LISTERIA MONOCYTOGENES INFECTION OF FREE-LIVING WESTERN EUROPEAN HEDGEHOGS (ERINACEUS EUROPAEUS).

Listeria monocytogenes is a ubiquitous environmental bacterium that causes disease in a wide range of species. Infection with this pathogen is most frequently diagnosed in ruminant livestock, but is also known to infect people and occasionally wildlife. Postmortem examinations of Western European hedgehogs (Erinaceus europaeus) in Great Britain (2011-2017) identified five (5/266, 2%, 95% confidence interval: 0.8-4.3%) animals with L. monocytogenes infection. The L. monocytogenes isolates comprised three serogroup 1/2a and two serogroup 4 from three multilocus sequence types (2, 37, and 121), all of which were different by single-nucleotide polymorphism analysis, indicating they were distinct and epidemiologically unrelated. These findings are consistent with hedgehogs contracting sporadic infection from the environment, perhaps through eating soil-dwelling invertebrates. Examination of data from scanning surveillance programs focused on other British wildlife species indicates that the hedgehog is one of the wildlife species from which L. monocytogenes has been most frequently identified to date in Great Britain. However, further studies of multiple taxa with comparable sampling efforts are required to assess the relative frequency of L. monocytogenes infection in different wildlife species. The bacterium was isolated from extraintestinal sites in multiple hedgehogs, which may indicate septicemia. However, histological examination was limited and could not discriminate subclinical infection from disease (i.e., listeriosis). Although L. monocytogenes is a zoonotic pathogen, disease in people is typically contracted from the ingestion of contaminated foods. The risk to immunocompetent people of contracting listeriosis from hedgehogs is considered very low to negligible.

Real-time PCRs and fingerprinting assays for the detection and characterization of Salmonella Genomic Island-1 encoding multidrug resistance: application to 445 European isolates of Salmonella, Escherichia coli, Shigella, and Proteus.

Salmonella Genomic Island-1 (SGI-1) harbors a cluster of genes encoding multidrug resistance (MDR). SGI-1 is horizontally transmissible and is therefore of significant public health concern. This study presents two novel realtime PCRs detecting three SGI-1 protein-coding genes and a SGI-1 fingerprinting assay. These assays were applied to 445 European enterobacterial isolates. Results from real-time PCRs were comparable to those obtained from gelbased PCRs used for the detection of SGI-1, but were rapid to perform and suitable for large-scale screening. Furthermore, real-time PCRs also detected SGI-1 even when only part of the island was present in bacterial isolates. No trace of SGI-1 was detected in isolates other than Salmonella enterica. The fingerprints showed that regions of SGI-1 outside the MDR region exhibited genomic variations between isolates. In conclusion, the realtime PCRs described here are suitable for the detection of SGI-1 in bacterial isolates. Further studies are necessary to elucidate divergence in its non-MDR region.

Detection and genotyping by real-time PCR/RFLP analyses of Giardia duodenalis from human faeces.

A nested PCR assay (TPILC-PCR) was developed to detect and distinguish between Giardia duodenalis assemblages A and B from human faeces by analysis of the triose phosphate isomerase gene (tpi). The assay comprised an initial multiplexed block-based amplification. This was followed by two separate real-time PCR assays specific for assemblages A and B using a LightCycler and SYBR Green I to identify PCR products by melting-point analysis. RFLP analysis was applied to distinguish G. duodenalis assemblage A groups I and II. The real-time nested PCR was evaluated using DNA extracted from purified giardial trophozoites, Cryptosporidium oocysts, whole faeces containing a range of potential pathogens (including G. duodenalis), faecal smears and bacterial suspensions. The assay was specific, sensitive, reproducible and rapid.

Polymerase chain reaction-based diagnosis of infection with Cryptosporidium in children with primary immunodeficiencies.

BACKGROUND: Patients with deficient cell-mediated immunity are prone to chronic biliary tract infection with Cryptosporidium, which can lead to the development of sclerosing cholangitis and acute cryptosporidiosis after bone marrow transplantation (BMT). The organism is very difficult to detect during asymptomatic periods. METHODS: PCR techniques were compared with standard microscopy for detecting the organism in such patients. Amplification targets were two fragments of the 18S ribosomal RNA gene (unnested) and part of the Cryptosporidium oocyst wall protein gene (nested and unnested). Twenty eight-patients with primary immunodeficiencies were studied including: CD40 ligand deficiency (13); undefined combined immunodeficiency (10); major histocompatibility complex II deficiency (2); and other defects (3). Samples analyzed included stool, bile and liver tissue. RESULTS: Of 25 patients tested prospectively, Cryptosporidium could be detected by PCR but not by microscopy in 12, only 3 of whom had a known history of infection. Five of this group had sclerosing cholangitis. Nine of the PCR-positive patients subsequently underwent BMT and 5 developed acute posttransplant diarrhea and cholangiopathy associated with Cryptosporidium excretion. Of the 13 PCR-negative patients, 3 had cholangiopathy (sclerosing cholangitis in 1 and minor changes in 2). Four of these underwent BMT and none developed cryptosporidiosis. In 3 patients, studied only after developing post-BMT cholangiopathy and diarrhea, Cryptosporidium was detected by PCR but not by microscopy. Genotyping and sequencing showed multiple types of Cryptosporidium in approximately one-third of positive cases. CONCLUSIONS: These results indicate that PCR-based procedures are more sensitive than microscopy for detecting Cryptosporidium in patients with immunodeficiencies.