Spoligotyping and exact tandem repeat (ETR) analysis ofAs a result, the official TB-free status was achieved starting from 1970 in some Italian areas such as the Autonomous Region of Trentino Alto Adige, Friuli Venezia Giulia Region (northeast Italy), and some provinces of northern and central Italy. In the rest of the national territory, TB still persists with different prevalence rates: 6.6% in Sicilia, 0.77% in Puglia, and 0.59% in Lazio and Campania. In northern Italy, where about the 70% of the cattle population is reared, TB is still present in the Piedmont (0.4%), Lombardy (0.15%), Veneto (0.1%), and Emilia-Romagna (0.08%) regions. In order to accelerate the achievement of a TB-free status, the authorities in these areas are implementing specific TB control measures, taking into account local situations and problems such as mean distance among herds, trade of animals from other territories, and common pasturing.In this respect, the origin of TB infection often remains undetermined despite its importance. Molecular typing of isolates has become a valuable tool in the study of M. bovis epidemiology, allowing investigators to better identify the sources of infection and achieve a wider knowledge of TB transmission routes. A genetic profile database collection of M. bovis isolates may help to confirm or reject hypotheses outlined by traditional epidemiological investigations.Genotyping of M. bovis probably lacks sufficiently informative methods. IS6110 restriction fragment length polymorphism (RFLP) typing has been considered a gold standard method for differentiation of M. tuberculosis strains for a long time; this has provided only limited discrimination among M. bovis populations where the majority of the isolates harbor only one or few IS copies (11). PCR-based spoligotyping (16) has been widely used to genotype M. bovis isolates (11); it is highly reproducible and rapid and represents the first universally recognized typing system for M. bovis populations. However, studies performed on M. bovis isolates in Northern Ireland (32,33), France (12), Australia, Canada, the Republic of Ireland, and Iran (7) showed a limited discrimination power of this method.Over the last 10 years several PCR-based genotyping methods have become available for rapid molecular epidemiology

BackgroundBat-borne virus surveillance is necessary for determining inter-species transmission risks and is important due to the wide-range of bat species which may harbour potential pathogens. This study aimed to monitor coronaviruses (CoVs) and paramyxoviruses (PMVs) in bats roosting in northwest Italian regions. Our investigation was focused on CoVs and PMVs due to their proven ability to switch host and their zoonotic potential. Here we provide the phylogenetic characterization of the highly conserved polymerase gene fragments.ResultsFamily-wide PCR screenings were used to test 302 bats belonging to 19 different bat species. Thirty-eight animals from 12 locations were confirmed as PCR positive, with an overall detection rate of 12.6% [95% CI: 9.3–16.8]. CoV RNA was found in 36 bats belonging to eight species, while PMV RNA in three Pipistrellus spp. Phylogenetic characterization have been obtained for 15 alpha- CoVs, 5 beta-CoVs and three PMVs; moreover one P. pipistrellus resulted co-infected with both CoV and PMV. A divergent alpha-CoV clade from Myotis nattereri SpA is also described. The compact cluster of beta-CoVs from R. ferrumequinum roosts expands the current viral sequence database, specifically for this species in Europe. To our knowledge this is the first report of CoVs in Plecotus auritus and M. oxygnathus, and of PMVs in P. kuhlii.ConclusionsThis study identified alpha and beta-CoVs in new bat species and in previously unsurveyed Italian regions. To our knowledge this represents the first and unique report of PMVs in Italy. The 23 new bat genetic sequences presented will expand the current molecular bat-borne virus databases. Considering the amount of novel bat-borne PMVs associated with the emergence of zoonotic infections in animals and humans in the last years, the definition of viral diversity within European bat species is needed. Performing surveillance studies within a specific geographic area can provide awareness of viral burden where bats roost in close proximity to spillover hosts, and form the basis for the appropriate control measures against potential threats for public health and optimal management of bats and their habitats.

In this rapid communication, a novel atypical bluetongue virus (BTV) strain detected in goats in the Piedmont region (north-western Italy) is described. This strain, BTV-Z ITA2017, is most related in Seg-2/VP-2 (83.8% nt/82.7% aa) to strain TOV of BTV-25. Reactive antisera of goats positive by cELISA for BTV antibodies failed to neutralize a chimeric virus expressing the outermost protein of TOV. Infected animals displayed low levels of RNAemia and absence of clinical signs consistent with bluetongue infection, a scenario described in animals infected with atypical BTV strains.

