Serological Survey and Molecular Typing Reveal New Leptospira Serogroup Pomona Strains among Pigs of Northern Italy

Abstract

Swine act as both maintenance and incidental hosts of pathogenic Leptospira spp. Here, a serological test was performed on 131,660 pig sera collected between 2002 and 2017 from 4715 farms in Northern Italy. A positivity rate of 13.05% was determined. Australis was the most frequently identified serogroup (77.29%), followed by Pomona (18.47%), Tarassovi (1.51%) and Icterohaemorrhagie (1.40%). Culture isolation and real-time Polymerase chain reaction (PCR) were carried out on 347 kidneys and 470 clinical samples, respectively. Overall, 133 strains were cultured successfully and 43 randomly chosen isolates were identified as serogroup Pomona. Multi-locus sequence typing (MLST) revealed that 41 isolates and 8 DNA extracted from biological samples belonged to sequence type 140. Using a multiple-locus, variable-number tandem repeat analysis, 43 samples produced identical profiles but, after 2014, three new Leptospira interrogans serogroup Pomona genotypes were observed. Interestingly, two isolates showed new MLST profiles and an unclassified identification by monoclonal antibodies. The 16S rRNA gene sequencing clustered them into L. kirschneri species and a core genome MLST analysis revealed an allelic identity of 96% compared with Mozdok strains. Genotyping allowed us to discriminate leptospires and to identify new emerging strains. The accurate identification of infective strains is required for formulating preventive methods and intervention strategies.

Detection of New Leptospira Genotypes Infecting Symptomatic Dogs: Is a New Vaccine Formulation Needed?

“…Leptospires having ST117 clustered with strain L. kirschneri Mozdok 5621, while those having ST198 clustered with strain 367/2012, a recently obtained isolated in Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna laboratory from a hedgehog [ 41 ], genotyped as L. interrogans serovar Australis and shared six out seven loci with ST24. The phylogenetic analysis indicated that ST289 is closely related to L. kirschneri serovar Mozdok, as recently reported [ 42 ].…”

“…ST24 was identified also in one hedgehog, one wild boar and one fox. ST289, which was first discovered in a pig in Northern Italy in 2014 [ 42 ], is a new genotype that has been identified in five dogs. ST117 was also found in a wolf and ST155 was responsible for the infection of one mouse and one horse.…”

“…Interestingly, five dogs were infected by leptospires genotyped as ST289, a new genotype recently found for the first time by IZSLER laboratory in a pig of 2014, living in Northern Italy [ 42 ]. It was typed using core genome MLST as very similar, but not identical, to L. kirschneri serovar Mozdok, within serogroup Pomona.…”

“…DNA extractions and real-time PCR targeting the lipL32 gene were carried out at IZSLER as previously described by Bertasio et al [ 42 ], while at IZSVE, different DNA extraction methods and kits were used in accordance with the variety of samples. A commercially available High Pure PCR Template Preparation kit (Roche Diagnostics, Mannheim, Germany) was used, in accordance with the manufacturer’s instructions, to extract 2 mL of urine or 1 cm 3 tissue homogenate.…”

Leptospira Infections in Domestic and Wild Animals

“…Using a Multi-Locus sequence typing (MLST) and Variable-Number Tandem Repeat (VNTR) analysis, 43 samples produced identical profiles but, after 2014, three new Leptospira interrogans serogroup Pomona genotypes were observed. Moreover, two isolates showed new MLST profiles and an unclassified identification by monoclonal antibodies [18].…”