Federica Marcer

Genetic and Morphological Approaches Distinguish the Three Sibling Species of the Anisakis simplex Species Complex, with a Species Designation as Anisakis berlandi n. sp. for A. simplex sp. C (Nematoda: Anisakidae)

Abstract:  Numerous specimens of the 3 sibling species of the Anisakis simplex species complex (A. pegreffii, A. simplex (senso stricto)), and A. simplex sp. C) recovered from cetacean species stranded within the known geographical ranges of these nematodes were studied morphologically and genetically. The genetic characterization was performed on diagnostic allozymes and sequences analysis of nuclear (internal transcribed spacer [ITS] of ribosomal [r]DNA) and mitochondrial (mitochondrial [mt]DNA cox2 and rrnS) genes. These markers showed (1) the occurrence of sympatry of the 2 sibling species A. pegreffii and A. simplex sp. C in the same individual host, the pilot whale, Globicephala melas Traill, from New Zealand waters; (2) the identification of specimens of A. pegreffii in the striped dolphin, Stenella coeruleoalba (Meyen), from the Mediterranean Sea; and (3) the presence of A. simplex (s.s.) in the pilot whale and the minke whale, Balaenoptera acutorostrata Lacépède, from the northeastern Atlantic waters. No F1 hybrids were detected among the 3 species using the nuclear markers. The phylogenetic inference, obtained by maximum parsimony (MP) analysis of separate nuclear (ITS rDNA region), combined mitochondrial (mtDNA cox2 and rrnS) sequences datasets, and by concatenated analysis obtained at both MP and Bayesian inference (BI) of the sequences datasets at the 3 studied genes, resulted in a similar topology. They were congruent in depicting the existence of the 3 species as distinct phylogenetic lineages, and the tree topologies support the finding that A. simplex (s.s.), A. pegreffii, and A. berlandi n. sp. (=A. simplex sp. C) represent a monophyletic group. The morphological and morphometric analyses revealed the presence of morphological features that differed among the 3 biological species. Morphological analysis using principal component analysis, and Procrustes analysis, combining morphological and genetic datasets, showed the specimens clustering into 3 well-defined groups. Nomenclatural designation and formal description are given for A. simplex species C: the name Anisakis berlandi n. sp. is proposed. Key morphological diagnostic traits are as follows between A. berlandi n. sp. and A. simplex (s.s.): ventriculus length, tail shape, tail length/total body length ratio, and left spicule length/total body length ratio; between A. berlandi n. sp. and A. pegreffii: ventriculus length and plectane 1 width/plectane 3 width ratio; and between A. simplex (s.s.) and A. pegreffii: ventriculus length, left and right spicule length/total body length ratios, and tail length/total body length ratio. Ecological data pertaining to the geographical ranges and host distribution of the 3 species are updated.

Dolphin Morbillivirus and Toxoplasma gondii coinfection in a Mediterranean fin whale (Balaenoptera physalus).

BackgroundAlthough Morbillivirus and Toxoplasma gondii have emerged as important pathogens for several cetaceans populations over the last 20 years, they have never been identified together in a Mysticete. In particular, morbilliviral infection has been never described in the Mediterranean fin whale population.Case presentationOn January 2011 an adult male of fin whale (Balaenoptera physalus) stranded along the Tyrrhenian coastline of Italy. During necropsy, tissue samples from heart, skeletal muscle, mesenteric lymph nodes, liver, spleen, lung, and kidney were collected and subsequently analyzed for Morbillivirus and Toxoplasma gondii by microscopic and molecular methods. Following the detailed necropsy carried out on this whale, molecular analysis revealed, for the first time, the simultaneous presence of a Dolphin Morbillivirus (DMV) and T. gondii infection coexisting with each other, along with high organochlorine pollutant concentrations, with special reference to DDT.ConclusionThis report, besides confirming the possibility for Mysticetes to be infected with DMV, highlights the risk of toxoplasmosis in sea water for mammals, already immunodepressed by concurrent factors as infections and environmental contaminants.

Detection of Babesia EU1 in Ixodes ricinus ticks in northern Italy.

Babesia EU1, a potentially important emerging zoonotic pathogen, already detected in ticks and wild ruminants of different European Countries, was found in three pools of Ixodes ricinus nymphs in three different sites located in a single District of north-eastern Italy. Totally 356 ticks (60 pools) were collected from the environment during a surveillance activity in the year 2006. Babesia EU1 estimated individual tick prevalence in the area is 0.85%. The finding that also in northern Italy the tick population is carrying Babesia EU1 suggests a wide geographical spreading of this zoonotic pathogen in Europe.

