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Leptospirosis is a growing public and veterinary health concern caused by pathogenic species of Leptospira. Rapid and reliable laboratory tests for the direct detection of leptospiral infections in animals are in high demand not only to improve diagnosis but also for understanding the epidemiology of the disease. In this work we describe a novel and simple TaqMan-based multi-gene targeted real-time PCR approach able to detect and differentiate Leptospira interrogans, L. kirschneri, L. borgpeteresenii and L. noguchii, which constitute the veterinary most relevant pathogenic species of Leptospira. The method uses sets of species-specific probes, and respective flanking primers, designed from ompL1 and secY gene sequences. To monitor the presence of inhibitors, a duplex amplification assay targeting both the mammal β-actin and the leptospiral lipL32 genes was implemented. The analytical sensitivity of all primer and probe sets was estimated to be <10 genome equivalents (GE) in the reaction mixture. Application of the amplification reactions on genomic DNA from a variety of pathogenic and non-pathogenic Leptospira strains and other non-related bacteria revealed a 100% analytical specificity. Additionally, pathogenic leptospires were successfully detected in five out of 29 tissue samples from animals (Mus spp., Rattus spp., Dolichotis patagonum and Sus domesticus). Two samples were infected with L. borgpetersenii, two with L. interrogans and one with L. kirschneri. The possibility to detect and identify these pathogenic agents to the species level in domestic and wildlife animals reinforces the diagnostic information and will enhance our understanding of the epidemiology of leptopirosis.

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Leptospirosis is a neglected zoonotic disease of worldwide distribution with a significant veterinary and public health impact. It is caused by pathogenic bacteria of the genus Leptospira. The availability of effective tools to accurately identify and type leptospires is of utmost importance for the diagnosis of the disease and for assessing its epidemiology. Several multi‐locus sequence typing (MLST) approaches were described for the typing of worldwide isolates of Leptospira but an extensive agreement towards the adoption of a unique consensus scheme for this agent is still lacking. Most genotyped strains originate from Asian and South American countries, with a minority originating from Europe (being most countries represented only by one or a few isolates). The knowledge of the diversity of circulating leptospires is the key to understanding the disease transmission and its zoonotic implications. In this study, we revisited the taxonomy of several isolates of pathogenic Leptospira obtained from domestic, wild and captive animals in Portugal, between 1990 and 2012. A selection of these isolates was genotyped using two previously published MLST schemes. A total of seven distinct sequence types (STs) were detected among the Portuguese isolates with two STs representing L. borgpetersenii (ST149 and ST152), two STs representing L. kirschneri (ST117 and ST100) and three STs representing L. interrogans (ST17, ST24 and ST140). Global widespread (and maybe more virulent) Leptospira genotypes seem to circulate in Portugal, particularly the L. interrogans ST17 isolates which are associated with several outbreaks of leptospirosis among humans and animals in different regions of the world. This study contributes to the enrichment of the global MLST databases with a new set of allele and sequence type information also providing novel data on circulating Leptospira serovars in Portugal.

Campylobacter is a major cause of human foodborne disease worldwide and has been associated with the consumption of contaminated poultry. The prevalence of Campylobacter species in broiler flocks from more than 200 producers widespread in mainland Portugal was assessed in 2008 in response to Commission Decision 2007/516/EC. Campylobacter isolates were obtained from 83.3% of 424 pooled cecal samples, with a higher prevalence of Campylobacter coli (61.2%) than Campylobacter jejuni (38.8%). Restriction fragment length polymorphism analysis of the flaA gene (flaA-RFLP) of 112 C. coli isolates and 67 C. jejuni isolates yielded 11 flaA-RFLP patterns with HinfI (Hunter Gaston diversity index [HGDI] = 0.62) and 48 flaA-RFLP patterns with DdeI (HGDI = 0.89), indicating a high level of genetic diversity. A wide geographic distribution of the most frequent restriction pattern was observed. MICs of five antimicrobials (ciprofloxacin, gentamicin, streptomycin, erythromycin, and tetracycline) were determined for 215 C. coli isolates and 136 C. jejuni isolates. The occurrence of non-wild-type isolates, exhibiting an acquired resistance phenotype, was higher for C. coli than C. jejuni for all antimicrobials tested. Sixty-three percent of C. jejuni and C. coli isolates were considered non-wild type based on their response to both ciprofloxacin and erythromycin, which are frequently used in the treatment of human infections. The high prevalence of antimicrobial-resistant Campylobacter strains detected supports the need for increased epidemiological surveillance and prevention in a country where large amounts of poultry meat are consumed.