Characterisation of the intracellular protozoan MPX in Scottish mussels, Mytilus edulis Linnaeus, 1758.

Ciliates have been reported as pathogens of many species of economically important bivalves. Mussel protozoan X (MPX), is an uncharacterised intracellular ciliate of mussels and has been widely reported in Mytilus spp. around the world. In order to characterise this ciliate, Mytilus edulis samples were collected from a site on the West coast of Scotland, and four different fixatives for histological examination were tested. Fresh preparations of mussel digestive glands were also examined by laser scanning confocal microscopy. Intracellular ciliates were prepared by laser capture microdissection and partial sequences of small subunit ribosomal RNA gene and of large subunit ribosomal RNA gene were generated, using Phyllopharyngea primers. Methacarn solution proved to be the best fixative for both histological and molecular characterisation. The morphological and molecular investigations confirmed that this ciliate belongs to the class Phyllopharyngea, order Rhynchodida. However, this organism does not belong to any known family, genus or species, therefore, a new description is necessary, following further morphological analyses. Most mussel samples containing MPX displayed mild to moderate infections, with no signs of necrosis or haemocytic response, although a single sample displayed a severe infection (∼103 ciliates per section). The localisation of this ciliate in tissues other than the digestive gland, the presence of necrosis in infected tissue of the most severely infected mussel and the binary fission of this ciliate have been observed here for the first time. We also report the first observation of the live ciliate isolated from tissue. Although MPX remains of unknown significance to the mussel industry, tools and protocols described here will be useful in further characterising these and other ciliates (subclass Rhynchodia) known as pathogens for bivalves.

Bacterial and viral pathogens detected in sea turtles stranded along the coast of Tuscany, Italy.

During 2014, six loggerhead turtles, Caretta caretta and one green turtle, Chelonia mydas, found stranded on the Tuscany coast of Italy, were examined for the presence of specific bacterial and viral agents, along with their role as carriers of fish and human pathogens. Thirteen different species of bacteria, 10 Gram negative and 3 Gram positive, were identified. Among them, two strains of Vibrio parahaemolyticus and one strain of Lactococcus garviae were recovered and confirmed by specific PCR protocols. No trh and tdh genes were detected in V. parahaemolyticus. The first isolation of L. garviae and the first detection of Betanodavirus in sea turtles indicate the possibility for sea turtles to act as carriers of fish pathogens. Furthermore, the isolation of two strains of V. parahaemolyticus highlights the possible role of these animals in human pathogens' diffusion.

Skin lesion-associated pathogens from Octopus vulgaris: first detection of Photobacterium swingsii, Lactococcus garvieae and betanodavirus.

The common octopus Octopus vulgaris Cuvier, 1798 is extremely important in fisheries and is a useful protein source in most Mediterranean countries. Here we investigated pathogens associated with skin lesions in 9 naturally deceased specimens that included both cultured and wild common octopus. Within 30 min after death, each octopus was stored at 4°C and microbiologically examined within 24 h. Bacterial colonies, cultured from swabs taken from the lesions, were examined using taxonomical and biochemical analyses. Vibrio alginolyticus and V. parahaemolyticus were only isolated from cultured animals. A conventional PCR targeting the 16S ribosomal RNA (rRNA) gene and sequencing were performed on 2 bacterial isolates that remained unidentified after taxonomical and biochemical analysis. The sequence results indicated that the bacteria had a 99% identity with Lactococcus garvieae and Photobacterium swingsii. L. garvieae was confirmed using a specific PCR based on the 16S-23S rRNA internal transcribed spacer region, while P. swingsii was confirmed by phylogenetic analyses. Although all animals examined were found to be infected by the protozoan species Aggregata octopiana localised in the intestines, it was also present in skin lesions of 2 of the animals. Betanodavirus was detected in both cultured and wild individuals by cell culture, PCR and electron microscopy. These findings are the first report of L. garvieae and betanodavirus from skin lesions of common octopus and the first identification of P. swingsii both in octopus skin lesions and in marine invertebrates in Italy.

Trichinellosis outbreak caused by meat from a wild boar hunted in an Italian region considered to be at negligible risk for Trichinella.

