The S-palmitoylome and DHHC-PAT interactome of Drosophila melanogaster S2R+ cells indicate a high degree of conservation to mammalian palmitoylomes.

Protein S-palmitoylation, the addition of a long-chain fatty acid to target proteins, is among the most frequent reversible protein modifications in Metazoa, affecting subcellular protein localization, trafficking and protein-protein interactions. S-palmitoylated proteins are abundant in the neuronal system and are associated with neuronal diseases and cancer. Despite the importance of this post-translational modification, it has not been thoroughly studied in the model organism Drosophila melanogaster. Here we present the palmitoylome of Drosophila S2R+ cells, comprising 198 proteins, an estimated 3.5% of expressed genes in these cells. Comparison of orthologs between mammals and Drosophila suggests that S-palmitoylated proteins are more conserved between these distant phyla than non-S-palmitoylated proteins. To identify putative client proteins and interaction partners of the DHHC family of protein acyl-transferases (PATs) we established DHHC-BioID, a proximity biotinylation-based method. In S2R+ cells, ectopic expression of the DHHC-PAT dHip14-BioID in combination with Snap24 or an interaction-deficient Snap24-mutant as a negative control, resulted in biotinylation of Snap24 but not the Snap24-mutant. DHHC-BioID in S2R+ cells using 10 different DHHC-PATs as bait identified 520 putative DHHC-PAT interaction partners of which 48 were S-palmitoylated and are therefore putative DHHC-PAT client proteins. Comparison of putative client protein/DHHC-PAT combinations indicates that CG8314, CG5196, CG5880 and Patsas have a preference for transmembrane proteins, while S-palmitoylated proteins with the Hip14-interaction motif are most enriched by DHHC-BioID variants of approximated and dHip14. Finally, we show that BioID is active in larval and adult Drosophila and that dHip14-BioID rescues dHip14 mutant flies, indicating that DHHC-BioID is non-toxic. In summary we provide the first systematic analysis of a Drosophila palmitoylome. We show that DHHC-BioID is sensitive and specific enough to identify DHHC-PAT client proteins and provide DHHC-PAT assignment for ca. 25% of the S2R+ cell palmitoylome, providing a valuable resource. In addition, we establish DHHC-BioID as a useful concept for the identification of tissue-specific DHHC-PAT interactomes in Drosophila.

A conserved Neurite Outgrowth and Guidance motif with biomimetic potential in neuronal Cell Adhesion Molecules.

The discovery of conserved protein motifs can, in turn, unveil important regulatory signals, and when properly designed, synthetic peptides derived from such motifs can be used as biomimetics for biotechnological and therapeutic purposes. We report here that specific Ig-like repeats from the extracellular domains of neuronal Cell Adhesion Molecules share a highly conserved Neurite Outgrowth and Guidance (NOG) motif, which mediates homo- and heterophilic interactions crucial in neural development and repair. Synthetic peptides derived from the NOG motif of such proteins can boost neuritogenesis, and this potential is also retained by peptides with recombinant sequences, when fitting the NOG sequence pattern. The NOG motif discovery not only provides one more tile to the complex puzzle of neuritogenesis, but also opens the route to new neural regeneration strategies via a tunable biomimetic toolbox.

Occurrence and Identification of Ixodes ricinus Borne Pathogens in Northeastern Italy.

In Europe, Ixodes ricinus is the main vector for tick-borne pathogens (TBPs), the most common tick species in Italy, particularly represented in pre-alpine and hilly northern areas. From 2011 to 2017, ticks were collected by dragging in Belluno province (northeast Italy) and analyzed by molecular techniques for TBP detection. Several species of Rickettsia spp. and Borrelia spp. Anaplaspa phagocitophilum, Neoerlichia mikurensis and Babesia venatorum, were found to be circulating in the study area carried by I. ricinus (n = 2668, all stages). Overall, 39.1% of screened pools were positive for at least one TBP, with a prevalence of 12.25% and 29.2% in immature stages and adults, respectively. Pathogens were detected in 85% of the monitored municipalities, moreover the presence of TBPs varied from one to seven different pathogens in the same year. The annual TBPs prevalence fluctuations observed in each municipality highlights the necessity of performing continuous tick surveillance. In conclusion, the observation of TBPs in ticks remains an efficient strategy for monitoring the circulation of tick-borne diseases (TBDs) in a specific area.

Real-time PCR assay for screening Pneumocystis in free-living wild squirrels and river rats in Italy.

We used a real-time PCR (rtPCR) targeting a 150-bp amplicon of the mitochondrial small subunit of ribosomal RNA (mtSSU rRNA) to screen for Pneumocystis DNA in lungs of wild squirrels ( Callosciurus finlaysonii, n = 85) and river rats ( Myocastor coypus, n = 43) in Italy. The rtPCR revealed Pneumocystis DNA in 20 of 85 (24%) squirrels and in 35 of 43 (81%) river rats, and was more sensitive than a nested PCR that targets a portion of the mtSSU rRNA and the mitochondrial large subunit of rRNA (mtLSU rRNA). Phylogenetic analysis based on mtSSU rRNA and mtLSU rRNA sequences showed distinct Pneumocystis sequence types in these rodents. The rtPCR assay should be reliable for screening large populations for this potential pathogen, thereby allowing cost-effective monitoring of the disease in wild animals.