Listeria monocytogenes, Toxoplasma gondii and Brucella spp. can infect a wide range of species, including humans. In cetaceans, meningoencephalitis has been associated with T. gondii and Brucella spp. infection, whereas to our knowledge, L. monocytogenes infection has not previously been reported. Meningoencephalitis and L. monocytogenes, T. gondii and Brucella spp. were identified by means of both direct and indirect laboratory techniques in an adult female striped dolphin Stenella coeruleoalba found stranded in January 2015 on the Ligurian Sea coast, northwestern Italy. The animal was emaciated, and histopathology disclosed severe meningoencephalitis. The nature of the inflammatory response and intra-lesional protozoa were consistent with a mixed infection by L. monocytogenes, T. gondii and Brucella spp. We believe this is an unprecedented case of infection by 3 zoonotic pathogens and also the first bacteriologically confirmed case report of neurolisteriosis in cetaceans. Cerebral toxoplasmosis and neurobrucellosis may have led to the animal's disorientation and stranding, with L. monocytogenes having likely exacerbated the coinfection leading to the demise of this dolphin. KEY WORDS: Meningoencephalitis · Toxoplasmosis · Listeriosis · Brucellosis · Stenella coeruleoalbaResale or republication not permitted without written consent of the publisher

Abstract. The current study describes the development of a set of 5 multiplex polymerase chain reaction (mPCR) assays for the simultaneous detection of abortive infection agents in bovine fetal tissues, including Brucella spp., Leptospira spp., and Campylobacter fetus (mPCR1); Hammondia heydorni, Neospora caninum, and Toxoplasma gondii (mPCR2); Coxiella burnetii and Chlamydophila psittaci (mPCR3); Mycoplasma bovis, Mycoplasma bovigenitalium, and Ureaplasma diversum (mPCR4); and Bovine viral diarrhea virus (BVDV) and Bovine herpesvirus-1 (BoHV-1; mPCR5). The protocol was tested on different tissue samples collected from 50 aborted bovine fetuses, and it showed that out of the 50 fetuses, 7 (14%, mPCR2) were PCR-positive for N. caninum, 4 (8%, mPCR5) were PCR-positive for BVDV, and 2 (4%, mPCR4) were PCR-positive for U. diversum. The results obtained by using each multiplex PCR were 100% concordant with those obtained by using the respective PCR assays targeting single genes on the same specimens. Moreover, all multiplex PCR assays on clinical samples were compared with reference methods, obtaining a perfect accordance in all samples and confirming the validity of the set of multiplex PCR assays. The proposed set of multiplex PCR assays is, therefore, suitable for the simultaneous detection of the main infectious agents responsible for bovine abortion.

Brucella ceti infections have been increasingly reported in cetaceans, although a very limited characterization of Mediterranean Brucella spp. isolates has been previously reported and relatively few data exist about brucellosis among cetaceans in Italy. To address this gap, we studied 8 cases of B. ceti infection in striped dolphins (Stenella coeruleoalba) stranded along the Italian coastline from 2012 to 2018, investigated thanks to the Italian surveillance activity on stranded cetaceans. We focused on cases of stranding in eastern and western Italian seas, occurred along the Apulia (N = 6), Liguria (N = 1) and Calabria (N = 1) coastlines, through the analysis of gross and microscopic findings, the results of microbiological, biomolecular and serological investigations, as well as the detection of other relevant pathogens. The comparative genomic analysis used whole genome sequences of B. ceti from Italy paired with the publicly available complete genomes. Pathological changes consistent with B. ceti infection were detected in the central nervous system of 7 animals, showing non-suppurative meningoencephalitis. In 4 cases severe coinfections were detected, mostly involving Dolphin Morbillivirus (DMV). The severity of B. ceti-associated lesions supports the role of this microbial agent as a primary neurotropic pathogen for striped dolphins. We classified the 8 isolates into the common sequence type 26 (ST-26). Whole genome SNP analysis showed that the strains from Italy clustered into two genetically distinct clades. The first clade comprised exclusively the isolates from Ionian and Adriatic Seas, while the second one included the strain from the Ligurian Sea and those from the Catalonian coast. Plotting these clades onto the geographic map suggests a link between their phylogeny and topographical distribution. These results represent the first extensive characterization of B. ceti isolated from Italian

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The wide geographical distribution and genetic diversity of bat-associated lyssaviruses (LYSVs) across Europe suggest that similar viruses may also be harboured in Italian insectivorous bats. Indeed, bats were first included within the passive national surveillance programme for rabies in wildlife in the 1980s, while active surveillance has been performed since 2008. The active surveillance strategies implemented allowed us to detect neutralizing antibodies directed towards European bat 1 lyssavirus in six out of the nine maternity colonies object of the study across the whole country. Seropositive bats were Myotis myotis, M. blythii and Tadarida teniotis. On the contrary, the virus was neither detected through passive nor active surveillance, suggesting that fatal neurological infection is rare also in seropositive colonies. Although the number of tested samples has steadily increased in recent years, submission turned out to be rather sporadic and did not include carcasses from bat species that account for the majority of LYSVs cases in Europe, such as Eptesicus serotinus, M. daubentonii, M. dasycneme and M. nattereri. A closer collaboration with bat handlers is therefore mandatory to improve passive surveillance and decrypt the significance of serological data obtained up to now.