Detection of Leishmania sp. kDNA in questing Ixodes ricinus (Acari, Ixodidae) from the Emilia-Romagna Region in northeastern Italy

To date, sand flies (Phlebotominae) are the only recognized biological vectors of Leishmania infantum, the causative agent of human visceral leishmaniasis, which is endemic in the Mediterranean basin and also widespread in Central and South America, the Middle East, and Central Asia. Dogs are the main domestic reservoir of zoonotic visceral leishmaniasis, and the role of secondary vectors such as ticks and fleas and particularly Rhipicephalus sanguineus (the brown dog tick) in transmitting L. infantum has been investigated. In the present paper, the presence of Leishmania DNA was investigated in questing Ixodes ricinus ticks collected from 4 rural areas included in three parks of the Emilia-Romagna Region (north-eastern Italy), where active foci of human visceral leishmaniasis have been identified. The analyses were performed on 236 DNA extracts from 7 females, 6 males, 72 nymph pools, and 151 larvae pools. Four samples (1.7%) (i.e., one larva pool, 2 nymph pools, and one adult male) tested positive for Leishmania kDNA. To the best of our knowledge, this is the first report of the presence of Leishmania kDNA in questing I. ricinus ticks collected from a rural environment. This finding in unfed larvae, nymphs, and adult male ticks supports the hypothesis that L. infantum can have both transstadial and transovarial passage in I. ricinus ticks. The potential role of I. ricinus ticks in the sylvatic cycle of leishmaniasis should be further investigated

A survey on zoonotic helminths in farmed fish to evaluate the safety of Italian aquaculture products

Introduction: Several zoonotic helminths can be transmitted to humans by consumption of raw, undercooked, marinated or cold smoked fish products, representing a relevant public health concern. Although up to now in Italy the presence of zoonotic helminths has been documented only in wild fish populations, extensive epidemiological surveys are needed in order to assess the possible zoonotic risks linked to consumption of national aquaculture products. At this purpose, a wide parasitological survey has being undertaken on rainbow trout Oncorhynchus mykiss (RBT), gilthead seabream Sparus aurata (GSB) and European seabass Dicentrarchus labrax (ESB) farmed in Italy. Methodology: From spring 2016 to spring 2017 a total of 2347 fish have been examined. In particular, 738 RBT from 4 Italian freshwater trout farms (two farms located in the area endemic for diphyllobothriasis in Northwestern Italy, 1 in the area endemic for opisthorchiasis in Central Italy and 1 in a highly productive area of Northeastern Italy) were subjected to methods aimed to search for larval stages of diphyllobothriid cestodes (visual inspection and candling) and opisthorchiid digeneans (muscular compression/artificial digestion followed by microscopic examination). Furthermore, 781 GSB and 828 ESB from 4 marine farms (3 cage systems and 1 inland farm located in Tyrrhenian and Adriatic seas) were examined for anisakid larvae by UV-press method and artificial digestion. A seasonal periodicity has been applied, sampling 65 specimens/fish species/farm/season in order to reach a statistically significant amount of fish at the end of the survey (258 fish/farm). Results: No zoonotic parasites have been found in all the examined fish at the level of confidence of 99% with a margin of error of 5%. Conclusions: The results so far obtained during this survey are encouraging and seem to confirm that the risks linked to zoonotic helminths in Italian aquacultured fish species are null or negligible, when good farming practices are applied along the production chain

Metacestodes of elasmobranch tapeworms in octopus vulgaris (Mollusca, cephalopoda) from central mediterranean—SEM and molecular data

Cephalopods are intermediate/paratenic hosts in the life cycle of elasmobranch tapeworms, nevertheless most records of infection in this group of mollusks are outdated and fragmentary. The present work aimed to investigate the cestode fauna of the common octopus Octopus vulgaris from the Tyrrhenian Sea (Central Mediterranean). The parasitic stages were characterized by light and Scanning Electron Microscopy (SEM) and sequencing of 28S rDNA. Three cestode taxa were identified to the genus level: the onchoproteocephalidean Acanthobothrium sp. (prevalence 28%), the “tetraphyllidean” Anthobothrium sp. (prevalence 13%) and the trypanorhynch Nybelinia sp. (prevalence 3%). The remarkable prevalence observed for gastrointestinal cestodes highlight a possible important role of O. vulgaris in the transmission of elasmobranch tapeworms, particularly Onchoproteocephalideans. Furthermore, the present work provides, for the first time, detailed morphological (SEM) and molecular support to confirm the occurrence of Anthobothrium sp. in cephalopod hosts. In order to gain higher taxonomic resolution for the identified taxa, we stress the need to collect further morphological and molecular data of adult cestodes infecting their elasmobranch definitive hosts