Ticks are more suitable than red foxes for monitoring zoonotic tick-borne pathogens in northeastern Italy.

BACKGROUND: Northeastern Italy is a hotspot for several tick-borne pathogens, transmitted to animals and humans mainly by Ixodes ricinus. Here we compare the results of molecular monitoring of ticks and zoonotic TBPs over a six-year period, with the monitoring of red foxes (Vulpes vulpes) in an endemic area. RESULTS: In the period 2011-2016, 2,578 ticks were collected in 38 sites of 20 municipalities of Belluno Province. Individual adults (264), pooled larvae (n = 330) and nymphs (n = 1984) were screened for tick-borne encephalitis virus, Borrelia burgdorferi (s.l.), Rickettsia spp., Babesia spp., Anaplasma phagocytophilum and "Candidatus Neoehrlichia mikurensis" by specific SYBR green real-time PCR assays and sequencing. The spleens of 97 foxes, culled in the period 2015-2017 during sport hunting or population control programs, were also screened. Overall, nine different pathogens were found in I. ricinus nymph and adult ticks: Rickettsia helvetica (3.69%); R. monacensis (0.49%); four species of the B. burgdorferi (s.l.) complex [B. afzelii (1.51%); B. burgdorferi (s.s.) (1.25%); B. garinii (0.18%); and B. valaisiana (0.18%)]; A. phagocytophilum (3.29%); "Candidatus N. mikurensis" (1.73%); and Babesia venatorum (0.04%). Larvae were collected and screened in the first year only and two pools (0.6%) were positive for R. helvetica. Tick-borne encephalitis virus was not found in ticks although human cases do occur in the area. The rate of infection in ticks varied widely according to tick developmental stage, site and year of collection. As expected, adults were the most infected, with 27.6% harboring at least one pathogen compared to 7.3% of nymphs. Pathogens with a minimum infection rate above 1% were recorded every year. None of the pathogens found in ticks were detectable in the foxes, 52 (54%) of which were instead positive for Babesia cf. microti (also referred to as Babesia microti-like, "Theileria annae", "Babesia annae" and "Babesia vulpes"). CONCLUSIONS: The results show that foxes cannot be used as sentinel animals to monitor tick-borne pathogens in the specific epidemiological context of northeastern Italy. The high prevalence of Babesia cf. microti in foxes and its absence in ticks strongly suggests that I. ricinus is not the vector of this pathogen.

Prevalence and molecular characterization of canine and feline hemotropic mycoplasmas (hemoplasmas) in northern Italy.

BACKGROUND: Hemotropic mycoplasmas (hemoplasmas), the agents of infectious anemia, have been reported in dogs and cats. Little data are available on hemoplasma infections in Italy. The aim of this study was to evaluate the species of hemoplasmas and their prevalence in dogs and cats of northern Italy. METHODS: Blood samples were obtained from 117 candidate blood donor dogs, 278 free-roaming dogs and 227 free-roaming cats in 2014 and 2015. Samples were first screened for hemoplasmas with a SYBR green real time PCR. The positive samples were confirmed by a second SYBR green real time PCR and sequencing. Co-infections were detected using species-specific SYBR green real time PCR. RESULTS: The overall prevalence in dogs was 4.5% (18/395). Among the donors only one dog was positive for Mycoplasma haemocanis (0.8%). The overall prevalence of infection in free-roaming dogs was 6.1% (17/278), which was significantly higher than in candidate donors (P < 0.05). Both M. haemocanis (13/278; 4.7%) and "Candidatus M. haematoparvum" (4/278; 1.4%) were identified. In dogs, no significant association was found between hemoplasma infection and gender, age or origin. The overall prevalence in cats was 13.2% (30/227). All three feline hemoplasma species were detected, i.e. "Candidatus Mycoplasma haemominutum" (28; 12.3%), "Candidatus Mycoplasma turicensis" (11; 4.8%) and Mycoplasma haemofelis (9; 4.0%). Half of the infected cats were co-infected (15; 6.6%) with different species of hemoplasmas. Risk factor analysis confirmed that older age, male gender and FIV positivity are predisposing factors for hemoplasma infection in cats. CONCLUSION: This study found that candidate blood donor dogs in northern Italy show a negligible risk for hemoplasma infection, confirming the appropriateness of the candidate selection criteria and the low prevalence in the study area. Accordingly, testing for hemoplasma should be considered optional for canine blood donor screening. Hemoplasma infection was instead common in free-roaming cats, and is expected to be non-negligible in owned cats with outdoor access. Feline candidates for blood donation will therefore need to be carefully selected.

First report outside Eastern Europe of West Nile virus lineage 2 related to the Volgograd 2007 strain, northeastern Italy, 2014.

