Palms of the past: Can morphometric phytolith analysis inform deep time evolution and palaeoecology of Arecaceae?

BACKGROUND AND AIMS: Palm fossils are often used as evidence for warm and wet palaeoenvironments, reflecting the affinities of most modern palms. However, several extant palm lineages tolerate cool and/or arid climates, making a clear understanding of the taxonomic composition of ancient palm communities important for reliable palaeoenvironmental inference. However, taxonomically identifiable palm fossils are rare and often confined to specific facies. Although the resolution of taxonomic information they provide remains unclear, phytoliths (microscopic silica bodies) provide a possible solution because of their high preservation potential under conditions where other plant fossils are scarce. We thus evaluate the taxonomic and palaeoenvironmental utility of palm phytoliths. METHODS: We quantified phytolith morphology of 97 modern palm and other monocot species. Using this dataset, we tested the ability of five common discriminant methods to identify nine major palm clades. We then compiled a dataset of species' climate preferences and tested if they were correlated with phytolith morphology using a phylogenetic comparative approach. Finally, we reconstructed palm communities and palaeoenvironmental conditions at six fossil sites. KEY RESULTS: Best performing models correctly identified phytoliths to their clade of origin only 59% of the time. Although palms were generally distinguished from non-palms, few palm clades were highly distinct, and phytolith morphology was weakly correlated with species' environmental preferences. Reconstructions at all fossil sites suggested that palm communities were dominated by Trachycarpeae and Areceae, with warm, equable climates, and high, potentially seasonal rainfall. However, fossil site reconstructions had high uncertainty and often conflicted with other climate proxies. CONCLUSIONS: While phytolith morphology provides some distinction among palm clades, caution is warranted. Unlike prior spatially-restricted studies, our geographically and phylogenetically broad study indicates phytolith morphology may not reliably differentiate most palm taxa in deep time. Nevertheless, it reveals distinct clades, including some likely to be palaeoenvironmentally informative.

The impact of public transport on the health of work commuters: a systematic review.

Although the public transport (PT) commute can form a substantial part of the working day, there is a significant gap in our understanding of how it influences health of those who engage in it. The purpose of this systematic review was to therefore generate evidence from 1972 about the extent to which the PT commute (involving train, bus, subway, tram, or metro) impacts on the mental health, physical health and well-being of the working people. We identified 47 studies in English worldwide involving an empirical quantitative focus which met the inclusion criteria. Of these, 23 studies involved over 500 participants. Although initial multi-modal comparisons showed impact on sickness rate, self-rated health complaints, perceived stress level and reduction in sleep, a more homogeneous analysis of rail commuters showed elevation in salivary cortisol, perceived stress, and affective reactions to crowding. Findings also revealed a bias towards use of endogenous self-report measures. On this basis, we argue that it would be of benefit to test theoretical models to account for more objective measures of job and commuting stress. Recommendations were made for flexible working agendas.

Endemic caseous lymphadenitis in a wild Caprinae population.

Between 2010 and 2013, an outbreak of caseous lymphadenitis (CLA) occurred in a captive stock of Iberian ibexes (Capra pyrenaica, Schinz 1838) maintained for conservation purposes in the National Game Reserve 'Ports de Tortosa i Beseit' (PTB), northeastern Spain. The aim of this study was to assess the CLA status in the free-ranging donor population of ibexes. An ELISA test to detect antibodies to CLA was performed in 360 hunter-harvested ibexes between 2007 and 2013. A spatial analysis and recursive partitioning approaches with regression tree models were used to explore CLA-associated risk factors. Nineteen per cent of ibexes had antibodies to CLA. Significant differences in seroprevalence were observed among game management areas but no clear trends of CLA occurrence were observed over the study period. Ibexes from areas of higher population density and living close to aggregation points displayed a higher probability of testing positive to CLA. These results suggest that CLA is endemic in the Iberian ibex population inhabiting PTB and density-dependent risk factors. To the best of our knowledge, this is the first record of CLA maintenance in a free-ranging wild Caprinae population.

Serological Follow-up of Tuberculosis in a Wild Boar Population in Contact with Infected Cattle.