Morphological and molecular characterization of two gastrointestinal parasites in Mustelus mustelus (Linnaeus, 1758) from Adriatic Sea

Specimens of common smooth-hound, Mustelus mustelus, fished as bycatch in the Northwestern Adriatic Sea, were subjected to a complete parasitological examination. Parasites were processed for morphological and morphometric analyses aimed at identifying them to the species level. Parasite identity was confirmed through amplification and sequencing of 28S rDNA. The nematode Acanthocheilus rotundatus was recovered from the stomach and the proximal intestine, while the cestode Calliobothrium verticillatum was collected from the spiral valve. Our results confirm the occurrence of C. verticillatum in M. mustelus from the Mediterranean Sea and provide the first sequence data of A. rotundatus from the same area. Updated knowledge on the parasite fauna of M. mustelus from Adriatic Sea contributes to elucidating aspects of the ecology, biology and health status of elasmobranchs from the Mediterranean Sea, providing useful information for their proper management and conservation

Parasitic infection in the scyphozoan Rhizostoma pulmo (Macri, 1778)

: Very little information is reported for parasites of cnidarians, therefore, the present work aimed to investigate parasitic infections in one of the most widespread jellyfish in the Mediterranean Sea, Rhizostoma pulmo. The goals were to determine prevalence and intensity of parasites in R. pulmo, identify the species involved through morphological and molecular analysis, test whether infection parameters differ in different body parts and in relation to jellyfish size. 58 individuals were collected, 100% of them infected with digenean metacercariae. Intensity varied between 18.7 ± 6.7 per individual in 0-2 cm diameter jellyfish up to 505 ± 50.6 in 14 cm ones. Morphological and molecular analyses suggest that the metacercariae belonged to the family Lepocreadiidae and could be possibly assigned to the genus Clavogalea. Prevalence values of 100% suggest that R. pulmo is an important intermediate host in the life cycle of lepocreadiids in the region. Our findings also support the hypothesis that R. pulmo is an important part in the diet of teleost fish, which are reported as definitive hosts of lepocreadiids, since trophic transmission is necessary for these parasites to complete their life cycles. Parasitological data may therefore be useful to investigate fish-jellyfish predation, integrating traditional methods such as gut contents analysis

An ISH technique for the early detection of Enterospora nucleophila, an intranuclear microsporidian causing emaciative disease in gilthead sea bream

Enterospora nucleophila is a microsporidian parasite causing serious emaciative disease in cultured gilthead sea bream (GSB), Sparus aurata, and closely related to Enterocytozoon hepatopenaei from shrimp. The parasite can be found within the nuclei of enterocytes and rodlet cells of the fish intestinal epithelium, and occasionally in the cytoplasm of phagocytes in more advanced infections. However, spores are the only stage that can be unmistakably identified but even this may require thorough histopathological examination and use of chitin fluorescent stains for a proper confirmatory diagnosis, due to their minute size and intranuclear location. In the absence of spores, the infection can be suspected from a remarkable hypercelullarity and the presence of altered nuclei in the epithelial layer. This results in a very poor correlation of disease signs with diagnosis of E. nucleophila infection, and to current uncertainty about its real impact in GSB culture. This work describes the development and application of an in situ hybridization (ISH) technique as a powerful tool to overcome current diagnostic limitations for this species, and to decipher basic data on the infection and disease onset. We designed DIG-labelled oligonucleotide probes targeting unique regions of the (+) strand of E. nucleophila rDNA gene, and we developed an ISH protocol that results in good staining of infected host cells prior to the development of spores or other conspicuous stages. On clinically infected samples, numerous ISH-positive cells are present which are not stained with fluorescent whiteners that bind to parasite spores. In contrast, poor staining of spores was obtained with the DNA probes, due to their limited penetration in these stages and the low number of target gene copies that they harbour. These aspects might be improved using specific steps (e.g., chitinase or more aggressive permeabilization strategies) but they are not necessary in a clinical diagnostic context. Due to the relevance of E. nucleophila infections it is essential to decipher unknown aspects of the parasite\u2019s biology and course of infection using proper diagnostic tools. Their cryptic nature makes this a challenging task for microsporidians like E. nucleophila, which can benefit from the method hereby presented

