Ricerca di Rickettsia del gruppo "Spotted Fever" in zecche antropofile raccolte in Toscana e Liguria

Abstract Several tick-borne rickettsiae cause human diseases and, in the last years, the increased use of molecular-based identification methods has resulted in new spotted fever group rickettsiae being characterized in ixodid ticks throughout Europe. To ascertain the Rickettsia Spotted Fever Group (SFG) that threatens people’s health, during March 2012- September 2012 the infection by SFG rickettsiae in tick species that bit humans and in host-seeking ticks was investigated in several areas of Tuscany and Liguria coast, Italy. After the identification of the tick, the DNA was extracted and the samples were investigated for gltA and rompA gene. Successful DNA extraction was verified using PCR for mitochondrial 16S rDNA sequences of hard and soft ticks. Specie identification was performed by sequence analysis and alignment with existing sequences in GenBank. Ticks belonging to following species were analysed: Dermacentor marginatus, Haemaphysalis punctata, Hyalomma marginatum, Ixodes ricinus, Ixodes (Pholeoixodes) hexagonus, Rhipicephalus sanguineus and R. turanicus. Of 76 ticks removed from patients in Liguria and Tuscany hospitals, 18 (32.14%, 95% C. I. 19.91–44.37) resulted positive for Rickettsia SFG DNA. R. monacensis was the most amplified (n=8, 14.29%, 95% C. I. 5.12–23.45), followed by R. helvetica (n=5, 8.93%, 95% C. I. 1.96–16.40) and Rickettsia sp. CyRtu43S (n=1). A total of 442 host-seeking tick specimens were collected by dragging vegetation, corresponding to 444 nymphs (92.12%), 24 females (4.98%), and 11 males (2.28%). The adults were analysed singularly while the nymphs were analysed in pool of three subjects of the same species. In total of 186 samples amplified, 60 resulted positive for R. monacensis (32.26%, 95% C. I. 25.54–38.98) and 9 for R. helvetica (4.84%, 95% C. I. 1.75–7.92). Two male of D. marginatus resulted positive for R. slovaca and one female of R. turanicus for R. massiliae. R. bellii was amplified from an I. ricinus nymph. Of 404 I. ricinus nymphs analysed, 22.89% (95% C. I. 18.78–26.99) resulted positive for Rickettsia SFG DNA. The southern provinces of Tuscany (Leghorn and Pisa) showed the highest prevalence, respectively 41.52%, (I.C. 95% 29.05–53.99) and 35.63% (I.C. 95% 21.64–49.63). Riassunto Molte rickettsie, trasmesse da morso di zecca, causano malattie nell’uomo e negli ultimi anni l’aumento dell’utilizzo di metodiche diagnostiche basate sull’analisi molecolare ha permesso la caratterizzazione di nuove rickettsie del gruppo Spotted Fever (SFG) in zecche della famiglia Ixodidae in tutta Europa. Per accertare le rickettsie SFG che minacciano la salute pubblica, tra marzo e settembre 2012, sono stati analizzati per ricerca di DNA di Rickettsia SFG campioni di zecche raccolte nella zona costiera delle regioni Toscana e Liguria e provenienti da tre flussi: flusso centrale (zecche derivate da pazienti dal pronto soccorso), monitoraggio ambientale (zecche raccolte in habitat) e flusso periferico (zecche raccolte da utenza sensibilizzata). La presenza di DNA di Rickettsia SFG nei campioni di zecca e stata ricercata utilizzando primer specifici per un frammento della regione gltA codificante per il gene della citrato sintetasi e per un frammento della regione rompA codificante per l’antigene di superficie OmpA. Come controllo di processo è stato utilizzato un frammento del DNA ribosomiale 16S specifico per le zecche. L’identificazione di specie è stata effettuata mediante analisi delle sequenze e allineamento con le sequenze presenti in GenBank. Le zecche analizzate appartenevano alle seguenti specie: Dermacentor marginatus, Haemaphysalis punctata, Hyalomma marginatum, Ixodes ricinus, Ixodes (Pholeoixodes) hexagonus, Rhipicephalus sanguineus and R. turanicus. Dei 76 campioni prelevati da paziente negli ospedali della Liguria e della Toscana, 18 (32,14%, I. C. 95% 19,91–44,37) sono risultati positivi per DNA di Rickettsia SFG. R. monacensis è stata, tra le diverse specie di Rickettsia SFG presenti in questi campioni, quella più frequentemente rilevata mediante PCR (n=8, 14,29%, I.C. 95% 5,12–23,45), seguita da R. helvetica (n=5, 8,93%, I.C. 95% 1,96–16,40) e Rickettsia sp. CyRtu43S (n=1). Un totale di 442 campioni di zecca in cerca di ospite sono stati raccolti mediante dragging, corrispondenti a 444 ninfe (92.12%), 24 femmine (4.98%) e 11 maschi (2.28%). Gli adulti sono stati analizzati singolarmente mentre le ninfe sono state analizzate in pool di tre soggetti della stessa specie. In totale dei 186 campioni amplificati, 60 sono risultati positivi per R. monacensis (32,26%, I. C. 95% 25,54–38,98) e 9 per R. helvetica (4,84%, I. C. 95% 1,75–7,92). Due maschi di D. marginatus sono risultati positive per R. slovaca e una femmina di R. turanicus è risultata positiva per R. massiliae. Un campione di I. ricinus ninfa ha presentato amplificato di R. bellii per il gene gltA. Delle 404 ninfe di I. ricinus analizzate il 22.89% (I. C. 95% 18.78–26.99) è risultato positive per Rickettsia SFG. Le province di Livorno e Pisa hanno presentato la prevalenza più alta, rispettivamente del 41.52% (I. C. 95% 29,05–53,99) e del 35,63% (I.C. 95% 21,64–49,63)

