Genomic and epidemiological evidence for the emergence of a L. infantum/L. donovani hybrid with unusual epidemiology in northern Italy.

Leishmania (L.) infantum is one of the main causative agents of animal and human leishmaniasis across many endemic areas in South America, Europe, North Africa, and Asia. Despite its clinical significance, little is known about the genetic diversity of L. infantum circulating in a given endemic area. Here, we investigate this important open question by applying a comparative genomics approach to seven L. infantum isolates from different hosts and Italian regions, including the northern part of the country (Emilia-Romagna, RER), Sicily, and Sardinia, as an initial attempt to explore the breadth of parasite genetic heterogeneity in Italy. Additionally, microsatellite analysis was carried out to compare the isolates from RER with other 70 L. infantum strains from the same region as well as 65 strains belonging to the L. donovani complex from other countries. We revealed important karyotypic instability and identified strain-specific changes in gene dosage, which affected important virulence factors such as amastins and surface antigen-like proteins. Single nucleotide polymorphism-based clustering analysis of these genomes together with over 80 publicly available L. infantum and L. donovani genomes placed the Italian isolates into three geographically distinct clusters within the Mediterranean basin and uncovered three isolates clustering with putative L. infantum/L. donovani hybrids isolated in Cyprus. As judged by microsatellite profiling, these hybrid isolates are representative of a sub-population of parasites circulating in northern Italy that preferentially infect humans but not dogs. Our results place Italy at the crossroads of L. infantum infection in the Mediterranean and call attention to the public health risk represented by the introduction of non-European Leishmania species.IMPORTANCEThis study closes important knowledge gaps with respect to Leishmania (L.) infantum genetic heterogeneity in a given endemic country, as exemplified here for Italy, and reveals genetic hybridization as a main cause for re-emerging human leishmaniasis in northern Italy. The observed high diversity of Leishmania parasites on the Italian peninsula suggests different geographical origins, with genomic adaptation to various ecologies affecting both pathogenicity and transmission potential. This is documented by the discovery of a putative L. infantum/L. donovani hybrid strain, which has been shown to preferentially infect humans but not dogs. Our results provide important information to health authorities, which need to consider the public health risk represented by the introduction of new Leishmania species into EU countries due to population displacement or travel from countries where exotic/allochthonous parasite species are endemic.

Clinical and Microbiological Characteristics of Visceral Leishmaniasis Outbreak in a Northern Italian Nonendemic Area: A Retrospective Observational Study.

Background. Visceral leishmaniasis (VL) caused by Leishmania infantum is endemic in the Mediterranean area. In the last decades a northward spread of the parasite has been observed in Italy. This paper describes a VL outbreak in Modena province (Emilia-Romagna, Northern Italy) between 2012 and 2015. Methods. Retrospective, observational study to evaluate epidemiological, microbiological characteristics, and clinical management of VL in patients referring to Policlinico Modena Hospital. Results. Sixteen cases of VL occurred in the study period. An immunosuppressive condition was present in 81.3%. Clinical presentation included anemia, fever, leukopenia, thrombocytopenia, and hepatosplenomegaly. Serology was positive in 73.3% of cases, peripheral blood PCR in 92.3%, and bone marrow blood PCR in 100%. Culture was positive in 3/6 cases (50%) and all the isolates were identified as L. infantum by ITS1/ITS2 sequencing. The median time between symptom onset and diagnosis was 22 days (range 6-131 days). All patients were treated with liposomal amphotericin b. 18.8% had a VL recurrence and were treated with miltefosine. Attributable mortality was 6.3%. Conclusions. VL due to L. infantum could determine periodical outbreaks, as the one described; thus it is important to include VL in the differential diagnosis of fever of unknown origin, even in low-endemic areas.

Sequence types and pleuromutilin susceptibility of Brachyspira hyodysenteriae isolates from Italian pigs with swine dysentery: 2003-2012.