There is an increasing concern in several European countries over the role that tuberculosis (TB)-infected wild boar may play in the progress of bovine TB eradication campaigns. In 2004, as a consequence of the detection of a TB focus in wild boar from a National Game Reserve (NGR) located in southern Catalonia, a surveillance programme based on post-mortem inspection for detection of macroscopic TB-like lesions (TBLL) was initiated in the affected area. The source of infection for wild boar was linked to a tuberculous cattle herd located in the same area. Besides, the results of the surveillance programme in wild boar were used for the validation of an indirect enzyme-linked immunosorbent assay (ELISA) specific for Mycobacterium tuberculosis complex (MTBC) IgG antibodies. Using this ELISA, a seven-year serological study of MTBC in wild boar from the NGR was conducted in 173 animals (93 adults, 44 juveniles-yearlings and 36 piglets) culled between 2004 and 2010. ELISA results and presence of TBLL showed excellent agreement for adult and juvenile wild boar (Kappa index = 0.85; 95% CI: 0.76-0.95). Of the thirty-eight adults, yearlings and juveniles classified as positives by the ELISA, 34 (89%) showed TBLL at necropsy. In contrast, none of the ELISA-positive wild boar piglets (n = 20) showed TBLL, suggesting the detection of early antibody responses to the infection. Overall, this study contributes to the knowledge of wild boar humoral responses to MTBC. The results also highlight the usefulness of this serological test for wild boar TB surveillance.

Post-Natal Persistent Infection With Classical Swine Fever Virus in Wild Boar: A Strategy for Viral Maintenance?

In this study, fifteen wild boar piglets were intranasally inoculated <10 h after birth with the moderately virulent classical swine fever virus (CSFV) strain Catalonia 01. At 5 days post-inoculation, seven other animals within 48 h of birth were put in contact with them. Viral replication and innate and specific immune responses were evaluated. Of the inoculated animals, 46.67% remained post-natally persistently infected and were apparently healthy with neither humoral nor cellular immunological responses specific to CSFV and with high viral loads in their blood, organs and body secretions. Moreover, the present data extend the time period to 48 h after birth when a moderately virulent CSFV strain could lead to post-natal persistent infection given the generation of persistently infected wild boars in the contact group (33.33%). The innate immune response to the virus, as measured by type I IFN-α in serum, was mostly not impaired in the persistently infected wild boars. Interestingly, a decrease and lack of IFN-γ-producing cells against CSFV and PHA was observed. In endemic countries where wild swine species are increasing and low and moderate virulence CSFV strains are prevalent, the possible generation of this form of disease cannot be ruled out.

Spillover Events of Infection of Brown Hares (Lepus europaeus) with Rabbit Haemorrhagic Disease Type 2 Virus (RHDV2) Caused Sporadic Cases of an European Brown Hare Syndrome-Like Disease in Italy and Spain.

Rabbit haemorrhagic disease virus (RHDV) is a lagovirus that can cause fatal hepatitis (rabbit haemorrhagic disease, RHD) with mortality of 80-90% in farmed and wild rabbits. Since 1986, RHDV has caused outbreaks in rabbits (Oryctolagus cuniculus) in Europe, but never in European brown hares (Lepus europaeus, EBH). In 2010, a new RHDV-related virus, called RHDV2, emerged in Europe, causing extended epidemics because it largely overcame the immunity to RHDV present in most rabbit populations. RHDV2 also was identified in Cape hare (Lepus capensis subsp. mediterraneus) and in Italian hare (Lepus corsicanus). Here, we describe two distinct incidents of RHDV2 infection in EBH that occurred in Italy (2012) and Spain (2014). The two RHDV2 strains caused macroscopic and microscopic lesions similar to European brown hare syndrome (EBHS) in hares, and they were genetically related to other RHDV2 strains in Europe. EBHs are common in Europe, often sharing habitat with rabbits. They likely have been exposed to high levels of RHDV2 during outbreaks in rabbits in recent years, yet only two incidents of RHDV2 in EBHs have been found in Italy and Spain, suggesting that EBHs are not a primary host. Instead, they may act as spillover hosts in situations when infection pressure is high and barriers between rabbits and hares are limited, resulting in occasional infections causing EBHS-like lesions. The serological survey of stocked hare sera taken from Italian and Spanish hare populations provided an understanding of naturally occurring RHDV2 infection in the field confirming its sporadic occurrence in EBH. Our findings increase the knowledge on distribution, host range and epidemiology of RHDV2.

Perfluorinated alkyl substances (PFASs) in northern Spain municipal solid waste landfill leachates.