BACKGROUND: West Nile virus (WNV) is a Flavivirus transmitted to vertebrate hosts by mosquitoes, maintained in nature through an enzootic bird-mosquito cycle. In Europe the virus became of major public health and veterinary concern in the 1990s. In Italy, WNV re-emerged in 2008, ten years after the previous outbreak and is currently endemic in many areas of the country. In particular, the northeastern part of Italy experience continuous viral circulation, with human outbreaks caused by different genovariants of WNV lineage 1, Western-European and Mediterranean subcluster, and WNV lineage 2, Hungarian clade. Alongside the WNV National Surveillance Program that has been in place since 2002, regional surveillance plans were implemented after 2008 targeting mosquitoes, animals and humans. FINDINGS: In July and September 2014, West Nile virus lineage 2 was detected in pools of Culex pipiens s.l. mosquitoes from northeastern Italy. Whole genome sequencing and phylogenetic analysis of two representative samples identified the presence of WNV lineage 2 related to the Volgograd 2007 strain (99.3 % nucleotide sequence identity), in addition to WNV lineage 2 Hungarian clade. CONCLUSIONS: This is the first evidence of the circulation of a WNV lineage 2 strain closely related to the Volgograd 2007 outside Eastern Europe, where it has caused large human outbreaks. This strain may pose a new threat to animal and human health in Italy.

Occurrence and identification of risk areas of Ixodes ricinus-borne pathogens: a cost-effectiveness analysis in north-eastern Italy.

BACKGROUND: Ixodes ricinus, a competent vector of several pathogens, is the tick species most frequently reported to bite humans in Europe. The majority of human cases of Lyme borreliosis (LB) and tick-borne encephalitis (TBE) occur in the north-eastern region of Italy. The aims of this study were to detect the occurrence of endemic and emergent pathogens in north-eastern Italy using adult tick screening, and to identify areas at risk of pathogen transmission. Based on our results, different strategies for tick collection and pathogen screening and their relative costs were evaluated and discussed. METHODS: From 2006 to 2008 adult ticks were collected in 31 sites and molecularly screened for the detection of pathogens previously reported in the same area (i.e., LB agents, TBE virus, Anaplasma phagocytophilum, Rickettsia spp., Babesia spp., "Candidatus Neoehrlichia mikurensis"). Based on the results of this survey, three sampling strategies were evaluated a-posteriori, and the impact of each strategy on the final results and the overall cost reductions were analyzed. The strategies were as follows: tick collection throughout the year and testing of female ticks only (strategy A); collection from April to June and testing of all adult ticks (strategy B); collection from April to June and testing of female ticks only (strategy C). RESULTS: Eleven pathogens were detected in 77 out of 193 ticks collected in 14 sites. The most common microorganisms detected were Borrelia burgdorferi sensu lato (17.6%), Rickettsia helvetica (13.1%), and "Ca. N. mikurensis" (10.5%). Within the B. burgdorferi complex, four genotypes (i.e., B. valaisiana, B. garinii, B. afzelii, and B. burgdorferi sensu stricto) were found. Less prevalent pathogens included R. monacensis (3.7%), TBE virus (2.1%), A. phagocytophilum (1.5%), Bartonella spp. (1%), and Babesia EU1 (0.5%). Co-infections by more than one pathogen were diagnosed in 22% of infected ticks. The prevalences of infection assessed using the three alternative strategies were in accordance with the initial results, with 13, 11, and 10 out of 14 sites showing occurrence of at least one pathogen, respectively. The strategies A, B, and C proposed herein would allow to reduce the original costs of sampling and laboratory analyses by one third, half, and two thirds, respectively. Strategy B was demonstrated to represent the most cost-effective choice, offering a substantial reduction of costs, as well as reliable results. CONCLUSIONS: Monitoring of tick-borne diseases is expensive, particularly in areas where several zoonotic pathogens co-occur. Cost-effectiveness studies can support the choice of the best monitoring strategy, which should take into account the ecology of the area under investigation, as well as the available budget.

Occurrence of Rickettsia felis in dog and cat fleas (Ctenocephalides felis) from Italy.

Rickettsia felis is an obligate intracellular bacterium belonging to the spotted fever group, suspected to cause a murine typhus-like illness in humans, with a cosmopolitan distribution. This study was designed to estimate presence and occurrence of this pathogen in fleas collected from dogs and cats in different areas of Italy. Two species of fleas were identified, Ctenocephalides felis (80.3%) and Ctenocephalides canis (19.7%).Overall, 320 fleas (257 C. felis and 63 C. canis) collected from 117 animals (73 dogs and 44 cats) were tested. Thirty-eight (11.9%) C. felis fleas, 13 from cats (17.6%) and 25 from dogs (10.2%) were positive for R. felis. No C. canis was positive. Fleas from cats showed a tendency to be more positive than fleas from dogs. Prevalence of R. felis among areas and within provinces of the same area was extremely variable, ranging from 0 to 35.3%. Overall, prevalence in north-eastern Italy (23.2%) was significantly higher than in south-western Italy (7.1%). This study confirmed the occurrence of R. felis in cat and dog fleas (C. felis) from Italy, similar to other European countries. The results also suggest that R. felis should be considered in the human differential diagnosis of any spotted-like fever in Italy, especially if the patient is known to have been exposed to flea bites.