Identification of Aichivirus in a Pet Rat (Rattus norvegicus) in Italy.

We investigated the occurrence of eight potential zoonotic viruses in 91 exotic companion mammals from pet shops in southern Italy via real-time PCR and end-point PCR. The animals were screened for aichivirus, sapovirus, astrovirus, hepatitis A, noroviruses (GI and GII), rotavirus, circovirus, and SARS-CoV-2. Among the nine species of exotic pets studied, only one rat tested positive for aichivirus. The high sequence similarity to a murine kobuvirus-1 strain previously identified in China suggests that the virus may have been introduced into Italy through the importation of animals from Asia. Since exotic companion mammals live in close contact with humans, continuous sanitary monitoring is crucial to prevent the spread of new pathogens among domestic animals and humans. Further investigations on detecting and typing zoonotic viruses are needed to identify emerging and re-emerging viruses to safeguard public health.

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.

Molecular Survey of Toxoplasma gondii in Wild Mammals of Southern Italy.

Systematic wildlife surveillance is important to aid the prevention of zoonotic infections that jeopardize human health and undermine biodiversity. Toxoplasma gondii is an opportunistic zoonotic protozoan that can infect all endothermic vertebrates, causing severe disease in immunocompromised humans and cases of congenital transmission. Humans can be infected by ingestion of raw meat containing bradyzoites or water contaminated by oocysts. In our study, we assessed the potential circulation of Toxoplasma gondii in wild mammals by performing surveillance in the Campania region (southern Italy) and surveyed its presence from 2020 to 2022 within the framework of the Regional Plans for Wildlife Surveillance. In detail, 211 individuals belonging to five wild mammals (wolf, fox, wild boar, badger, and roe deer) underwent necropsy and the organs were analyzed by real-time PCR for the detection of the parasite. Toxoplasma gondii was found in 21.8% (46/211) of the subjects examined. No statistically significant differences were noticed between the prevalence and the host's trophic level or age, rejecting the hypotheses that Toxoplasma gondii will have a higher prevalence in top predators and adult individuals, respectively. Our work emphasized the high circulation of Toxoplasma gondii in wildlife and remarked on the critical role of anthropized areas where domestic cats and wildlife may come into contact, urging a systematic surveillance.

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.

A correlation of Mycobacterium bovis SB0134 infection between cattle and a wild boar (Sus Scrofa) in Campania region.

A case of Mycobacterium bovis infection is described in a death adult female wild boar in the province of Avellino, Campania Region (Southern Italy). The carcass was sent to the Istituto Zooprofilattico Sperimentale del Mezzogiorno (IZSM) of Portici, Naples, Italy, where postmortem examination was performed. At necropsy, a disseminated granulomatous infection was observed, with involvement of various lymph node districts, spleen and lungs. Therefore, all lymph nodes were collected, together with spleen and lung lesions, in order to carry out bacteriological and molecular analyses that confirmed an uncommon disseminated Mycobacterium bovis infection. Subsequently, an analysis of the spoligotype, performed by the National Reference Center of Mycobacterium bovis in Brescia (Northern Italy), resulted in the spoligotype SB0134, previously identified in bovine outbreaks in the same area where the wild boar was found.

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.

First identification of porcine parvovirus 3 in a wild boar in Italy by viral metagenomics - Short communication.

Metagenomic analysis revealed the presence of porcine parvovirus 3 (PPV3) in the pool of the internal organs of a wild boar found dead in Southern Italy. Phylogenetic analysis based on the complete coding sequences showed that the newly detected virus is most closely related to those found also in wild boars in Romania during 2010-2011. Even though the death could not be associated with this virus, PPV3 could have contributed to lowering the host's immunological defences.

Coxiella burnetii in Infertile Dairy Cattle With Chronic Endometritis.