Viral nervous necrosis outbreaks caused by the RGNNV/SJNNV reassortant betanodavirus in gilthead sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax)

Mediterranean marine aquaculture has suffered significant economic losses due to viral nervous necrosis (VNN) outbreaks mainly caused by different RGNNV betanodavirus strains. In recent years, the marine aquaculture sector has experienced the emergence of the RGNNV/SJNNV reassortant betanodavirus, harbouring the RNA1 segment of RGNNV genotype and the RNA2 segment of SJNNV genotype. So far, the reassortant strains caused massive mortality outbreaks in gilthead sea bream (Sparus aurata) larvae sparing the European sea bass (Dicentrarchus labrax). In this study, multiple mortality outbreaks occurred in one Italian marine hatchery involving both European sea bass and gilthead sea bream at different life stages were investigated through a complete microbiological and molecular analysis. Gilthead sea bream larvae and juveniles have recorded the highest mortality rates, however, both European sea bass and gilthead sea bream incurred a RGNNV/SJNNV reassortant betanodavirus persistent infection, able to act as asymptomatic carriers and viral source for susceptible fish. These new epidemiological data on nervous necrosis virus (NNV) reassortant infection provide precious advice on how to manage fish to reduce VNN spread in Mediterranean aquaculture. Evidence of interspecies transmission of RGNNV/SJNNV reassortant strains and the persistent infection in both European sea bass and gilthead sea bream, point out the importance to enforce a wide surveillance and a strict biosecurity programme addressing both RGNNV and reassortant RGNNV/SJNNV betanodaviruses in Mediterranean European sea bass and gilthead sea bream farms. Furthermore, the presence assessment of betanoviruses in all newly-introduced fish batches in the farm, regardless of the species and a strict segregation between European sea bass and gilthead sea bream batches within farms can significantly reduce the risk of NNV transmission. Finally, surviving fish can act as carrier fish, and thereby must be segregated from other batches and protected from stress conditions that could trigger a new clinical phase

Roe deer (Capreolus capreolus) are a novel potential reservoir for human visceral leishmaniasis in the Emilia-Romagna region of northeastern Italy

Leishmaniasis is a complex human disease caused by intracellular parasites of the genus Leishmania, predominantly transmitted by the bite of sand flies. In Italy, leishmaniasis is caused exclusively by Leishmania infantum, responsible for the human and canine visceral leishmaniases (HVL and CVL, respectively). Within the Emilia-Romagna region, two different foci are active in the municipalities of Pianoro and Valsamoggia (both in the province of Bologna). Recent molecular studies indicated that L. infantum strains circulating in dogs and humans are different, suggesting that there is an animal reservoir other than dogs for human visceral leishmaniasis in the Emilia-Romagna region. In this work, we analyzed specimens from wild animals collected during hunts or surveillance of regional parks near active foci of human visceral leishmaniasis for L. infantum infection in the province of Bologna. Out of 70 individuals analyzed, 17 (24%) were positive for L. infantum. The infection prevalence in hedgehogs (Erinaceus europaeus), roe deer (Capreolus capreolus), badgers (Meles meles), and bank voles (Myodes glareolus) was 80, 33, 25, and 11%, respectively. To distinguish the two strains of L. infantum we have developed a nested PCR protocol optimized for animal tissues. Our results demonstrated that most (over 90%) of L. infantum infections in roe deer were due to the strain circulating in humans in the Emilia-Romagna region