The wild boar is an important source of trichinellosis for people in European countries as a large number of hunted animals escape veterinary control. In November 2012, uncooked sausages made with meat from wild boar were consumed by 38 persons in a village of the Lucca province (Tuscany region, Italy). Of them, 34 were serologically positive, 32 developed clinical signs and symptoms of trichinellosis, and two were asymptomatic. Trichinella britovi larvae were detected in vacuum-packed sausages made with the same batch of sausages consumed raw which had been prepared with meat from wild boar hunted in the Lucca province. As no case of trichinellosis had been reported in this region during the last 20 years, the regional public health authority considered the risk for this zoonosis to be negligible and put in place a surveillance programme on Trichinella spp. in indicator animals (mainly foxes and including wild boar for private consumption), by testing only a percentage of heads. The experience from this outbreak shows that the definition of a region with a negligible risk for Trichinella infection is not applicable to wild boar and stresses the need to test all Trichinella-susceptible wild animals intended for human consumption and to implement risk communication to consumers and hunters.

Postmonorchis sp. inq. (Digenea: Monorchiidae) metacercariae infecting natural beds of wedge clam Donax trunculus in Italy.

The wedge clam Donax trunculus Linnaeus, 1758 is one of the most common bivalve molluscs inhabiting the sandy shores of the Mediterranean Sea and is considered an important commercial resource. In this study, we report the first molecular, morphological and histopathological descriptions of metacercariae from a trematode belonging to the genus Postmonorchis (Digenea: Monorchiidae) that infects D. trunculus in natural beds of the Italian Tyrrhenian coast (Campania, Lazio and Tuscany). Morphological analysis of the parasite revealed a combination of features that exist in the 3 previously identified species of Postmonorchis, viz. P. donacis, P. variabilis and P. orthopristis, with the addition of new, distinctive morphological characteristics. The pathogen exhibited a predilection for the gill; however, it was also present in the labial palp and mantle in addition to the gut, kidney epithelium and foot. The inflammatory response was characterised by either a focal or diffuse haemocyte infiltration followed by the formation of multiple, large multi-layered capsules associated with tissue destruction. The prevalence of the pathogen ranged from 75 to 100%, while the infection intensity fluctuated among the study areas. Further studies regarding the life cycle of this parasite and the identification of other larval and adult stages and their respective hosts may confirm the identification of a new species of Postmonorchis that infects wedge clams in Mediterranean waters. The study of the parasite is completed by molecular analysis of the ITS1 and ITS2 rDNA sequences.

Detection of Cyprinid herpesvirus 2 in association with an Aeromonas sobria infection of Carassius carassius (L.), in Italy.

Sixteen specimens of female crucian carp, Carassius carassius (L.), during the breeding season, were investigated for post-mortem and full diagnostic examination during a mortality outbreak in a tributary stream of the Arno River in Tuscany in 2011. Necropsy highlighted the presence of a swollen anus and widespread haemorrhages in the body, fins, gills and eyes. Haemorrhages in internal organs and spleen granulomas were also observed. Bacteria isolated from the brain, kidney and spleen of affected fish were identified as A. sobria. Microscopic lesions observed in gills were characterized by necrosis of the secondary lamellae, congestion and multifocal lamellar fusion. The kidney showed necrosis, oedema, fibrin exudation and areas of haemorrhages, while in the spleen the main lesions were by multifocal necrosis of the lymphoid tissue. In the gills, transmission electron microscopy revealed herpesvirus-like particles, subsequently identified as Cyprinid herpesvirus-2 (CyHV-2) with a nested PCR protocol. Although it was not possible to attribute a pathogenic role to CyHV-2 in this mortality event, the identification of this herpesvirus in crucian carp increases the concern about its potential role in this species.

Coccidiosis of wild and captive European mouflons (Ovis aries) living in a natural reserve of central Italy.

Between December 2005 and November 2006, a survey on coccidiosis of wild and captive mouflons living in the Orecchiella Natural Reserve (Tuscany, Italy) was performed on faecal samples collected approximately every two months. A total of 96 samples were collected, 55 of which from wild mouflons and 41 from captive mouflons. On these faecal samples qualitative and quantitative parasitological analyses were performed; moreover, faecal cultures were made in order to identify the involved Eimeria species. Significantly higher prevalence (%) and mean intensity (mean OPG +/- SD) of coccidian infection resulted for captive mouflons (73.17% and 814.6 +/- 1297.2 OPG) comparing to wild mouflons (36.73% and 112.7 +/- 268.7 OPG). Eimeria parva, E ovinoidalis, E. bakuensis, E. ahasata, E. intricata, E. crandallis, E. granulosa, E. faurei and an Eimeria species very similar to the descriptions previously reported for E. arloingi and E. yakimoffmatschoulsky, were isolated.

Isolation of Scopulariopsis spp. fungi from Psoroptes cuniculi body surface and evaluation of their entomopathogenic role.

