First molecular detection of Babesia vulpes and Babesia capreoli in wild boars from southern Italy.

Introduction: Following the increase of wild boar (Sus scrofa) populations in Europe, a potential risk of emerging infections by vector-borne pathogens may occur. Despite this, the circulation of piroplasmid species in these ungulates is still a neglected topic, particularly in the Mediterranean basin. Therefore, this study aimed to investigate the presence of Babesia/Theileria spp. in wild boars from southern Italy to assess the epidemiological role of these ungulates in the circulation of piroplasmids. Methods: By using a citizen science approach among hunters and veterinarians, wild boar spleen samples were collected in the Campania region (southern Italy) between 2016 and 2022. A combined semi-nested PCR/sequencing analysis targeting the V4 hyper-variable region of 18S rRNA was run to detect Babesia/Theileria spp. DNA. Results: Out of 243 boars, 15 (i.e., 6.2, 95% CI: 3.4-9.9) tested positive to Babesia/Theileria spp., Babesia vulpes (n = 13, 5.3, 95% CI: 3.1-8.9) the most prevalent, followed by Babesia capreoli (n = 2, 0.8, 95% CI: 0.2-2.9). Three different B. vulpes sequence types were identified (i.e., ST1, ST2, ST3), with the most representative as ST1 (60%), and a single B. capreoli sequence type. No statistically significant difference (p > 0.05) were found between the presence of the pathogens and boar age, sex, province and sample collection year. Discussion: Data demonstrate for the first time the occurrence of B. vulpes and B. capreoli in wild boars, which may play a role in the biological cycle of piroplasmids. We emphasize the importance of monitoring these ungulates to prevent potential foci of infection. The engagement of hunters in epidemiological scientifically based surveys can constitute a technically sound control strategy of piroplasmids in a One Health perspective.

Canine Distemper Virus in Autochtonous and Imported Dogs, Southern Italy (2014-2021).

This study aims to investigate the presence of canine distemper virus (CDV) infection in 949 autochthonous or illegally imported dogs from Southern Italy, over a period of eight years (2014-2021). CDV RNA was detected in 6.8% (65/949) of the animals tested, with no detection of CDV in dogs sampled in 2020-2021. The frequency of CDV detection was higher in imported dogs (19/103, 18.3%) with respect to stray (27/365, 7.4%) and household dogs (19/481, 3.9%). On sequence and phylogenetic analyses of selected strains, the analyzed viruses belonged to the Arctic clade, which has already been reported in Italy and in Europe. The results of our study may suggest a reduction of CDV circulation in Southern Italy, while at the same time highlighting the need for strict controls on dog importation, in order to prevent the introduction of viruses from endemic countries.

Parasite Load and STRs Genotyping of Toxoplasma gondii Isolates From Mediterranean Mussels (Mytilus galloprovincialis) in Southern Italy.

Toxoplasmosis is a zoonotic food-borne disease caused by Toxoplasma gondii, a land-derived protozoan parasite that infects a broad range of terrestrial and aquatic hosts. T. gondii may reach coastal waters via contaminated freshwater runoff and its oocysts may enter into the marine food web. Marine invertebrates as mussels being filter feeders are exposed and may concentrate T. gondii oocysts representing a potential source of infection for animals and humans. The present works investigated the prevalence, parasite burden and genotypes of T. gondii in the Mediterranean mussels (Mytilus galloprovincialis) from southern Italy. We sampled a total of 382 individual Mediterranean mussels from May to August 2018 from seven production sites in the Gulf of Naples (Campania region). An additional sample including 27 farmed Mediterranean mussels was obtained in February 2018 from a mollusk depuration plant in Corigliano Calabro (Calabria region). T. gondii DNA was detected in 43 out of 409 (10.5%) Mediterranean mussels from seven out of eight sampling sites. The number of T. gondii copies/g in the digestive gland ranged from 0.14 to 1.18. Fragment analysis of Short Tandem Repeats (STRs) at 5 microsatellite loci was performed from 10 T. gondii PCR positive samples revealing the presence of five distinct genotypes including one corresponding to type I and four atypical genotypes. These findings suggest potential implications of epidemiological importance for human and animal health because both type I and atypical genotypes could be highly pathogenic.

Molecular Detection of Babesia spp. (Apicomplexa: Piroplasma) in Free-Ranging Canids and Mustelids From Southern Italy.

Babesiosis is an emerging tick-borne disease caused by apicomplexan parasites with widespread geographical distribution and various wildlife species as reservoir hosts. The aims of this study were to investigate the prevalence and assess the role of free-ranging canids and mustelids in the maintenance of Babesia spp. in southern Italy. PCR analysis of splenic samples targeting the 18S rRNA gene revealed the presence of Babesia spp. in 36 of 82 (43.9%) red foxes (Vulpes vulpes) including 29 (58%) from Campania region and seven (21.8%) from Calabria region, in seven of 13 (53.8%) Eurasian badgers (Meles meles), and in one of 13 (7.7%) gray wolves (Canis lupus). Samples from other host species including 9 Eurasian otters (Lutra lutra), 1 stone marten (Martes foina), 1 least weasel (Mustela nivalis), and 1 European polecat (Mustela putorius) tested Babesia spp. negative. Sequence analysis of the 18S rRNA gene demonstrated the presence of B. vulpes in the red fox and two sequence types of badger-associated Babesia spp. in the Eurasian badger. The Babesia sp. sequence detected in the gray wolf was identical to a badger-associated Babesia sp. This study shows that the number of Babesia spp. infecting free-ranging carnivores in Italy is higher than currently believed, and suggests that these hosts may play an important role in the maintenance of the sylvatic cycle of these parasites. It is the first report of badger-associated Babesia spp. in Italy and in a gray wolf.