A Parasitological Survey of European Sea Bass (Dicentrarchus Labrax) and Gilthead Sea Bream (Sparus Aurata) Cultured in Italy

M.L. Fioravanti∗, M. Caffara, D. Florio, A. Gustinelli and F. Marcer Department of Veterinary Public Health and Animal Pathology, Faculty of Veterinary Medicine, University of Bologna, Ozzano Emilia (BO), Italy ∗Correspondence: Dipartimento di Sanità Pubblica Veterinaria e Patologia Animale – Facoltà di Medicina Veterinaria, via Tolara di Sopra, 50 – 40064, Ozzano Emilia (BO), Italy E-mail: fioravanti@vet.unibo.it

First evidence of the European wildcat (Felis silvestris silvestris) as definitive host of Angiostrongylus chabaudi

Angiostrongylus chabaudi (Strongylida, Angiostrongylidae) is a parasitic nematode described for the first time last century from the pulmonary arteries of six European wildcats (Felis silvestris silvestris) in central Italy. Since then, this parasite remained practically unknown until recently, when immature A. chabaudi have been reported from one wildcat in Germany and two domestic cats (Felis silvestris catus) in Italy. The present report describes the first record of A. chabaudi in Greece and, most importantly, the first known case of patent infection by A. chabaudi. The necropsy of a road-killed F. s. silvestris found near the lake Kerkini, in the municipality of Serres (Macedonia, Greece), revealed the presence of nematodes of both sexes in the right ventricle and the pulmonary artery of the heart. All parasites were mature adults and numerous eggs were present in the uteruses of females. The morphological characteristics of the parasites were consistent with those of A. chabaudi. Moreover, Angiostrongylus-like first stage larvae (L1) were present in the faeces of the animal that was negative for any other cardio-pulmonary parasite. Genetic examination of adult parasites and L1 confirmed the morphological identification as A. chabaudi. Histopathological examination of the lungs showed severe, multifocal to coalescing, chronic, interstitial granulomatous pneumonia due to the presence of adult parasites, larvae and eggs. These findings demonstrate for the first unequivocal time that this nematode reproduces in the European wildcat which should be ultimately considered a definitive host of A. chabaudi. Finally, the L1 of A. chabaudi are described here for the first time, opening new prospects for further studies on this neglected parasite.

Comparative field study to evaluate the performance of three different traps for collecting sand flies in northeastern Italy

ABSTRACT: Three standard methods for collecting sand flies (sticky trap, CDC light trap, and CO2 trap) were compared in a field study conducted from June to October, 2012, at a site located in the center of a newly established autochthonous focus of canine leishmaniasis in northeastern Italy. Six traps (two sticky traps, two CDC light traps, and two CO2 traps) were activated at the same time for a single night every two weeks during the season of sand fly activity. A total of 5,667 sand flies were collected and 2,213 identified, of which 82.1% were Phlebotomus perniciosus, 17.4% P. neglectus, 0.3% Sergentomya minuta, and 0.2% P. mascitti. The performances of all traps were influenced by their position inside the site, increasing with proximity to the animal shelters. CO2 traps were more attractive for females of P. perniciosus and P. neglectus. CDC light traps showed an intermediate efficiency and were more attractive for P. neglectus, compared to other two traps. Results suggest that in northern Italy the CO2 trap is a suitable sampling method for sand fly monitoring programs that include transmitted pathogen surveillance.

Post-mortem investigations on a leatherback turtle Dermochelys coriacea stranded along the Northern Adriatic coastline

Leatherback sea turtles Dermochelys coriacea are regularly reported in the Mediterranean Sea but rarely reach the northern Adriatic Sea. In the summer of 2009, a well-preserved carcass of an adult female of this species was found dead along the coast of Lido di Venezia. A complete necropsy was carried out, along with evaluation of levels of tissue trace elements. The the post-mortem revealed acute severe bacterial gastroenteritis caused by Photobacterium damselae ssp. piscicida, an opportunistic agent that infected an apparently debilitated animal weakened by ingested plastic debris. High levels of heavy metals (Hg, Pb, Cd and As) found in the liver and kidneys might have contributed to the animals demise. These findings support previous indications that marine debris is one of the major threats to marine animals, particularly for critically endangered species such as the leatherback turtle.

