The demand for rapid methods for the quantification of pathogens is increasing. Among these methods, those based on nucleic acids amplification (quantitative PCRs) are the most widespread worldwide. Together with the qPCR, a new approach named digital PCR (dPCR), has rapidly gained importance. The aim of our study was to compare the results obtained using two different dPCR systems and one qPCR in the quantification of three different bacterial pathogens: Listeria monocytogenes, Francisella tularensis and Mycobacterium avium subsp. paratuberculosis. For this purpose, three pre-existing qPCRs were used, while the same primers and probes, as well as PCR conditions, were transferred to two different dPCR systems: the QX200 (Bio-Rad) and the Quant Studio 3D (Applied Biosystems). The limits of detection and limits of quantification for all pathogens, and all PCR approaches applied, were determined using genomic pure DNAs. The quantification of unknown decimal suspensions of the three bacteria obtained by the three different PCR approaches was compared through the Linear Regression and Bland and Altman analyses. Our results suggest that, both dPCRs are able to quantify the same amount of bacteria, while the comparison among dPCRs and qPCRs, showed both over and under-estimation of the bacteria present in the unknown suspensions. Our results showed qPCR over-estimated the amount of Mycobacterium avium subsp. paratuberculosis and F. tularensis cells. On the contrary, qPCR, compared to QX200 dPCR, under-estimated the amount of L. monocytogenes cells. However, the maximum difference among PCRs approaches was less than 0.5 Log10, while cultural methods underestimated the number of bacteria by one to two Log10 for Francisella tularensis and Mycobacterium avium subsp. paratuberculosis. On the other hand, cultural and PCRs methods quantified the same amount of bacteria for L. monocytogenes, suggesting for this last pathogen, PCRs approaches can be considered as a valid alternative to the cultural ones.

Leptospirosis in dogs has been largely described worldwide, and epidemiological studies have been mainly based on serological data. This study aims to detect and genotype leptospires affecting symptomatic dogs in Northeast Italy between 2013 and 2019. Overall, 1631 dogs were tested using real-time PCR, and leptospires from 193 dogs were subjected to Multilocus Sequence Typing and a Multiple Loci Variable-number Tandem Repeat Analysis. Leptospires were successfully isolated from 15 symptomatic dogs. Six distinct Sequence Types (STs) were found for 135 leptospires, with 3 STs characterizing Leptospira interrogans (ST17, ST198 and ST24), 2 STs characterizing Leptospira kirschneri (ST117 and ST289) and 1 ST characterizing Leptospira borgpetersenii (ST155), revealing the circulation of the serogroups Icterohaemorrhagiae, Australis, Sejroe and Pomona. The Multiple Loci Variable-number Tandem Repeat Analysis of 17 samples did not result in any additional discrimination. Genotypes were compared with those of strains present in the historical internal database, and possible transmission chains were identified from rat, mouse, hedgehog and pig. This work highlights the importance of molecular methods in revealing and identifying circulating Leptospira strains, and it also encourages the evaluation of the ability of commercially available vaccines to reduce the disease burden among dogs.

Leptospirosis is an infectious disease widely reported in veterinary practice and a worldwide zoonosis. In Northeastern Italy, different serogroups and genotypes of Leptospira have been described in ill dogs, the most commonly detected being Icterohaemorragiae (ICT) ST 17, Australis (AUS) ST 24 and ST 198, Pomona (POM) ST 117 and ST 289, and Sejroe (SEJ) ST 155. However, there is little information available on the environmental exposure to Leptospira of wild and synanthropic animals. The aim of this study was to identify the circulating genotypes in potential reservoirs to fill this gap of knowledge. Between 2015 and 2022, 681 animal carcasses collected by the Public Veterinary Service were analyzed for Leptospira with a real-time PCR-based screening test, while positive samples were genotyped by multi-locus sequence typing analysis. To carry out our study, we tested 330 hedgehogs, 105 red foxes, 108 Norway rats, 79 mice, 22 coypus, 10 bank voles, 13 grey wolves, 5 common shrews and 9 greater mouse-eared bats. Five sequence types (STs) common in dogs were also found in wild animals: ST 24, ST 198, ST 17 and ST 155 in hedgehogs, ST 17 and ST 24 in foxes, ST 17 in rats, ST 17 and ST 155 in mice, and ST 117 in a wolf. In addition, to the best of the authors’ knowledge, this is the first Italian report of SEJ ST 197 in a bank vole. Furthermore, this study described a previous survey conducted in 2009 on coypus (30 animals from the province of Trento and 41 from the province of Padua), referring to a serological positivity (L. Bratislava) without any molecular detection of Leptospira. This study on Leptospira in synanthropic and wild animals highlighted the importance of increasing our epidemiological knowledge of leptospirosis and its zoonotic risks.

In aquaculture, Lactococcus garvieae is a common fish pathogen that can cause significant economic losses in several fresh and saltwater species. Despite the extensive range of hosts, L. garvieae infection in sea bass (Dicentrarchus labrax) has rarely been reported. During the summer of 2023, an outbreak occurred in an inland farm in the Gulf of Follonica (Tuscany, Italy). Fish of various sizes were affected, showing apathy, inappetence, erratic swimming and eye lesions, while the mortality was low (2–3% per month). Anatomopathological examinations suggested a septicaemic infection characterised by melanosis, diffuse redness (skin and fins), paleness (gills and internal organs), haemorrhages and splenomegaly. Seventy swabs from the viscera of 14 subjects were collected and colonies similar to Streptococcus spp. grew from all the samples. Lactococcus garvieae was identified via the biochemical tests, API20STREP, MALDI-TOF, 16S rDNA and whole genome sequencing. Genetical characterisation revealed remarkable differences between this isolate and the strains previously isolated in Italian fish farms. Feed treatments with flumequine and erythromycin were ineffective. Considering the limited effects of antimicrobials, preventive measures, such as vaccination and biosecurity, should be implemented.