Landfill leachates have been recognized as significant secondary sources of poly- and perfluoroalkyl substances (PFASs). This study presents data on the occurrence and concentration of 11 perfluoroalkyl carboxylates (PFCAs) and 5 perfluoroalkyl sulfonates (PFSAs) in leachates from 4 municipal solid waste landfill sites located across northern Spain. To the best of our knowledge, this is the first report of the presence of PFASs in Spanish landfill leachates. Two of the landfill sites applied on-site treatment using membrane bioreactors (MBR), and its effect on PFASs occurrence is also reported. Total PFASs (∑PFASs) in raw leachates reached 1378.9 ng/L, while in treated samples ∑PFASs was approximately two-fold (3162.3 ng/L). PFCAs accounted for the majority of the detected PFASs and perfluorooctanoic acid (PFOA) was the dominant compound in raw leachates (42.6%), followed by shorter chain PFHxA (30.1%), PFPeA and PFBA. The age of the sites might explain the PFASs pattern found in raw leachates as all of them were stabilized leachates. However, PFASs profile was different in treated samples where the most abundant compound was PFHxA (26.5%), followed by linear perfluorobutane sulfonate (L-PFBS) (18.7%) and PFOA (17.7%). The overall increase of the PFASs content as well as the change in the PFASs profile after the MBR treatment, could be explained by the possible degradation of PFASs precursors such as fluorotelomer alcohols or fluorotelomer sulfonates. Using the volume of leachates generated in the landfill sites, that served 1.8 million people, the discharge of 16 ∑PFASs contained in the landfill leachates was estimated as 1209 g/year.

The European hare (Lepus europaeus) as a potential wild reservoir for ruminant pestiviruses.

Ruminant pestiviruses cause important economic losses in livestock and the epidemiological role of free-ranging sympatric wildlife is of special interest for the implementation of pestivirus eradication plans. Moreover, the emergence of high mortality outbreaks of pestivirus in Pyrenean chamois (Rupicapra pyrenaica pyrenaica) since 2001 in the border between Spain and France has increased the value of knowing the hosts that role pestivirus infection. In the present study, pestivirus infection was assessed in 94 sera from wild hunted European hares (Lepus europaeus) collected in two different areas: Pyrenees (alpine and subalpine ecosystems) versus Non Pyrenees (non alpine and subalpine ecosystems). The presence of antibodies against Border Disease Virus (BDV) and Bovine Viral Diarrhea Virus (BVDV) was evaluated by means of the Virus Neutralization Test and the presence of viral RNA in sera samples was assessed by Reverse transcription-polymerase chain reaction (RT-PCR). A total of 34 out of 94 (36.2%; CI95 0.26-0.46) sera presented neutralizing antibodies against ruminant pestiviruses, and significant differences between BDV4 and BVDV1 titres were found in 7 hares. In the Pyrenean area not statistically significant seroprevalence was observed when comparing with the Non Pyrenean area. RT-PCR analysis of sera samples resulted all negative. The results of the present study indicate that the European hare is susceptible to pestivirus infection and that could be involved in the epidemiology of ruminant pestiviruses. To the authors' knowledge, this is the third wild non-artiodactyl with reported antibodies against ruminant pestivirus after the rabbit and Bennet's wallaby.

Pestivirus in alpine wild ruminants and sympatric livestock from the Cantabrian Mountains, Spain.

Bovine viral diarrhoea virus (BVDV) and Border disease virus (BDV) were investigated at the wildlife-livestock interface in the distribution area of chamois in the Cantabrian Mountains, North-Western Spain. From 2010 to 2014, sera from sympatric wild (n=167) and domestic (n=272) ruminants were analysed for pestivirus antibodies by cELISA, virus neutralisation test (VNT) and for the presence of pestiviral RNA using a reverse transcription (RT)-PCR. Results showed a higher seroprevalence in cattle (59.4 per cent, 13/13 of herds) than in domestic small ruminants (5.9 per cent sheep, 2/8 of flocks; 0 per cent goats of 4 flocks) and wildlife (10.8 per cent in red deer, 0 per cent in roe deer and 0 per cent in Cantabrian chamois). High VNT titres were detected in two cattle herds, suggesting the circulation of BVDV-1 and BVDV-2 strains. BVDV-1 RNA was detected in one cattle calf by RT-PCR and sequencing. Conversely to other similar grazing systems, sheep flocks did not play a relevant role in the pestivirus epidemiology in this region. Pestivirus infections in wild ruminants were sporadic and most probably dependent on a domestic source.