In this study, some in vitro trials were carried out to evaluate the association between Scopulariopsis spp. fungi with Psoroptes cuniculi (Acari: Psoroptidae) and their potential pathogenicity to this mite species. After cultivation on Sabouraud dextrose agar with chloroamphenicol at 26 degrees C for 20 days and macro- and microscopical examinations, from P. cuniculi mites taken from some infested rabbits fungi belonging to the genus Scopulariopsis were isolated. Investigations were carried out to evaluate in vitro the potential pathogenic role of Scopulariopsis to P. cuniculi; to this aim, Scopulariopsis brevicaulis isolated from a cat was tested. In several culture media, the dose-dependent P. cuniculi mortality with different concentrations of S. brevicaulis and the ability of S. brevicaulis to penetrate inside the body of infected mites were evaluated. Results obtained demonstrated that, in the rabbit, Scopulariopsis fungi can be associated with P. cuniculi, and that S. brevicaulis can be an entomopathogen for P. cuniculi in a dose-dependent manner. A more rapid mortality of the mites and a quicker S. brevicaulis growth in plates containing rabbit serum with respect to all other media used were also found. S. brevicaulis demonstrated the ability to invade the body of P. cuniculi.

Efficacy of an essential oil of Eugenia caryophyllata against Psoroptes cuniculi.

The acaricidal activity of Eugenia caryophyllata essential oil was evaluated in vitro and in vivo on Psoroptes cuniculi, a mange mite. In vitro, different concentrations of the oil were tested and the observed mites mortality was compared with that observed in untreated and treated (Acacerulen R) controls. In vivo, six P. cuniculi infected rabbits were topically treated with the oil diluted at 2.5% and compared with untreated and treated control groups of six rabbits each. In vitro, up to the concentration of 0.10% the oil gave highly significant (P<0.01) percentages of mite mortality respect to the untreated controls, but only up to 0.16% it showed the same efficacy of Acacerulen R. In vivo, the treatment with the essential oil cured all infested rabbits and no statistical differences were observed respect to the treated control group. The untreated rabbits remained infested.

Efficacy of an essential oil of Cinnamomum zeylanicum against Psoroptes cuniculi.

The aim of the present study was to investigate the in vitro and in vivo acaricidal effects of an essential oil of Cinnamomum zeylanicun leaves on Psoroptes cuniculi, a mange mite. In vitro, 2.5 ml of the essential oil diluted at different concentrations, from 10% to 0.03%, in paraffin oil were added to Petri dishes containing all motile stages of P. cuniculi. Mites mortality observed in these dishes was compared with that observed in untreated and treated (AcaCerulen R) control plates. In vivo, one group of six P. cuniculi infected rabbits was topically treated two times at seven days interval with two ml of the essential oil at the concentration of 2.5% in paraffin oil and compared with untreated and treated (AcaCerulen R) control groups of six rabbits each. After 24 h of contact, all concentrations of essential oil between 0.10 and 10% showed a good in vitro acaricidal efficacy if compared with the untreated controls (p<0.01), but only the concentrations between 0.16 and 10% turned out as active as the drug. In vivo, the treatment with the essential oil cured all infested rabbits and no statistical differences were observed with the treated control group.

Efficacy of mangiferin against Cryptosporidium parvum in a neonatal mouse model.

The inhibitory activity of mangiferin (50 mg/kg/die and 100 mg/kg/die) on Cryptosporidium parvum was evaluated in a neonatal mouse model and its activity was compared with that of paromomycin (100 mg/kg/die). At 4 days of age, neonatal Swiss conventional outbred mice were experimentally infected by oral administration of 10(4) oocysts/animal of C. parvum and treated orally for 10 consecutive days, starting 7 days after the experimental infection. One group of mice was left untreated. To evaluate the efficacy of mangiferin, from euthanised mice, 3-mum-thick tissue sections of the intestine were stained with haematoxylin-eosin and periodic acid Schiff. Immunohistochemistry was also used by employing a monoclonal anti-C. parvum antibody. Oocysts were counted and results were expressed as mean oocysts number/intestine. Results obtained show that mangiferin at 100 mg/kg/die has a significant anticryptosporidial activity and that its activity is similar to that showed by the same dose (100 mg/kg/die) of paromomycin. However, both mangiferin and paromomycin were not able to completely inhibit intestinal colonization of C. parvum but only to reduce it. This reduction was calculated at over 80% for both mangiferin and paromomycin with respect to the untreated control. A significant activity was found also for mangiferin at 50 mg/kg/die only after the end of treatment.