New insights into the epidemiology of bovine piroplasmoses in Italy

Few studies have been published on bovine piroplasmoses in Italy, and therefore a clear picture of the epidemiology of these infections is difficult to obtain. Vertebrate and invertebrate hosts in Central and Northern Regions of Italy were investigated in 2005 and 2006, when microscopy, molecular tools and serological tests were applied to 468 blood samples drawn from cattle in order to evaluate the presence of these protozoa and identify possible risk factors. Ticks were also collected, identified and analyzed by molecular techniques. Microscopy identified 6.5% of the animals as positive, whereas PCR detected piroplasm DNA in 21.6%. BLAST analysis showed 67 amplicons (17.0%) referable to the Theileria sergenti/buffeli/orientalis group, 17 (4.3%) to Theileria annae, and 1 to Babesia divergens. Serology evidenced a prevalence of 45.4% for Babesia bovis, 17.4% for Babesia bigemina, and 34.9% for B. divergens. The 127 collected ticks were identified as belonging to 5 species, mostly represented by Rhipicephalus bursa, Hyalomma marginatum and Ixodes ricinus. Molecular analyses evidenced the presence of B. bovis and B. bigemina, in 3 and 5 ticks, respectively. Our findings suggest that different species of piroplasms are circulating in bovine populations in Central and Northern Italy, and provide new insights into the complex epidemiology of bovine piroplasmoses in Italy.

Mitochondrial haplotypes of Aelurostrongylus abstrusus and Troglostrongylus brevior (Nematoda, Metastrongyloidea) from domestic and wild felids

The lungworm Aelurostrongylus abstrusus is the most important respiratory parasite of domestic cats. Pulmonary aelurostrongylosis has been reported in wild felids, though unequivocally evidence of wildlife infection by A. abstrusus is scant. Recently, Troglostrongylus brevior, a lungworm usually infecting wild felids, has been described in domestic cats from Mediterranean areas. The present work evaluates the sequence variation of an informative region within the gene encoding the mitochondrial cytochrome c oxidase subunit 1 of A. abstrusus and T. brevior, in order to provide novel information on the genetic make-up of these lungworms. Parasitic stages of A. abstrusus and T. brevior were collected from domestic and wild hosts (i.e., domestic cat, European wildcat, caracal, serval, and lion) from Italy, Greece, and South Africa. Five (HI-HV) and four (HI-HIV) haplotypes were recorded for A. abstrusus and T. brevior, respectively, mostly shared between domestic and wild felids in different geographical areas. The phylogenetic analysis showed that all haplotypes of A. abstrusus and T. brevior clustered as monophyletic groups with a strong nodal support, indicating that all haplotypes identified were distinct from each other. All sequence types represent two distinct species, A. abstrusus and T. brevior, and these genetic convergences are also detected within and among populations of these nematodes, irrespective of their hosts and geographical origin. The occurrence of A. abstrusus and T. brevior haplotypes in different hosts from the same regions and between different countries indicates that the same lungworm populations circulate in domestic and wild hosts under the same routes of transmission.

Development of Culture-Based Serological Assays to Diagnose Babesia divergens Infections

Babesioses are hematic tick-borne diseases that induce malaria-like disorders in domestic, wild animals, and humans. Although indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA) commercial kits are available to test the presence of antibodies against most Babesia species, no kit exists to serologically diagnose the infections due to Babesia divergens, one of the most important zoonotic species. To fill this gap and to develop assays to detect animal and human infections, in vitro cultures (microaerophilous stationary phase system) of B. divergens were organized. Infected erythrocytes were adsorbed as corpuscular antigen (CA) on IFAT slides and ELISA microwells. The supernatant medium of the cultures (metabolic antigen, MA) was collected and employed in ELISA and western blot (WB) assays. B. divergens was also used to produce positive sera in Meriones unguiculatus and to infect a calf. Serological tests were set up with sera from experimentally/naturally infected animals, and possible cross-reactions were evaluated using heterologous sera from cattle positive to other piroplasms. Sera from clinically healthy people at risk of infection were also tested. As expected, assays based on the purified MAs from in vitro cultures proved more sensitive and specific than CA-IFAT and CA-ELISA. In fact, MA-ELISA provided satisfactory performances (even if 8.4%-15.7% cross-reactions were evidenced), and the WB developed proved totally sensitive and specific. WB indicated as immunodominant antigens two major protein bands at 33 and 37 kDa, which were also evidenced in 2.2% of the human sera tested, proving the parasite transmission to humans also in Italy.