Genome Sequence of a Leptospira montravelensis Strain Isolated from Donkey Urine during a Leptospirosis Outbreak in Sardinia Island, Italy.

Leptospirosis is a neglected zoonosis caused by pathogenic spirochetes classified within the genus Leptospira. Among saprophytic species, Leptospira montravelensis was previously reported only from environmental samples. Here, we report the whole-genome sequence and annotation of a strain of Leptospira montravelensis that was isolated from donkey urine during a leptospirosis outbreak in Sardinia, Italy.

Co-Infection by Leptospira montravelensis and Leptospira interrogans Serovar Pomona in Urine Samples of Donkeys and Pigs in Sardinia, Italy.

Saprophytic leptospires are spirochetes enclosed within the non-pathogenic clade of the genus Leptospira, which in turn is subdivided into two subclades S1 and S2. To date, the microorganisms included in these subclades have been isolated from the environment in various parts of the world, and are believed to have no known animal reservoirs. After a case of Leptospira interrogans serovar Pomona was notified to the owner of a farm in Sardinia, all of the farm animals (11 pigs and 3 donkeys) were examined for the presence of Leptospira. Sera of all tested animals resulted positive for antibodies to Leptospira using a microscopic agglutination test (MAT). Moreover, nine (82%) kidney samples from pigs and three urine samples collected from donkeys (100%) tested positive for Leptospira DNA after qPCR. Results obtained after MLST analysis and sequencing of rrs, rpoB, and secY genes, performed on six Leptospira strains isolated in culture, revealed the presence of the genomospecies L. interrogans serovar Pomona in the kidney samples. Conversely, whole-genome sequencing combined with mean nucleotide identity revealed the presence of the saprophytic L. montravelensis in the urine samples. Our results report, for the first time, the isolation of a saprophytic species from mammalian urine, suggesting a new ecological specialization for these bacteria, with a possible transition from free-living to a symbiotic lifestyle. Further studies will have to be conducted to understand the evolution of virulence of these bacteria, potential infectivity, and possible public health implications.

Molecular Survey of Hepatozoon canis Infection in Domestic Dogs from Sardinia, Italy.

Tick-borne protozoans of the genus Hepatozoon have been associated with infections of domestic and wild animals over the world. The occurrence of these apicomplexan agents in Sardinia has been poorly explored so far. In this study, the occurrence of Hepatozoon spp. has been investigated in domestic dogs from nine cities of Sardinia, Italy. Blood samples from each dog were collected and tested molecularly for the presence of Hepatozoon and Babesia/Theileria DNAs. Out of fifty-one dogs, nine were positive for Hepatozoon species based on the molecular detection of the parasite in blood samples. The phylogenetic relationships of strains detected here were also established. The PCR for amplification of the 18S rRNA fragment gene of Babesia/Theileria spp. did not give amplicons in any of the analyzed samples. Our results report the first molecular confirmation of Hepatozoon canis in Sardinian pet dogs and contribute to better understand the presence of these protozoans on the island. This study highlights the importance of recognizing and predicting the risk levels for the canine population, thus increasing the development of specific control measures. Also, since the distribution of hepatozoonosis is closely related to that of the definitive tick host, Rhipicephalus sanguineus, more accurate studies on Rhipicephalus ticks will be needed due to increasing the epidemiological knowledge of Hepatozoon species on the island.

Origin, Genetic Variation and Molecular Epidemiology of SARS-CoV-2 Strains Circulating in Sardinia (Italy) during the First and Second COVID-19 Epidemic Waves.

Understanding how geography and human mobility shape the patterns and spread of infectious diseases such as COVID-19 is key to control future epidemics. An interesting example is provided by the second wave of the COVID-19 epidemic in Europe, which was facilitated by the intense movement of tourists around the Mediterranean coast in summer 2020. The Italian island of Sardinia is a major tourist destination and is widely believed to be the origin of the second Italian wave. In this study, we characterize the genetic variation among SARS-CoV-2 strains circulating in northern Sardinia during the first and second Italian waves using both Illumina and Oxford Nanopore Technologies Next Generation Sequencing methods. Most viruses were placed into a single clade, implying that despite substantial virus inflow, most outbreaks did not spread widely. The second epidemic wave on the island was actually driven by local transmission of a single B.1.177 subclade. Phylogeographic analyses further suggest that those viral strains circulating on the island were not a relevant source for the second epidemic wave in Italy. This result, however, does not rule out the possibility of intense mixing and transmission of the virus among tourists as a major contributor to the second Italian wave.

