Parasitic infections in sea turtles and cetaceans in Mediterranean Sea waters, with a focus on the Adriatic Sea

Parasitic infections contribute to natural mortality in population of free ranging sea turtles and cetaceans worldwide. Notwithstanding all host-dependant factors influencing the outcomes of parasitic infections, some parasites, spanning from protozoan to helminthic taxa, have a high pathogenic potential and can cause fatal disease in these animals. Sea turtles blood flukes (Digenea: Spirorchiidae) are a main factor in mortality of populations of green and loggerhead turtles in Atlantic and Pacific Ocean. In order to explore presence and pathology of spirorchiidiasis in the Mediterranean area, data from 168 loggerhead turtles (Caretta caretta) stranded dead along North Western Adriatic coast were collected during a six-years period. Parasitological study and pathological findings revealed the presence of eggs and adult flukes of Hapalotrema mistroides and Neospirorchis – Neogen 11, with prevalence of 15.5% and 6.0% respectively. Mild lesions were observed, including multifocal granulomatous enteritis and chronic focal vasculitis of the major vessels. Egg emboli were found widespread in the tissues, mostly attributable to H. mistroides. Sequencing of rDNA markers (28S and ITS-2) and phylogenetic analyses revealed the identity of the Mediterranean isolates with Floridian specimens. Genetic analysis on the hosts led to the conclusion that the life cycles of both genera can be entirely supported by the Mediterranean ecosystem. To estimate the infection intensity, a new rapid method was set up. Correlation between spleen egg burden and fecal egg output was studied by means of statistical tests applied to splenic tissue and fecal material, revealing the absence of correlation and the unreliability of fecal burden to assess severity of the disease in vivo. Gastrointestinal helminth community of loggerheads was also studied, revealing a species diversity typical of demersal stage turtles and similar to that observed in other neritic areas of the Mediterranean. As for cetaceans, crassicaudosis is considered one of most severe helminthic infections in these animals. Nevertheless, knowledge on the epidemiology of Crassicauda species is scarce, due to constraints of sampling free-ranging wild host species and to the difficulty of obtaining entire specimens suitable for morphologic studies. Presence and pathology of Crassicauda boopis were investigated in fin whales (Balaenoptera physalus) stranded along Italian coasts. Severe lesions linked to the presence of adult nematodes in circulatory system and kidneys were detected in five rorquals (5/7), associated with renal function impairment and arteritis of the mesenteric vessels linked to spirurid larvae migrans. Morphological studies enabled the identification of the adult nematodes as C. boopis. Sequencing of molecular barcode regions was performed on adult and larval nematodes from the whales; additionally, specimens of Crassicauda spp. isolated from toothed whales were morphologically and molecularly studied. A multigene analysis made on the barcode regions of rDNA and on the mtDNA revealed the ITS-2 spacer to be the most efficient marker in species differentiation inside the genus Crassicauda. This analysis opened new insights on the identity of the isolated larvae migrans and on the life cycles of these poorly known nematodes. Furthermore, during the parasitological examination, immature elements of the genus Pennella were recovered from one fin whale and described from a morphological point of view for the first time. Preliminary molecular data were obtained, with the aim of clarifying the taxonomy of the genus Pennella. The coccidian Toxoplasma gondii was detected in the tissues of one rorqual as well, likely associated with a chronic infection. In conclusion, these parasitological surveys highlighted the importance of a continue monitoring of the health of these endangered species avoiding underestimating the potential impact of parasitic diseases in natural mortality

Factors influencing gastrointestinal parasites in a colony of Alpine ibex (Capra ibex) interacting with domestic ruminants

Parasitic infections in populations of wild herbivores can affect the individual fitness and population dynamics of their hosts. In this study, the ecology of gastrointestinal (GI) parasites was investigated in an Alpine ibex colony of the Marmolada massif, eastern Alps. Both individual and environmental factors were studied to reveal their relationship with parasite prevalence and burden. In a four-year period, 414 individual faecal samples of ibex were collected on a monthly basis during summer and autumn and were subjected to quali-quantitative examinations for the identification and quantification of oocysts and eggs. Domestic ungulates grazing in the same area were also sampled to investigate the risk of GI parasite transmission. Negative binomial regression models were developed to study the influence of physiological and environmental factors on parasite burdens. The results of the qualitative examination were in agreement with the few data already present in the literature for Alpine ibex, showing high prevalence values for Coccidia and GI strongyles, low values for Cestoda and sporadic presence of whipworms. Higher burdens in kids were found for Coccidia and Cestoda. Analysis of the GI endoparasite community of cattle and sheep suggested a negligible risk of parasite transmission to the Alpine ibex population. The sanitary risk represented by Coccidia and Cestoda in ibex kids and some peculiarities of the endoparasite distribution patterns in the Marmolada ibex population call for future in-depth ecological studies to investigate their influence on the limited growth rate shown by the ibex colony during the last decade