The Eurasian otter (Lutra lutra) as a potential host for rickettsial pathogens in southern Italy.

Canine monocytic ehrlichiosis and rickettsiosis are zoonotic tick-borne diseases of canids caused by the intracellular obligate bacteria Ehrlichia canis and Rickettsia species respectively. In this study, we investigated using standard and real-time PCR and sequencing, the occurrence and molecular characterization of E. canis and Rickettsia species in the Eurasian otter (Lutra lutra) from the southern Italian population. Samples were screened by using molecular assays also for Neospora caninum, Toxoplasma gondii, Clamydophyla spp., Coxiella burnetii, Leishmania spp., Cryptosporidium spp., and Giardia spp. detection, and helminths were studied by traditional methods. Out of six carcasses tested, three were positive for E. canis and co-infection with Rickettsia sp. occurred in one of those. Sequences of the 16S rRNA E. canis gene were identical to each other but differed from most of those previously found in red foxes (Vulpes vulpes) and wolves (Canis lupus) from southern Italy. Helminths included just cystacanths of Sphaerirostris spp. from the intestine of two Eurasian otters and the nematode Angiostrongylus vasorum from the lungs of a single Eurasian otter. None of the samples was positive for the other investigated selected pathogens. This study is the first report on the evidence of infection by rickettsial pathogens in the Eurasian otter. The present result prompts some inquiries into the pathogenic role of those bacteria for the isolated sub-populations of the endangered Eurasian otter in southern Italy.

PCR detection of Neospora caninum in water buffalo foetal tissues.

The seroprevalence of Neospora caninum was surveyed by an ELISA kit on two water buffalo herds of Southern Italy. Seropositive samples were detected in 47% and 59% of individuals, respectively, thus indicating high level of exposure to the parasite even if the possibility of vertical transmission cannot be excluded. Tissue samples collected from three aborted fetuses from the same herds were investigated for N. caninum presence by PCR assays targeting the 18S and the Nc5 DNA sequences, respectively. Both methods have shown the presence of N. caninum DNA in heart and brain. Sequencing of the Nc5 genomic DNA confirmed the presence of N. caninum in the samples; phylogenetic analysis of the obtained sequences showed high homology among the Neospora recovered from different samples. The present study suggests an important role of N. caninum as a possible abortive agent for water buffaloes.

Integrin receptors play a role in the internalin B-dependent entry of Listeria monocytogenes into host cells.

Listeria monocytogenes enters non-phagocytic cells by binding its surface proteins inlA (internalin) and inlB to the host's E-cadherin and Met, respectively. The two internalins play either separate or cooperative roles in the colonization of infected tissues. Here, we studied bacterial uptake into HeLa cells using an L. monocytogenes mutant strain (DeltainlA) carrying a deletion in the gene coding for inlA. The DeltainlA mutant strain showed the capability to invade HeLa cells. The monoclonal anti-beta(3)- and anti-beta(1)-integrin subunit antibodies prevented bacterial uptake into the cells, while the anti-beta(2)- and anti-beta(4)-integrin subunit antibodies failed to affect L. monocytogenes entry into HeLa cells. Three structurally distinct disintegrins (kistrin, echistatin and flavoridin) also inhibited bacterial uptake, showing different potencies correlated to their selective affinity for the beta(3)- and beta(1)-integrin subunits. In addition to inducing Met phosphorylation, infection of cells by the L. monocytogenes DeltainlA mutant strain promoted the tyrosine phosphorylation of the focal adhesion-associated proteins FAK and paxillin. Our findings provide the first evidence that beta(3)- and beta(1)-integrin receptors play a role in the inlB-dependent internalization of L. monocytogenes into host cells.

Isolation and characterisation of two novel coliphages with high potential to control antibiotic-resistant pathogenic Escherichia coli (EHEC and EPEC).

Two newly isolated virulent coliphages (MVBS and MVSS) showed ability to lyse (form plaque on) a high percentage of pathogenic Escherichia coli strains of various serotypes and origins (292/310; 94.2%), whilst displaying low lytic (plaque-forming) capacity on non-pathogenic ECOR strains (MVBS and MVSS lysed 10/72 (13.9%) and 15/72 (20.8%) strains, respectively). In comparison, a higher percentage (196/310, 63.2%) of tested isolates exhibited resistance to a broad range of antibiotics. It was also observed that, in contrast to antibiotics, phage treatment did not induce Shiga toxin production. These findings suggest that the newly isolated bacteriophages have potential for biocontrol and as therapeutic agents for pathogenic E. coli (EHEC and EPEC) strains.