Streptococcus caprae sp. nov., isolated from Iberian ibex (Capra pyrenaica hispanica).

Biochemical and molecular genetic studies were performed on a novel Gram-stain-positive, catalase-negative, coccus-shaped organism isolated from tonsil samples of two Iberian ibexes. The micro-organism was identified as a streptococcal species based on its cellular, morphological and biochemical characteristics. 16S rRNA gene sequence comparison studies confirmed its identification as a member of the genus Streptococcus, but the organism did not correspond to any species of this genus. The nearest phylogenetic relative of the unknown coccus from ibex was Streptococcus porci 2923-03T (96.6 % 16S rRNA gene sequence similarity). Analysis based on rpoB and sodA gene sequences revealed sequence similarity values lower than 86.0 and 83.8 %, respectively, from the type strains of recognized Streptococcus species. The novel bacterial isolate was distinguished from Streptococcus porci and other Streptococcus species using biochemical tests. Based on both phenotypic and phylogenetic findings, it is proposed that the unknown bacterium be classified as representing a novel species of the genus Streptococcus, for which the name Streptococcus caprae sp. nov. is proposed. The type strain is DICM07-02790-1CT ( = CECT 8872T = CCUG 67170T).

Nutrient and plant secondary compound composition and iron-binding capacity in leaves and green stems of commonly used plant browse (Carolina willow; Salix caroliniana) fed to zoo-managed browsing herbivores.

Plant secondary compounds are diverse structurally, and associated biological effects can vary depending on multiple factors including chemical structure and reaction conditions. Phenolic compounds such as tannins can chelate dietary iron, and supplementation of animal species sensitive to iron overload with tannins may prevent/treat iron overload disorder. We assessed the nutrient and phenolic composition and iron-binding capacity of Carolina willow (Salix caroliniana), a plant fed to zoo-managed browsing herbivores. Based on studies in other plant species and the chemical structures of phenolic compounds, we hypothesized that the concentration of condensed tannins in willow would be inversely related to the concentration of phenolic glycosides and directly related to iron-binding capacity. Our results indicated that willow nutrient composition varied by year, season, and plant part, which could be taken into consideration when formulating animal diets. We also found that the predominant plant secondary compounds were condensed tannins with minimal phenolic glycosides. Instead of binding to iron, the willow leaf extracts reduced iron from the ferric to ferrous form, which may have prooxidative effects and increase the bioavailability of iron depending on animal species, gastrointestinal conditions, and whole animal processes. We recommend identifying alternative compounds that effectively chelate iron in vitro and conducting chelation therapy trials in vivo to assess potential effects on iron balance and overall animal health.

Campylobacter shared between free-ranging cattle and sympatric wild ungulates in a natural environment (NE Spain).

Campylobacter infections are a public health concern and an increasingly common cause of food-borne zoonoses in the European Union. However, little is known about their spill-over from free-ranging livestock to sympatric wild ungulates, especially in regards to uncommon Campylobacter species. In this study, we aim to determine the prevalence of C. coli, C. jejuni and other C. spp. in game ungulates (wild boar Sus scrofa and Iberian ibex Capra pyrenaica) and free-ranging sympatric cattle in a National Game Reserve in NE Spain. Furthermore, we explore the extent to which Campylobacter species are shared among these co-habiting hosts. Faecal samples from Iberian ibex (n = 181) were negative for C. spp. By direct plating, two wild boars out of 150 were positive for C. coli (1.3%, 95% CI 0.16-4.73), and one was positive for C. jejuni (0.67%, 95% CI 0.02-3.66). The latter was predominant in cattle: 5.45% (n = 55, 95% CI 1.14-5.12), while C. coli was not isolated from this host. C. lanienae was the most frequent species in wild boar at 10% (95% CI 5.7-15.96), and one cow cohabiting with positive wild boars in the same canyon also carried C. lanienae. Four enrichment protocols (using Bolton or Preston broth combined with either mCCDA or CFA) were added for 172 samples (57 from wild boars, 55 cattle and 60 Iberian ibexes) to increase the number of isolates obtained allowing the detection of statistically significant differences. The prevalence of C. lanienae was statistically significantly higher in wild boar than in cattle (P < 0.01), but the prevalence of C. jejuni was higher in the latter (P = 0.045). These results suggest that wild boar and cattle carry their own predominant Campylobacter species, while Iberian ibex do not seem to play an important role in the epidemiology of Campylobacter. However, there is a potential spill-over of C. spp., and thus, further research is needed to elucidate the factors determining inter-species transmission.