Cardiovascular flukes (Trematoda: Spirorchiidae) in Caretta caretta Linnaeus, 1758 from the Mediterranean Sea

Background: The northern Adriatic Sea represents one of the most important neritic foraging grounds for the loggerhead sea turtle Caretta caretta L. in the Mediterranean Sea. Four genera of blood flukes with variable prevalence and pathogenic impact have been reported worldwide in this species. Hapalotrema Looss, 1899 and Amphiorchis Price, 1934 are the only two genera reported in Mediterranean waters; however, updated data describing spirorchiidiasis in the central and eastern Mediterranean and infection prevalence are still lacking. This work aimed to investigate the presence and pathology of spirorchiidiasis in C. caretta in the Mediterranean Sea. Methods: One hundred sixty-eight animals stranded along the northwestern Adriatic coast between 2009 and 2015 were submitted to necropsy and subsequent analyses for the detection of adult flukes, detection of eggs in the faeces and spleen and histopathology. Molecular analyses were carried out on hosts (mitochondrial D-loop) and parasites (28S gene and ITS2 spacer) to trace the turtle origins and identify the fluke phylogenetic relationships. Results: Spirorchiidiasis was detected in 16.7% of the animals. Hapalotrema mistroides (Monticelli, 1899) and Neospirorchis sp. were found in twenty-six and ten cases, respectively. Adult flukes were found in six cases, while eggs were detectable through copromicroscopic examination for all infected turtles, and the results for the detection of eggs in the spleen agreed with the copromicroscopic analysis. Only mild lesions were observed. Eggs of types 1 and 3 were grossly visible in the gastrointestinal mucosa, vasculitis was rarely observed in the heart and great vessels, and multifocal granulomas were widespread in the tissues. Molecular identification unambiguously assigned the spirorchiid samples to H. mistroides and Neospirorchis sp. Genetic characterization of loggerhead mtDNA pointed to a Mediterranean origin of the turtle hosts. Conclusion: This survey provides new data on the spread of spirorchiidiasis in the Mediterranean loggerhead sea turtle population and reports for the first time the presence of Neospirorchis spp. in this basin. The infections did not have a causal effect on the death nor a strong impact on the general health status of the animals

Oceanic giants in the Mediterranean: first mitochondrial analysis of leatherback turtles (Dermochelys coriacea) in the Adriatic and Tyrrhenian seas

The leatherback turtle Dermochelys coriacea (Vandelli, 1761) is an occasional trophic visitor of the Mediterranean basin. So far, very few individuals have been genetically analysed from this sea and none from Italy. We sequenced a mitochondrial DNA fragment of three specimens of D. coriacea found in recent years along the North-Adriatic and Tyrrhenian shores. They were all females approaching adult stage. Causes of death were attributable to the main threats for sea turtles in Mediterranean waters, all related to human activities (collisions with boats, entanglement in fishing nets and plastic debris ingestion). Two different mitochondrial haplotypes were observed, with the two North-Adriatic turtles sharing the same one. Compared to known Dermochelys sequences and previous genetic characterization of rookeries, these results suggest that the most probable origin of at least two of the three leatherbacks was the western Atlantic

Molecular survey of Cytauxzoon spp. and Hepatozoon spp. in felids using a novel real-time PCR approach

Tick-transmitted apicomplexans of the genera Cytauxzoon and Hepatozoon affect a wide range of felids worldwide, but little is known about them. Recently, several studies addressed the species circulating in Europe, their distribution, and their hosts. Molecular assays are the method of choice for their detection. Unfortunately, conventional PCRs already described are time- and cost-consuming and specific for either Hepatozoon or Cytauxzoon detection. This study was developed to evaluate (i) the occurrence of Cytauxzoon and Hepatozoon in felids using a fast and cost-saving real-time PCR capable of detecting both protozoa simultaneously, (ii) the distribution of Cytauxzoon and Hepatozoon species in north-eastern Italy, and (iii) the involvement of other susceptible felid hosts in the same area. An SYBR® Green-based real-time PCR with primers targeting the 18S-rRNA was validated and applied to 237 felid samples, i.e., whole blood from 206 domestic cats and 12 captive exotic felids, and tissues from 19 wildcats. Positive results were obtained by melting temperature curve analysis due to the specific melting peak (i.e., 81°C Cytauxzoon spp.; 78–78.5°C Hepatozoon spp.). Positive samples were subjected to conventional PCR, followed by sequencing for species identification. Phylogenetic analyses were performed to assess relatedness among European isolates. Data on domestic cats (age class, sex, origin, management, and lifestyle) were recorded, and statistical analyses were performed to identify potential risk factors. A total of 31 (15%) domestic cats were positive for Hepatozoon spp. (i.e., 12 for H. felis, 19 for H. silvestris), while six (2.9%) for C. europaeus. The prevalence of Hepatozoon felis was significantly (p < 0.05) higher in domestic cats, while H. silvestris was higher in strays and animals from the Eastern region (i.e., Friuli- Venezia Giulia). Cytauxzoon europaeus was detected only in stray cats from Friuli-Venezia Giulia (province of Trieste). Among captive felids, one tiger was infected with H. felis and another with H. silvestris; eight out of 19 (42%) wildcats were positive for Hepatozoon spp. (i.e., six with H. felis, two with H. silvestris) and four out of 19 (21%) for Cytauxzoon europaeus. Outdoor lifestyle and origin (i.e., Friuli-Venezia Giulia region) were the most relevant risk factors for H. silvestris and C. europeus infections. Conversely, H. felis was most frequently isolated from domestic cats, suggesting different modes of transmission