Eimeria legionensis and Eimeria kofoidi (Apicomplexa: Eimeriidae) infection and associated lesions in naturally infected red-legged partridges (alectoris rufa)

With the aim to identify the Eimeria species responsible for coccidiosis in 50 deceased red-legged partridges (Alectoris rufa), individual faecal samples were collected, dissolved in 2.5% K2Cr2O7 solution and maintained at room temperature to allow sporulation of the oocysts. Morphology and dimensions of sporulated oocysts were microscopically evaluated. To assess Eimeria intestinal localisation, faecal samples and scrapings taken from the different intestinal segments of each deceased animal were examined by fresh smears and flotation test, while the intestines were examined for gross lesions, then fixed in 10% formalin and processed for histopathological analysis. From scrapings and morphological analysis, Eimeria kofoidi and Eimeria legionensis were identified in the small intestine and in the caecum and colon, respectively. Histopathological analysis confirmed the presence of two distinct Eimeria species. In particular, E. kofoidi macrogamonts were found in epithelial cells of jejunum and ileum, between the basal lamina and the nucleus of the infected intestinal cells. This latter was flattened and displaced above. E. legionensis macrogamonts were instead found localised between the nucleus and the luminal surface of the infected caeca and colonic cells and these macrogamonts were larger than those of E. kofoidi. Chronic enteritis and severe displacement of the deep crypts of the small intestine, large areas of caeca and colonic epithelial necrosis associated to thickened wall and mononuclear cells infiltration diffused in a transmural manner, were the main histopathological lesions

Oocyst excretion pattern of three intestinal Eimeria species in female rabbits

[EN] The dynamic change in faecal Eimeria oocyst excretion was evaluated in 10 naturally infected female rabbits, starting from their weaning at 33 d of age until about 1 mo after their second parturition. Faecal samples collected from examined animals were quali-quantitatively analysed to evaluate presence and number of Eimeria oocysts. In addition, isolated Eimeria oocysts were identified at the species level following sporulation. Animals were found to be infected by Eimeria perforans, Eimeria exigua and Eimeria magna and shed Eimeria oocysts after weaning and after parturition. In particular, at 33 d of age all female rabbits examined were negative, while the discharge of Eimeria oocysts started at 39th day of age and peaked between 46th and 53rd day of age. From 81-109 d of age until the first parturition and from 25 d of age of the litters born at the first parturition to the second parturition, all animals resulted negative. After parturition, Eimeria oocyst output occurred from 6th to 12th day after the first parturition and from 7th to 13th day after the second parturition, while a second period of oocyst excretion was observed from 18th to 24th day after both parturitions. These findings may indicate the existence of a relationship between the periparturient phase and Eimeria oocyst output and suggest an important role of the mothers in transmission of the infection to their litters.The authors thank the Italian Ministry of University (MIUR) for financing this studyPapeschi, C.; Fichi, G.; Perrucci, S. (2013). Oocyst excretion pattern of three intestinal Eimeria species in female rabbits. World Rabbit Science. 21(2):77-83. doi:10.4995/wrs.2013.1235.SWORD778321

