens studied (42%). A landscape alterations have occurred in the metropolitan area of comparison to other studies in the Iberian Peninsula shows a similar Murcia over the centuries, resulting in alterations to ecotones and prevalence, within the range observed in the central (52%, Gauss the creation of new and multiple human–­domestic–­wild interfaces, et al., 2003) and north-­eastern part of the Iberian Peninsula (37%, which have shaped changes in density and distribution of wildlife Miró et al., 2004). In these same studies, a statistically significant species (Mata Olmo & Fernández Muñoz, 2004). Under this per- higher prevalence in adults was found. A similar difference was spective, free-­roaming cats have the opportunity to consume wild found in our study, although not statistically significant. However, prey, which can be reservoirs of C. burnetii (Meredith et al., 2014). these studies are difficult to compare due to different epidemio- Other factors that may increase contact between conspecifics, and logical situations of the cat populations. The prevalence of T. gondii thus, the risk of disease transmission in urban areas is the smaller may differ between specific areas of a country and even within a (Dards, 1978) and overlapping home ranges of free-­roaming cats, single city (Barros et al., 2018; Dubey, 2010). This is because the due to the greater abundance of trophic resources, either because prevalence depends on factors such as the presence of intermediate of prey abundance (Fitzgerald & Karl, 1979; Lieber, 1980) or the hosts (mainly birds and small mammals), the availability of infected supplementary food provided to cat colonies by local residents prey (Hill & Dubey, 2002; although some studies did not detect a (Natoli et al., 2006). relationship between prevalence in rodents and infected cats—­see 306 | CANDELA et al. De Feo et al., 2002) and oocyst accumulation in the soil (Shapiro municipality. The GLM identified the percentage of water surface et al., 2019). In urban areas, the survival of oocysts can be influ- as a protective factor. C. felis seems to be highly unstable outside enced by the increase in green spaces (Aronson et al., 2017; Yilmaz the host and remains infectious for a short time in the environment & Hopkins, 1972) and by the patterns of defecation of cats. Cat col- (Halánová et al., 2019), although other Chlamydia-­like organisms are onies often defecate repeatedly in specific areas called latrines, and heat-­resistant and able to survive in the environment and in artificial areas favourable for cat defecation (such as urban parks and gar- water networks (Coulon et al., 2012). Considering the dry landscape dens) have been shown to increase the presence of oocysts in the of Murcia and the surrounding neighbourhood, most water areas are soil (Afonso et al., 2008; Simon et al., 2019). human-­made water supplies to agriculture. The presence of these In our study, T. gondii seroprevalence does not appear to be areas implies a reduction in biological and ecological diversity. Thus, influenced by urbanization and human population density, nor by they might be interpreted as a proxy of low animal density as well as the increased presence of intermediate hosts associated with peri-­ a low richness of potential host for C. felis. urban environments (Aronson et al., 2017; Barros et al., 2018; Hill & In our study, the prevalence rate for FIV was low and in accor- Dubey, 2002). The risk of T. gondii is medium to high (0.34–­0.67) in dance with those reported in other areas of Spain (Arjona et al., 2000; almost the entire municipality of Murcia, and based on results from Ravicini et al., 2016). Of the three cats positive for FIV, two were also the GLM analysis, the presence of orchard and farming areas was a seropositive for other pathogens. About 20% of free-­roaming cats decreasing factor to infection in peri-­urban environments. Studies tested were found to have two pathogens, and 15% had three patho- indicate that rural cats are more likely to be infected than urban cats gens, suggesting that some areas have higher exposure to various (Oi et al., 2015), because in rural areas, which have a higher percent- pathogens than others, and that there are risk factors that favour age of permanent crop areas, cats may have larger home ranges and these rates of pathogen contact in cats (Candela et al., 2009). thus less focused deposition places. The seroprevalence found in the To ensure public health in urban and peri-­urban landscapes, peri-­urban territory studied does not correlate with non-­irrigated preventive measures need to focus on cat colony management, cropland landscapes, and decreases in our case, with the existence environmental enrichment, conservation of dryland bush land- of trees and permanent crops in irrigated areas. However, further scapes and health education for sheep and goat farmers. To re- studies should be carried out to