An update and ecological perspective on certain sentinel helminth endoparasites within the Mediterranean Sea

The Mediterranean Sea is recognized as a marine biodiversity hotspot. This enclosed basin is facing several anthropogenic-driven threats, such as seawater warming, pollution, overfishing, bycatch, intense maritime transport and invasion by alien species. The present review focuses on the diversity and ecology of specific marine trophically transmitted helminth endoparasites (TTHs) of the Mediterranean ecosystems, aiming to elucidate their potential effectiveness as ‘sentinels’ of anthropogenic disturbances in the marine environment. The chosen TTHs comprise cestodes and nematodes sharing complex life cycles, involving organisms from coastal and marine mid/upper-trophic levels as definitive hosts. Anthropogenic disturbances directly impacting the free-living stages of the parasites and their host population demographies can significantly alter the distribution, infection levels and intraspecific genetic variability of these TTHs. Estimating these parameters in TTHs can provide valuable information to assess the stability of marine trophic food webs. Changes in the distribution of particular TTHs species can also serve as indicators of sea temperature variations in the Mediterranean Sea, as well as the bioaccumulation of pollutants. The contribution of the chosen TTHs to monitor anthropogenic-driven changes in the Mediterranean Sea, using their measurable attributes at both spatial and temporal scales, is proposed.publishedVersio

Parasitic infections in sea turtles and cetaceans in Mediterranean Sea waters, with a focus on the Adriatic Sea