Implementing a low-starch biscuit-free diet in zoo gorillas: the impact on behavior.

In the wild, western lowland gorillas travel long distances while foraging and consume a diet high in fiber and low in caloric density. In contrast, gorillas in zoos typically consume a diet that is low in fiber and calorically dense. Some items commonly used in captive gorilla diets contain high levels of starch and sugars, which are present at low levels in the natural diet of gorillas. Diet items high in simple carbohydrates are associated with obesity and heart disease in humans. Typical captive gorilla diets may also encourage undesirable behaviors. In response to these issues, we tested the behavioral impact of a diet that was biscuit-free, had low caloric density, and which was higher in volume at five institutions. We hypothesized that this diet change would reduce abnormal behaviors such as regurgitation and reingestion (R/R), decrease time spent inactive, and increase time spent feeding. The biscuit-free diet significantly reduced (and in the case of one zoo eliminated) R/R and may have reduced hair-plucking behavior. However, an increase in coprophagy was observed in many individuals following the diet change. The experimental diet caused a general increase in time the gorillas spent feeding, but this increase did not occur across all institutions and varied by individual. Interestingly, the overall time gorillas spent inactive actually increased with this diet change. Future research will examine these behavioral changes in a greater number of individuals to determine if the results remain consistent with these preliminary findings. Additionally, future research will examine the physiological impact of this diet change.

Implementing a low-starch biscuit-free diet in zoo gorillas: the impact on health.

In the wild, western lowland gorillas consume a diet high in fiber and low in caloric density. In contrast, many gorillas in zoos consume a diet that is high-calorie and low in fiber. Some items commonly used in captive gorilla diets contain high levels of starch and sugars, which are minimal in the natural diet of gorillas. There is a growing concern that captive gorillas may qualify as obese. Furthermore, the leading cause of death for adult male gorillas in zoos is heart disease. In humans, a diet that is high in simple carbohydrates is associated with both obesity and the incidence of heart disease. In response to these issues, we implemented a biscuit-free diet (free of biscuits and low in fruit) and measured serum biomarkers of obesity and insulin resistance pre- and post-diet change at three institutions: North Carolina Zoological Garden, Cleveland Metroparks Zoo, and Columbus Zoo and Aquarium. We also added a resistant starch supplement to gorilla diets at two of the above institutions. We anticipated that these diet changes would positively affect biomarkers of obesity and insulin resistance. Both diet manipulations led to a reduction in insulin. Resistant starch also decreased overall serum cholesterol levels. Future research will examine these health changes in a greater number of individuals to determine if the results remain consistent with these preliminary findings.

Lack of evidence of spill-over of Salmonella enterica between cattle and sympatric Iberian ibex (Capra pyrenaica) from a protected area in Catalonia, NE Spain.

Salmonella enterica is a zoonotic agent of worldwide importance found in a wide range of wild hosts. However, its prevalence in many popular game species has never been assessed. Iberian ibex (Capra pyrenaica) is the main game caprinae of the Iberian Peninsula and around two thousand individuals are hunted every year for trophy or for home consumption. In this work, 313 Iberian ibexes from the Ports de Tortosa i Beseit National Game Reserve (NE Spain) were tested for Salmonella enterica in faeces, and anti microbial susceptibility was determined. The exact location of shooting or capture was recorded with a GPS device to study the links of Salmonella infection with cattle presence and human proximity. Additionally, samples were taken from cattle grazing inside this reserve (n = 73). Only three Iberian ibexes (0.96%, 95% CI 0.2-2.8) were positive to Salmonella (serotype Enteritidis, Bardo and 35:r:z35), while prevalence was moderate in cattle: 21.92% (95% CI 13.10-33.14, serotype Meleagridis, Anatum, Kedougou and Othmarschen). All isolates were susceptible to the anti microbial agents tested. Moreover, a case of fatal septicaemic salmonellosis in an 11-year-old male Iberian ibex is described where Salmonella enterica serotype Enteritidis was isolated from the lung, liver and spleen samples. The low prevalence of Salmonella in Iberian ibex and the lack of shared serotypes suggest no association to cattle. Despite this, game meat aimed for human consumption should be examined, and it is strongly recommended that hunters and game keepers manipulate animals and carcasses under maximal hygienic conditions to avoid environmental contamination and human contagion.