INDAGINE PARASSITOLOGICA SULLA PRESENZA DI STADI LARVALI DI NEMATODI ANISAKIDAE IN PESCI MARINI ALLEVATI IN ITALIA

L\u2019anisakiasi \ue8 una zoonosi parassitaria di origine alimentare sostenuta da larve al terzo stadio (L3) di nematodi appartenenti alla famiglia Anisakidae ed in particolare al genere Anisakis. Soprattutto in seguito alla recente emergenza di sindromi allergiche da Anisakis, l\u2019EFSA nel 2010 ha pubblicato un parere scientifico sui rischi correlati alla presenza di parassiti zoonotici in prodotti ittici, considerando sicura la pratica d\u2019allevamento del salmone atlantico (Salmo salar) in quanto, se allevati partendo da avannotti prodotti in sistemi chiusi e alimentati sempre con mangime inerte, questi animali presenterebbero un rischio di infestazione da larve di Anisakis trascurabile. Su questa base, il Reg. UE n. 1276/2011, che modifica l\u2019allegato III del Reg. CE n. 853\2004, puntualizza la possibile deroga al trattamento di congelamento, obbligatorio per i prodotti della pesca destinati ad essere consumati sotto forma di preparazioni a rischio (crudi, marinati, affumicati a freddo, ecc.), per i prodotti ittici derivanti da allevamento quando vengono rispettati i criteri definiti dall\u2019EFSA analogamente al salmone atlantico. Appare quindi d\u2019importanza strategica fornire dati scientifici estesi ed attendibili sull\u2019assenza di parassiti zoonotici nei prodotti della maricoltura mediterranea non solo ai fini di una loro migliore qualificazione commerciale ma anche per una pi\uf9 facile applicabilit\ue0 di tale deroga da parte delle autorit\ue0 competenti. Nell\u2019ambito delle attivit\ue0 di ricerca previste dal progetto europeo H2020 ParaFishControl \u201cAdvanced Tools and Research Strategies for Parasite Control in European farmed fish\u201d, da marzo 2016 a giugno 2017 \ue8 stato condotto un esteso monitoraggio parassitologico in spigole (Dicentrarchus labrax) ed orate (Sparus aurata) allevate in Italia. Le attivit\ue0 di campionamento hanno coinvolto 4 allevamenti nazionali, di cui 3 in gabbie galleggianti ed uno in vasche a terra, prelevando almeno 258 esemplari per specie/allevamento suddivisi in 4 campionamenti a cadenza stagionale per un totale di 1040 spigole e 1090 orate. La numerosit\ue0 campionaria \ue8 stata calcolata in modo da ottenere almeno il 95% di probabilit\ue0 di evidenziare un esemplare positivo con prevalenza >2%. Per la ricerca di larve di nematodi Anisakidae sono stati associati: esame visivo diretto e mediante transilluminatore di visceri e porzioni muscolari, metodo della UV-press dei filetti e digestione con soluzione cloro- peptica del pacchetto viscerale. Tutti gli esemplari di spigola e orata esaminati sono risultati negativi per la presenza di stadi larvali di Anisakis spp. In un unico esemplare di spigola proveniente da un allevamento in gabbia \ue8 stata evidenziata la presenza di una larva di Hysterothylacium fabri (nematode non zoonotico) incistata sulla superficie del fegato ed identificata a livello di specie mediante PCR-RFLP. Alla luce della trasmissione di questo parassita a pesci predatori per via trofica, similmente ai nematodi Anisakidae zoonotici, nel prossimo futuro verranno condotte indagini mirate ad identificare i fattori di rischio che hanno condotto all\u2019infestazione da H. fabri in allevamento, seppur in un unico esemplare, ed individuare gli eventuali punti critici da monitorare. I risultati ottenuti da questa indagine indicano comunque come nelle spigole e nelle orate allevate in Italia il rischio d\u2019infestazione da Anisakis sia da ritenersi trascurabile, aprendo la via ad azioni di qualificazione dei prodotti della maricoltura mediterranea

Anisakid and Raphidascaridid parasites in Trachurus trachurus: infection drivers and possible effects on the host’s condition

This study investigated the distribution of nematode larvae of Anisakidae and Raphidascarididae (genera Anisakis and Hysterothylacium) in Trachurus trachurus (Linnaeus, 1758) in the Ligurian and central-northern Tyrrhenian Seas. The relationship between the number of parasites and the length and weight parameters of the fish was assessed, and the possible effect of the parasites on the condition factor was evaluated. A total of 190 T. trachurus specimens were collected in July 2019. Parasites were found in 70 individuals. A total of 161 visible larvae were collected in the viscera. Morphological analysis revealed the presence of Anisakis spp. in 55 fish and Hysterothylacium spp. in 15 fish, while 5 fish showed coinfection with both genera. The specimens subjected to PCR (n = 67) showed that 85% of the Anisakis larvae analyzed belonged to the species A. pegreffii, while the remaining 15% belonged to hybrids of A. pegreffii-A. simplex (s.s.). A total of 58% (n = 7) of the Hysterothylacium larvae analyzed belonged to the species H. fabri, while 42% belonged to the species H. aduncum. Our results support the hypothesis that infection with these parasites does not affect the condition of the fish host analyzed, and that body size and depth are major drivers in determining infection levels with Anisakid and Raphidascaridid nematodes