elucidate these issues. duce pathogen exposure in free-­roaming cats and cat colonies in The presence of trees and permanent crops in irrigated peri-­ the Municipality of Murcia, we recommended (a) enhancing san- urban areas, and shrubs and bushes in dryland areas, are landscape itary surveillance measures in cat colonies (serological testing, features that reduce the exposure of free-­roaming cats to pathogens complying with the principles of hygiene, deworming, therapy and such as T. gondii and C. burnetii. Conversely, urbanization and popu- vaccination of animals) because contact with free-­roaming cats or lation density increase the contact with C. burnetii. Therefore, sero- cats living in colonies could be a C. burnetii risk factor for keep- prevalence in cats in a given area may serve as a sentinel not only ers and veterinarians (Komiya et al., 2003); (b) free-­roaming cat for the prevalence of T. gondii infection in the definitive host (Dubey population surveillance, taking into account that these animals in et al., 1995), but also for anthropogenic disturbance for the mainte- urban landscapes are more likely to be infected with C. burnetii nance of T. gondii infections (Barros et al., 2018), bacteria and oocyst than indoor pet cats and are considered an important route to environmental contamination and, consequently, for the health risk transmission to humans (Ma et al., 2020); (c) implementing pop- to the human population in this area. ulation control measures in cat colonies, and controlling cat feed To our knowledge, very few studies have been carried out on support and over-­d ensification through planned captures of cats C. felis in the Iberian Peninsula. Specifically, Millán and Rodriguez for sterilization (Natoli et al., 2006), because high population den- (2009) reported a seroprevalence of 27% in wild cats in central sity may be associated with the spread of C. felis and with disease Spain, and Ravicini et al. (2016) found that 2.6% of stray cats were severity (Gonsales et al., 2016; Halánová et al., 2019); (d) eliminat- positive to C. felis with PCR in Catalonia. Several studies have re- ing free-­roaming cat feeding points that do not provide adequate ported C. felis prevalence in feline populations in other countries; sanitary and hygiene conditions to prevent C. felis human disease in Slovakia, an overall positivity of 17.1% was found by PCR in cats (Wu et al., 2013); and (e) measuring soil contamination by T. gon- living in different environments (Halánová et al., 2011); in central dii oocyst estimation in green areas near cat colonies (Afonso China, a seroprevalence of 5.9% was found in domestic cats (Wu et al., 2008). et al., 2013), and a 20% seroprevalence was found in Iran (Maazi Trees and permanent crops in irrigated peri-­urban areas, and et al., 2016). In our study, C. felis seroprevalence was lower (8%) in shrubs and bushes in dryland areas, are landscape features that comparison with most of these studies. Differences in C. felis expo- reduce the exposure of feral cats to pathogens such as T. gondii sure could be due to different environmental and climatic factors. and C. burnetii. Conversely, urbanization and population density Specifically, Murcia is characterized by the typical Mediterranean increase the presence of C. burnetii. To reduce the risk of human semi-­arid subtropical climate, with hot summers and mild win- exposure to these pathogens by increasing biodiversity, the en- ters, which could reduce the extra-­host survival of the bacterium vironmental restoration of vegetation loss in urban areas and (Gruffydd-­Jones et al., 2009), a pattern that was well depicted by its conservation in peri-­urban environments is likely necessary the risk map, with a low probability of C. felis occurrence in the whole (Aronson et al., 2017; Barros et al., 2018; Lepczyk et al., 2017). On CANDELA et al. | 307 the contrary, cats suspected of having been exposed to C. burnetii was partially funded by the City Council of Murcia through the at a positive farm might shed infective forms during parturition, OTRI research contract awarded to the Department of Animal becoming a potential and mobile vector for spreading the infec- Health of the University of Murcia, ‘Epidemiological study of para- tion to other conspecifics, nearby farms and homes in the area sitic and infectious agents affecting stray cats in the municipality (Cyr et al., 2021). In addition, health education of small ruminant of Murcia. Zoonotic implications of the cat and its role as an envi- farmers should be provided, especially regarding sheep vaccina- ronmental biomarker'. The authors would like to thank the official tion, indoor lambing, appropriate disposal of placentas and litter Veterinary Services and trappers at Centro Municipal de Control to prevent the spread of the pathogen to other domestic and de Zoonosis from the Municipality of Murcia (CMCZ) for providing wild species