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

Dolphins Stranded along the Tuscan Coastline (Central Italy) of the “Pelagos Sanctuary”: A Parasitological Investigation

Parasite monitoring is considered a necessary step for cetacean management and conservation. Between February 2013 and July 2015, 26 dolphins (15 Stenella coeruleoalba, 10 Tursiops truncatus, and one Grampus griseus) stranded along the Tuscan coastline of the protected marine area “Pelagos Sanctuary”, were examined. Organs, tissues, and faecal and blood samples taken from all animals were analysed by parasitological, immunological, and molecular techniques. Twenty-one out of 26 dolphins (80.77%) tested positive for at least one parasite species, and 13/15 (86.7%) S. coeruleoalba, 7/10 (70%) T. truncatus, and the single G. griseus were found positive. Identified parasites included the nematodes Skrjabinalius guevarai (7.69%, 2/26), Halocercus lagenorhynchi (3.85%, 1/26), Halocercus delphini (7.69%, 2/26), Stenurus ovatus (7.69%, 2/26), Crassicauda spp. (7.69%, 2/26); the trematodes Pholeter gastrophilus (26.92%, 7/26), Campula palliata (3.85%, 1/26); the cestodes Phyllobothrium delphini (42.31%, 11/26), Monorygma grimaldii (23.08%, 6/26), Tetrabothrium forsteri (7.69%, 2/26), Strobilocephalus triangularis (7.69%, 2/26), and the acanthocephalan Bolbosoma vasculosum (7.69%, 2/26). Moreover, 6/26 (23%) animals scored positive to Toxoplasma gondii at serology, but PCR confirmed the infection (T. gondii Type II genotype) in a single animal. In examined dolphins, obtained results showed a high prevalence of endoparasites, which included species considered as a cause of severe debilitation or deat

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.REF Compliant by Deposit in Stirling's Repositor

Dolphin Morbillivirus Associated with a Mass Stranding of Sperm Whales, Italy

In September 2014, 7 sperm whales stranded along the Adriatic Italian coastlines. Postmortem investigations on 3 dead females dead and in 1 fetus harbored by the largest one revealed molecular and immunoistochemical evidences of dolphin morbillivirus infection. A possible role of the virus in the stranding event was considered

A New Multilocus Sequence Typing Scheme and Its Application for the Characterization of Photobacterium damselae subsp. damselae Associated with Mortality in Cetaceans

Photobacterium damselae subsp. damselae (PDD) is a known pathogen of fish, humans and marine mammals. In this study, a Multilocus Sequence Typing (MLST) scheme based on six housekeeping genes (glp, gyrB, metG, pnt, pyrC and toxR) was developed to better understand the PDD population structure and used to type 73 PDD isolates from cetaceans, mainly striped dolphins (Stenella coeruleoalba) involved in mortality episodes, and from a few marine chelonians. Five reference ATCC strains were also included in the study. Typing allowed the discrimination of groups of PDD strains isolated from different host species, at different times and from different geographic areas, suggesting that a clonal PDD group may have spread in the Tyrrhenian sea at the time of an Unusual Mortality Event (UME) among cetaceans, mainly striped dolphins, occurred in early 2013 along the Italian western coasts

Loss of ap4s1 in zebrafish leads to neurodevelopmental defects resembling spastic paraplegia 52.

Autosomal recessive spastic paraplegia 52 is caused by biallelic mutations in AP4S1 which encodes a subunit of the adaptor protein complex 4 (AP-4). Using next-generation sequencing, we identified three novel unrelated SPG52 patients from a cohort of patients with cerebral palsy. The discovered variants in AP4S1 lead to reduced AP-4 complex formation in patient-derived fibroblasts. To further understand the role of AP4S1 in neuronal development and homeostasis, we engineered the first zebrafish model of AP-4 deficiency using morpholino-mediated knockdown of ap4s1. In this model, we discovered several phenotypes mimicking SPG52, including altered CNS development, locomotor deficits, and abnormal neuronal excitability