Isolation of Leptospira interrogans Serovar Canicola in a Vaccinated Dog without Clinical Symptoms.

More than one million cases of leptospirosis occur across the globe annually, resulting in about 59,000 deaths. Dogs are one of the most important reservoirs of Leptospira species and play an important role in transmitting the pathogen to humans. Many of these infections are controlled by routine vaccination that has reduced the possible reintroduction of leptospiral serovars into the human population. However, it is still not clear how a vaccinated dog can become infected with one or more Leptospira serovars contained in the vaccine formulation and thus against which it should be immunized. Here, we present the case of an asymptomatic dog who developed leptospiral infection despite being vaccinated. This unusual case emphasizes the substantial impact of immunization on mitigating the acute signs of the disease, even while providing limited protection against infection. Further studies will be required to better understand the role of dogs in the environmental circulation of leptospiral serovars in Sardinia. Asymptomatic leptospiral infection in vaccinated dogs should be considered to allow for better diagnosis and management of the infection. This will be essential for preventing Leptospira outbreaks in the future.

First Isolation and Molecular Typing of Pathogenic and Intermediate Leptospira Species from Urine of Symptomatic Dogs.

Aim of this study was to evaluate, the presence and diversity of Leptospira spp. in blood and urine samples collected from 175 owned-dogs from Sardinia, Italy. After determination of leptospiral infection by microscopic agglutination test (MAT), urine from MAT-positive dogs were examined by real-time polymerase chain reaction (lipL32 rt-PCR) and then isolated by culture. In order to characterize obtained serovars, positive cultures were then subjected to 16S rRNA and secY sequencing, phylogenetic analysis and Multilocus Sequence Typing (MLST). Results showed that seven dogs (4%; 95% CI: 0-55) had Leptospira DNAs in their urine and five strains were isolated from urine cultures. The three different sequence types (ST17, ST198 and ST24) belonging to Leptospira interrogans genomospecies identified by MLST analyses in this study, confirmed that the leptospiral infection was widespread in Sardinian dogs. We also reported the first characterization of a new Leptospira spp. isolated from urine of one dog living in the study area. Whole genome sequencing and phylogenetic analysis, confirmed that this genospecies was closely related to Leptospira hovindhougenii, an intermediate Leptospira spp. with unknown pathogenicity previously isolated from a rat in Denmark. Further studies are required to clarify whether healthy dogs that shed leptospires in their urine could represent a zoonotic risk for humans in this region.

Draft Genome Sequences of Four Different Strains Belonging to Leptospira interrogans Serovar Pomona Isolated from Mammals in the Island of Sardinia, Italy.

Leptospirosis is a zoonotic disease of global importance caused by a pathogenic group of bacteria belonging to the genus Leptospira. Here, we report four draft genome sequences of Leptospira interrogans serovar Pomona isolated in Sardinia (Italy) from four different species of mammals (i.e., dolphin, wild boar, bovine, and fox).

New Insights on Leptospira Infections in a Canine Population from North Sardinia, Italy: A Sero-Epidemiological Study.

Leptospirosis is a widespread zoonosis recognized as a re-emerging infectious disease in a wide variety of animal species, including humans and dogs. No data exist regarding the presence of Leptospira species in the canine population of Sardinia Island. This study reports the first sero-survey for leptospirosis in kennel and owned dogs from six areas of the north of Sardinia. Sera from 1296 dogs were tested by microscopic agglutination test (MAT) specific for nine different serovars that are known to be well widespread in the Mediterranean environment. Moreover, kidney homogenates from rodents collected from the study area were also analyzed by LipL32 real-time PCR and multi-locus sequence type (MLST) on the basis of the analysis of seven concatenated loci. A total of 13% of the examined sera (95%CI: 11-15) tested positive for one or more serovars of Leptospira MAT detected; antibodies for serogroup Icterohaemorrhagiae (57%; 95%CI: 49-65) were the most common, followed by serovars Bratislava (22%; 95%CI: 16-28), Canicola (14%; 95%CI: 9-19), and Grippotyphosa (7%; 95%CI: 3-11). MLST analyses on isolates from rodents identified L. interrogans and L. borgpetersenii genomospecies. Different serovars belonging to pathogenic Leptospira serogroups are circulating in dogs from the island. Moreover, data obtained from rodents, indicated that rodents likely act as reservoir of spirochetes. Further sero-epidemiological studies are needed in order to obtain data from other collection sites in Sardinia and to increase the information on Leptospira species circulating in this area.