that might consume these lambing remains. Wind us with euthanized animals according to European Union proto- spreading of manure infected with C. burnetii in unirrigated agri- cols for animal welfare and bioethics. We are also very grateful to cultural landscapes is a factor that can only be monitored and not Dr. Pedro Pérez Cutillas for his supervision, review and improve- prevented. Finally, tick prevention measures are recommended ment of the text describing the study area. Emmanuel Serrano (Egberink et al., 2013). is supported by the Spanish Ministerio de Ciencia, Innovación y Operational strategies to increase the effectiveness of zoonotic Universidades (MICINN) through a Ramon y Cajal agreement (RYC-­ pathogen detection and apply appropriate prevention and control 2016-­21120). João Carvalho was supported by a research con- measures should be focussed on those hotspots where pathogen tract (CEECIND/01428/2018) from the Fundação para a Ciência spread or risk factors have been detected (Ortega et al., 2020). The e a Tecnologia (FCT). Thanks for financial support are also due detection of seroprevalence in cats in a given area may serve as a to CESAM (UID/AMB/50017/2019), to FCT/MCTES through na- proxy not only for the prevalence of infection in the host (Dubey tional funds and co-­f unding by the FEDER, within the PT2020 et al., 1995), but also for anthropogenic disturbance for the main- Partnership Agreement and Compete 2020. Finally, we would also tenance of infections (Barros et al., 2018), bacteria and oocyst en- like to acknowledge the inspiring comments of two anonymous vironmental contamination, and consequently for the health risk to reviewers who helped to improve the understanding of the work humans in this area. Interdisciplinary collaboration of policy makers presented. and public health, veterinary and human health professionals is re- quired to improve our understanding on the dynamics of zoonoses C O N FL I C T O F I N T E R E S T in urban areas. The authors declare no conflict of interest. AU T H O R C O N T R I B U T I O N S 5 | CO N C LU S I O N Mónica G. Candela worked on the conceptualization, data cura- tion, formal analysis, investigation, methodology, supervision, Disease dynamics in urban and peri-­urban areas are complex, as validation, writing of the original draft, and reviewing and editing they involve the presence of varied hosts in a human–­domestic–­ of the manuscript. Angela Fanelli worked on the formal analysis, wild interface. Therefore, a One Health approach, bringing together validation and writing of the original draft. João Carvalho worked professionals from human medicine, veterinary medicine, biology, on the formal analysis, validation, and reviewing and editing of the geography and landscaping, is necessary to address the challenges manuscript. Emmanuel Serrano worked on the conceptualization, posed by the spread of zoonotic pathogens in areas with high human formal analysis, validation, writing of the original draft, and re- population density. Our study indicates that (a) free-­roaming cats are view and editing of the manuscript. Guillermo Domenech worked sentinels for detecting zoonotic pathogens in anthropized areas, (b) on data curation and investigation. Francisco Alonso worked on peri-­urban agricultural areas influence the contact of free-­roaming visualization, methodology, data curation and investigation. Carlos cats with C. felis, C. burnetii and T. gondii, and (c) urbanization is a Martínez-­C arrasco worked on the conceptualization, data curation, risk factor for C. burnetii infection in free-­roaming cats. These results methodology, supervision, validation, and reviewing and editing of should be the basis of further studies to deepen knowledge on the the manuscript. epidemiology of these pathogens. The findings of our study highlight the potential public health threat posed by cats in urban areas, if ANIMAL ETHICS proper preventive and control measures, as well as proper hygienic The authors declare no animal ethics conflict. Animal samples practices, are not applied. were from animals euthanized according to European Union pro- tocols regarding animal welfare and bioethics and provided by the AC K N OW L E D G E M E N T S Centro Municipal de Control de Zoonosis de la ciudad de Murcia Preliminary results were presented as an oral presentation at the (CMCZ). 37èmes Rencontres du GEEFSM. Etroubles, Vallee d'Aoste, Italia, 13-­16 Jun 2019. We thank the Department of Public Health of DATA AVA I L A B I L I T Y S TAT E M E N T Murcia and the official Veterinary Services at Centro Municipal de Data sharing is not applicable to this article as no new data were cre- Control de Zoonosis de la ciudad de Murcia (CMCZ). This research ated or analyzed in this study. 308 | CANDELA et al. ORCID of the aoudad (Ammotragus lervia). European Journal of Clinical Mónica G. 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