Parasitic infections contribute to natural mortality in population of free ranging sea turtles and cetaceans worldwide. Notwithstanding all host-dependant factors influencing the outcomes of parasitic infections, some parasites, spanning from protozoan to helminthic taxa, have a high pathogenic potential and can cause fatal disease in these animals. Sea turtles blood flukes (Digenea: Spirorchiidae) are a main factor in mortality of populations of green and loggerhead turtles in Atlantic and Pacific Ocean. In order to explore presence and pathology of spirorchiidiasis in the Mediterranean area, data from 168 loggerhead turtles (Caretta caretta) stranded dead along North Western Adriatic coast were collected during a six-years period. Parasitological study and pathological findings revealed the presence of eggs and adult flukes of Hapalotrema mistroides and Neospirorchis – Neogen 11, with prevalence of 15.5% and 6.0% respectively. Mild lesions were observed, including multifocal granulomatous enteritis and chronic focal vasculitis of the major vessels. Egg emboli were found widespread in the tissues, mostly attributable to H. mistroides. Sequencing of rDNA markers (28S and ITS-2) and phylogenetic analyses revealed the identity of the Mediterranean isolates with Floridian specimens. Genetic analysis on the hosts led to the conclusion that the life cycles of both genera can be entirely supported by the Mediterranean ecosystem. To estimate the infection intensity, a new rapid method was set up. Correlation between spleen egg burden and fecal egg output was studied by means of statistical tests applied to splenic tissue and fecal material, revealing the absence of correlation and the unreliability of fecal burden to assess severity of the disease in vivo. Gastrointestinal helminth community of loggerheads was also studied, revealing a species diversity typical of demersal stage turtles and similar to that observed in other neritic areas of the Mediterranean. As for cetaceans, crassicaudosis is considered one of most severe helminthic infections in these animals. Nevertheless, knowledge on the epidemiology of Crassicauda species is scarce, due to constraints of sampling free-ranging wild host species and to the difficulty of obtaining entire specimens suitable for morphologic studies. Presence and pathology of Crassicauda boopis were investigated in fin whales (Balaenoptera physalus) stranded along Italian coasts. Severe lesions linked to the presence of adult nematodes in circulatory system and kidneys were detected in five rorquals (5/7), associated with renal function impairment and arteritis of the mesenteric vessels linked to spirurid larvae migrans. Morphological studies enabled the identification of the adult nematodes as C. boopis. Sequencing of molecular barcode regions was performed on adult and larval nematodes from the whales; additionally, specimens of Crassicauda spp. isolated from toothed whales were morphologically and molecularly studied. A multigene analysis made on the barcode regions of rDNA and on the mtDNA revealed the ITS-2 spacer to be the most efficient marker in species differentiation inside the genus Crassicauda. This analysis opened new insights on the identity of the isolated larvae migrans and on the life cycles of these poorly known nematodes. Furthermore, during the parasitological examination, immature elements of the genus Pennella were recovered from one fin whale and described from a morphological point of view for the first time. Preliminary molecular data were obtained, with the aim of clarifying the taxonomy of the genus Pennella. The coccidian Toxoplasma gondii was detected in the tissues of one rorqual as well, likely associated with a chronic infection. In conclusion, these parasitological surveys highlighted the importance of a continue monitoring of the health of these endangered species avoiding underestimating the potential impact of parasitic diseases in natural mortality.Le infestazioni parassitarie contribuiscono alla mortalità naturale in popolazioni di tartarughe marine e cetacei in tutto il mondo. A prescindere dai diversi fattori dipendenti dall’ospite che possono influenzare l’esito di tali infestazioni, alcuni parassiti, dai protozoi agli elminti, possiedono un alto potenziale patogeno, tale da essere causa primaria di morte. Infestazioni da Trematodi Digenei del sistema cardiocircolatorio (Digenea:Spirorchiidae) sono un’ importante causa di morbidità in popolazioni di tartarughe marine degli oceani Pacifico e Atlantico. Per indagare la presenza e la patologia di questa infestazione nel Mediterraneo, sono stati raccolti dati da 168 tartarughe Caretta caretta spiaggiate lungo la costa adriatica nord occidentale in un periodo di sei anni. L’approccio parassitologico, sia tradizionale che molecolare, e lo studio anatomopatologico hanno rivelato la presenza di uova e adulti di Hapalotrema mistroides e Neospirorchis – Neogen 11 con prevalenze di 15.5% e 6.0% rispettivamente. Sono state riscontrate lesioni lievi, tra cui enterite granulomatosa multifocale e vasculite cronica focale a carico dei grossi vasi. Il sequenziamento dei markers genomici (28S e ITS-2) e l’analisi filogenetica hanno permesso di verificare l’identità dei nostri campioni con quelli isolati in Florida. Analisi genetiche effettuate sugli ospiti hanno portato a concludere che il ciclo vitale di questi parassiti può completarsi all’interno del Mediterraneo. Un nuovo metodo rapido per la quantificazione dell’intensità di infestazione è stato quindi messo a punto. È stata studiata la correlazione tra l’emissione di uova nelle feci e la presenza di uova nei tessuti attraverso l’applicazione di test statistici, che hanno rivelato l’inattendibilità del valore della carica di uova fecali al fine di stimare la gravità dell’infestazione in vivo. Per quanto riguarda i cetacei, la crassicaudosi è una delle più gravi elmintiasi in questi animali ma ciononostante i dati sull’epidemiologia di Crassicauda spp. sono ancora scarsi, a causa delle limitazioni poste dal campionare queste specie ospite e alla difficoltà di ottenere campioni parassitologici integri. La presenza e la patologia della crassicaudosi sono state studiate in balenottere comuni (Balaenoptera physalus) spiaggiate lungo le coste italiane. Gravi lesioni collegate alla presenza di nematodi adulti nel sistema circolatorio e nei reni sono state trovate in cinque animali (5/7), in presenza di insufficienza renale in un caso e arterite dei vasi mesenterici associata alla presenza di larvae migrans di nematodi spiruridi. Gli studi morfologici, comparati con le descrizioni in letteratura, hanno permesso di identificare i parassiti adulti come Crassicauda boopis. Sono state sequenziate le regioni di barcoding sui parassiti adulti e sulle larve e, parallelamente, altri individui di Crassicauda spp., isolati da odontoceti, sono stati studiati morfologicamente e molecolarmente. Un’analisi multigenica sulle regioni barcode ha rivelato che il segmento ITS-2 si è rivelato il più efficiente nella distinzione di specie all’interno del genere Crassicauda. Questa analisi apre nuove questioni riguardo l’identità delle larve migrans e il ciclo vitale di questi nematodi. Durante l’esame parassitologico, elementi immaturi di Pennella spp. sono stati isolati da una balenottera e descritti morfologicamente per la prima volta. Dati molecolari preliminari sono stati ottenuti al fine di chiarire la tassonomia del genere Pennella. Toxoplasma gondii è stato isolato da un altro esemplare, associato a infezione cronica. I dati parassitologici raccolti hanno rivelato l’importanza di un continuo monitoraggio della salute di queste specie che tenga in considerazione il potenziale effetto delle parassitosi sulla mortalità naturale