Swine dysentery is a mucohaemorrhagic colitis of pigs caused by infection with Brachyspira hyodysenteriae. The disease can be controlled by treatment with antimicrobial agents, with the pleuromutilins tiamulin and valnemulin being widely used. In recent years, the occurrence of B. hyodysenteriae with reduced susceptibility to these drugs has been increasing. The aim of this study was to determine temporal changes in genetic groups and pleuromutilin susceptibility amongst B. hyodysenteriae isolates from Italy. Multilocus sequence typing (MLST) was performed on 108 isolates recovered from 87 farms in different regions of Italy from 2003 to 2012, and their minimum inhibitory concentrations (MICs) for tiamulin and valnemulin were determined. Logistic regression was performed to assess associations between susceptibility to the two antimicrobial agents and genetic group, year and region of isolation. The isolates were allocated to 23 sequence types (STs), with five clonal clusters (Ccs) and seven singletons. More than 50% of isolates were resistant to both pleuromutilins (MIC >2.0 µg/mL for tiamulin and >1.0 µg/mL for valnemulin). All 10 isolates in ST 83 were resistant; these were first isolated in 2011 and came from nine farms, suggesting recent widespread dissemination of a resistant strain. Significant associations were found between the proportion of pleuromutilin susceptible isolates and the genetic group and year of isolation. Although resistant isolates were found in all Ccs, isolates in Ccs 2 and 7 were over five times more likely to be susceptible than those in the other Ccs. A significant trend in the reduction of susceptibility over time also was observed.

Environmental methicillin-resistant Staphylococcus aureus contamination in pig herds in relation to the productive phase and application of cleaning and disinfection.

The aim of the study is to better understand MRSA environmental contamination in pig holdings in relation to the productive phase and the application of environmental cleaning and disinfection practices (C&D). This study was carried out on six herds. Dust samples from farrowing crates, weaning boxes, growing and finishing pens were collected from each herd environment prior to and after C&D and cultured for MRSA. Ten samples were collected at the end of each productive phase with the animals present and 10 additional samples were collected after C&D, prior to introduction of a new stock. The proportion of positive samples prior to C&D was lower in fattening than in other phases. The proportion of positive samples prior to and after C&D was 121/240 (50%) and 46/240 (19%) respectively. In the mixed effects logistic regression analysis it was shown not only that both productive phase and C&D were associated with the probability of having a positive sample but also that the C&D effect was different in the different productive phases. In particular the effect of C&D was stronger in farrowing crates than in the other productive phases. The results of this study show that, although current practices of cleaning and disinfection reduce MRSA environmental contamination, they are likely to be inadequate to the elimination of the microorganism. However, a strict application of hygienic protocols can lead to a marked reduction of MRSA environmental contamination.

West Nile virus circulation in Emilia-Romagna, Italy: the integrated surveillance system 2009.

Following a large West Nile virus (WNV) epidemic in northeastern Italy in 2008, human and animal surveillance activities were implemented in Emilia Romagna. Human surveillance was performed by serology or genome detection on blood and cerebrospinal fluid for all suspected cases suffering from acute meningoencephalitis in the regional territory. Animal surveillance consisted of passive and active surveillance of horses and active surveillance of wild birds and mosquitoes. Between 15 June and 31 October 2009, nine of 78 possible cases of West Nile neuroinvasive disease were confirmed (three fatal). From May to October, 26 cases of neurological West Nile disease were confirmed among 46 horses. The overall incidence of seroconversion among horses in 2009 was 13%. In 2009, 44 of 1,218 wild birds yielded positive PCR results for WNV infection. The planned veterinary and entomological surveillance actions detected WNV activity from the end of July 2009, about 2-3 weeks before the onset of the first human neurological case. Passive surveillance of horses seems to be an early and suitable tool for the detection of WNV activity, but it will be less sensitive in the future, because an intensive programme of horse vaccination started in June 2009.

Survey on helminthofauna in pheasants from Eastern Europe.

In order to assess the parasitological status in imported pheasants, 51 birds (Phasianus colchicus) coming from Poland and Rumania and used in Italy in repopulation interventions for hunting purposes were examined. From each animal the trachea, oesophagus, crop and intestine were collected and examined for the presence of nematodes. The examination of the oesophagi and crops of 5 birds revealed the presence of parasites pertaining to the family Capillariidae: Eucoleus contortus was found in all of the 5 animals, E. annulatus was present along with the previous parasite in one animal coming from Poland. The examination of the tracheae revealed the presence of Syngamus trachea in 5 animals (9.80%). Adult or larval stages of Heterakis gallinarum (37.25% of birds) and Capillariidae (35.29%) were found in the intestinal tracts. Aonchotheca caudinflata was detected only in one bird coming from Poland; the capillarids found in all of the remaining pheasants exhibited morphological characteristics referable to Capillaria phasianina, a species never reported in Italy. The release of game from foreign countries, therefore, may always constitute a risk for the autochthonous one due to the spread of new parasitic infections.