Antimicrobial resistance in indicator Escherichia coli isolates from free-ranging livestock and sympatric wild ungulates in a natural environment (Northeastern Spain).

Antimicrobial resistance was assessed in indicator Escherichia coli isolates from free-ranging livestock and sympatric wild boar (Sus scrofa) and Iberian ibex (Capra pyrenaica) in a National Game Reserve in northeastern Spain. The frequency of antimicrobial resistance was low (0% to 7.9%). However, resistance to an extended-spectrum cephalosporin and fluoroquinolones was detected.

Cattle drive Salmonella infection in the wildlife-livestock interface.

The genus Salmonella is found throughout the world and is a potential pathogen for most vertebrates. It is also the most common cause of food-borne illness in humans, and wildlife is an emerging source of food-borne disease in humans due to the consumption of game meat. Wild boar is one of the most abundant European game species and these wild swine are known to be carriers of zoonotic and food-borne pathogens such as Salmonella. Isolation of the pathogen, serotyping and molecular biology are necessary for elucidating epidemiological connections in multi-host populations. Although disease management at population level can be addressed using a number of different strategies, such management is difficult in free-living wildlife populations due to the lack of experience with the wildlife-livestock interface. Herein, we provide the results of a 4-year Salmonella survey in sympatric populations of wild boar and cattle in the Ports de Tortosa i Beseit National Game Reserve (NE Spain). We also evaluated the effects of two management strategies, cattle removal and increased wild boar harvesting (i.e. by hunting and trapping), on the prevalence of the Salmonella serovar community. The serovars Meleagridis and Anatum were found to be shared by cattle and wild boar, a finding that was confirmed by 100% DNA similarity patterns using pulse field gel electrophoresis. Cattle removal was more efficient than the culling of wild boar as a means of reducing the prevalence of shared serotypes, which underlines the role of cattle as a reservoir of Salmonella for wild boar. To our knowledge, this is the first attempt to manage Salmonella in the wild, and the results have implications for management.

Evaluation of pulmonary function in European land tortoises using whole-body plethysmography.

The aim of this study was to evaluate the use of whole-body plethysmography as a non-invasive method to determine the respiratory parameters and profiles in two tortoise species belonging to the genus Testudo. Pulmonary functions and volumetric parameters were determined in 10 adults of Testudo hermanni and in seven Testudo marginata animals, using whole-body plethysmography. A profile pattern was regularly observed: an inspiratory flow peak, an expiratory peak, an apnoea phase and a second expiratory peak, previous to the beginning of the next respiratory cycle. Positive and significant correlation was observed between the inspiratory time, weight and length of the tortoises. Larger tortoises showed a higher time of inhalation. The peak of inspiratory flow was correlated with the sex, being longer in the females. T. marginata had an inspiratory time longer than that of T. hermanii. In T. hermanii, differences related to the sex were observed in the tidal volume, peak inspiratory flow, peak expiratory flow, expiratory flow of 50 per cent and enhanced pause, which could be related to the smaller size of males. The results suggest that additional information on new technologies currently used in pet medicine or even in human medicine should be developed and adjusted as alternative ways to support the rehabilitation of turtles and tortoises.

Presence of Toxoplasma gondii and Neospora caninum DNA in the brain of wild birds.

Toxoplasma gondii infections are prevalent in many avian species and can cause mortality in some bird hosts. Although T. gondii has been isolated from various species of birds, the role of many different species of wild birds in the epidemiology of T. gondii remains unknown. Neospora caninum, a closely related parasite to T. gondii, has been recently confirmed to infect domestic chickens and wild birds such as house sparrows (Passer domesticus). The present study reports the presence of T. gondii and N. caninum DNA by PCR in brain tissues of 14 species of wild birds from Spain. From a total of 200 samples analyzed, 12 samples (6%) were positive for T. gondii [5 Eurasian jays (Garrulus glandarius), 5 magpies (Pica pica), 1 black kite (Milvus migrans) and 1 Griffon vulture (Gyps fulvus)], while 3 samples (1.5%) were positive for N. caninum [2 magpies and 1 common buzzard (Buteo buteo)]. This is the first report of detection of T. gondii in magpies, griffon vulture and black kite and of N. caninum in common buzzard and magpies, extending the list of natural intermediate hosts for T. gondii and N. caninum infections to these species.