Coxiella burnetii is an obligate intracellular pathogen and the cause of Q fever in many animal species and humans. Several studies have reported the association between C. burnetii and abortion, premature delivery, stillbirth, and weak offspring. However, no solid evidence indicates that C. burnetii causes endometritis, subfertility, and retained fetal membranes. For this study, histopathological and PCR evaluation were performed on 40 uterine biopsies from dairy cattle with poor fertility. Uterine swabs were concurrently tested with microbiology assays. The endometrial biopsies of 30 cows did not have any significant lesions, and no pathogens were identified by aerobic bacterial culture and PCR. Ten cows were PCR-positive for C. burnetii and negative for other pathogens by aerobic bacterial culture and PCR. These 10 cases revealed a mild to severe chronic endometritis admixed with perivascular and periglandular fibrosis. Immunohistochemical evaluation of C. burnetii PCR-positive biopsies identified, for the first time, the presence of intralesional and intracytoplasmic C. burnetii in macrophages in the endometrium of cattle.

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.

Molecular survey of Ehrlichia canis and Coxiella burnetii infections in wild mammals of southern Italy.

Ehrlichiosis and Q fever caused by the intracellular bacteria Ehrlichia canis and Coxiella burnetii, respectively, are tick-borne diseases with zoonotic potential and widespread geographical distribution. This study investigated the prevalence of both infections in wild mammals in southern Italy. Tissue samples obtained from the red fox (Vulpes vulpes), European badger (Meles meles), gray wolf (Canis lupus), beech marten (Martes foina), and crested porcupine (Hystrix cristata) were processed for molecular detection of both pathogens. E. canis was detected in 55 out of 105 (52 %) red foxes and three out of six gray wolves. Four sequence types were identified, three of which were found in the spleen and liver samples of red foxes and wolves, and one in the kidney of a red fox. None of the examined mammals was positive to C. burnetii type. This represents the first report of E. canis in free-ranging wolves worldwide, as well as the first evidence of this pathogen in red foxes in the peninsular Italy. Our results suggest that E. canis infection is common in free-ranging canids in southern Italy and that a sylvatic life cycle of this pathogen may occur.

Identification of single nucleotide polymorphisms in Toll-like receptor candidate genes associated with tuberculosis infection in water buffalo (Bubalus bubalis).

BACKGROUND: Toll-like receptors play a key role in innate immunity by recognizing pathogens and activating appropriate responses. Pathogens express several signal molecules (pathogen-associated molecular patterns, PAMPs) essential for survival and pathogenicity. Recognition of PAMPs triggers an array of anti-microbial immune responses through the induction of various inflammatory cytokines. The objective of this work was to perform a case-control study to characterize the distribution of polymorphisms in three candidate genes (toll-like receptor 2, toll-like receptor 4, toll-like receptor 9) and to test their role as potential risk factors for tuberculosis infection in water buffalo (Bubalus bubalis). RESULTS: The case-control study included 184 subjects, 59 of which resulted positive to both intradermal TB test and Mycobacterium bovis isolation (cases) and 125 resulted negative to at least three consecutive intradermal TB tests. The statistical analysis indicated that two polymorphisms exhibited significant differences in allelic frequencies between cases and controls. Indeed, the TT genotype at TLR9 2340 C > T locus resulted significantly associated with susceptibility to bovine tuberculosis (P = 0.030, OR = 3.31, 95% CI = 1.05-10.40). One polymorphism resulted significantly associated with resistance to the disease, and included the CC genotype, at the TLR4 672 A > C locus (P = 0.01, OR = 0.26, 95% CI = 0.08-0.80). Haplotype reconstruction of the TLR2 gene revealed one haplotype (CTTACCAGCGGCCAGTCCC) associated with disease resistance (P = 0.04, OR = 0.51, 95% CI = 0.27-0.96), including the allelic variant associated with disease resistance. CONCLUSIONS: The work describes novel mutations in bubaline TLR2, TLR4 and TLR9 genes and presents their association with M. bovis infection. These results will enhance our ability to determine the risk of developing the disease by improving the knowledge of the immune mechanisms involved in host response to mycobacterial infection, and will allow the creation of multiple layers of disease resistance in herds by selective breeding.

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.