Molecular Typing of Pathogenic Leptospira Species Isolated from Wild Mammal Reservoirs in Sardinia.

Leptospirosis is a global zoonosis caused by pathogenic species of Leptospira that infect a large spectrum of domestic and wild animals. This study is the first molecular identification, characterization, and phylogeny of Leptospira strains with veterinary and zoonotic impact in Sardinian wild hosts. All samples collected were cultured and analyzed by multiplex real time polymerase chain reaction (qPCR). Sequencing, phylogenetic analyses (based on rrs and secY sequences), and Multilocus Sequence Typing (MLST) based on the analysis of seven concatenated loci were also performed. Results revealed the detection of Leptospira DNA and cultured isolates in 21% and 4% of the samples examined, respectively. Sequence analysis of Leptospira positive samples highlighted the presence of the interrogans and borgpetersenii genospecies that grouped in strongly supported monophyletic clades. MLST analyses identified six different Sequence Types (ST) that clustered in two monophyletic groups specific for Leptospirainterrogans, and L. borgpetersenii. This study provided about the prevalence of leptospires in wild mammals in Sardinia, and increased our knowledge of this pathogen on the island. Monitoring Leptospira strains circulating in Sardinia will help clinicians and veterinarians develop strategic plans for the prevention and control of leptospiral infections.

Presence of pathogenic Leptospira spp. in the reproductive system and fetuses of wild boars (Sus scrofa) in Italy.

Leptospirosis is a re-emerging and globally spread zoonosis caused by pathogenic genomospecies of Leptospira. Wild boar (Sus scrofa) are an important Leptospira host and are increasing in population all over Europe. The aim of this investigation was to evaluate Leptospira spp. infection in the reproductive systems of wild boar hunted in two Italian regions: Tuscany and Sardinia. From 231 animals, reproductive system tissue samples (testicles, epididymides, uteri) as well as placentas and fetuses were collected. Bacteriological examination and Real-Time PCR were performed to detect pathogenic Leptospira (lipL32 gene). Leptospires were isolated from the testicles and epididymides of one adult and two subadult wild boar. Four isolates from the two subadult males were identified as Leptospira interrogans serogroup Australis by MLST, whereas Leptospira kirschneri serogroup Grippotyphosa was identified from the adult testicles and epididymis. Using Real-Time PCR, 70 samples were positive: 22 testicles (23.16%) and 22 epididymides (23.16%), 10 uteri (7.35%), 3 placentas (6.66%), and 13 fetuses (28.88%). Amplification of the rrs2 gene identified L. interrogans and L. kirschneri species. The results from this investigation confirmed that wild boar represent a potential source of pathogenic Leptospira spp. Isolation of Leptospira serogroups Australis and Grippotyphosa from the male reproductive system and the positive Real-Time PCR results from both male and female samples could suggest venereal transmission, as already demonstrated in pigs. Furthermore, placentas and fetuses were positive for the lipL32 target, and this finding may be related to a possible vertical transmission of pathogenic Leptospira.

Isolation of Leptospira interrogans from a Bottlenose Dolphin (Tursiops truncatus) in the Mediterranean Sea.

The pathogenic Leptospira species are very widespread in nature, persisting in the renal tubules of many domestic and wild animal reservoirs. We report the isolation of Leptospira interrogans serovar Pomona in a bottlenose dolphin (Tursiops truncatus) stranded along the coast of Sardinia, Italy, in 2016.

Parallel synthesis of trisubstituted formamidines: a facile and versatile procedure.

A focused library of formamidines having several diversity points has been prepared. The use of isocyanate resin as scavenger may offer several practical advantages over conventional procedures, such as the ease of workup and availability. The technique described herein was performed in a parallel synthesis format. The whole protocol has potential use in high throughput synthesis for the preparation and purification of libraries of unsymmetrical formamidines containing sets of different aliphatic substituents.