Validation of PCR methods for confirmation and species identification of thermotolerant Campylobacter as part of EN ISO 10272 - Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter spp.

This article describes the outline and organisation of the validation of three multiplex PCR methods for species identification and/or confirmation of thermotolerant Campylobacter spp. The three PCR methods were validated against the reference method described in the EN ISO standard 10272:2017. The results of the PCR methods were compared against the reference method in a method comparison study and an interlaboratory study based on EN ISO 16140-6:2019. The performance, in terms of inclusivity and exclusivity, of each of the eight PCR targets were comparable to the performance of the reference method: close, equal, or better depending on the target. In total, all three PCR methods were concluded to be equally qualified as the reference method for molecular identification and/or confirmation of thermotolerant Campylobacter spp., C. jejuni, C. coli and C. lari isolated from the food chain and have been included in Amendment 1 of ISO 10272:2017.

Detection of Campylobacter species in different types of samples from dairy farms.

BACKGROUND: Livestock, domestic pets and wildlife can be intestinal carriers of thermotolerant Campylobacter species. These reservoirs can in turn contaminate the environment and food products, thus creating pathways to campylobacteriosis in human beings. The purposes of this study were to investigate sampling strategies applied for surveillance of Campylobacter on dairy cattle farms and to identify the presence and species of Campylobacter in different age groups. METHODS: Boot sock and faecal samples were collected from five dairy herds from three age groups-cows, heifers and calves younger than 12 months-and from milk filters. RESULTS: Campylobacter species were isolated in 152 of 250 samples, of which 93 isolates were identified as C jejuni, 51 as C hyointestinalis, two as C lari and one as C coli, whereas five isolates could not be identified to species level. Campylobacter species were isolated from 86 of 110 faecal samples, 60 of 97 sock samples and six of 43 milk filter samples. CONCLUSION: Faecal samples were the optimal sample type for detection of Campylobacter on dairy farms. However, taking multiple types of samples could be recommended in order to optimise the recovery rate and variety of Campylobacter species detected when investigating the presence of Campylobacter on dairy farms.

Brucella abortus: determination of survival times and evaluation of methods for detection in several matrices.

BACKGROUND: Brucella abortus is a highly pathogenic zoonotic agent, tempting for the development of a rapid diagnostic method to enable adequate treatment and prevent further spread. Enrichment of the bacteria is often used as a first step in diagnostics to increase the bacterial number above the detection limit of the real-time PCR. The enrichment of Brucella spp. takes at least 3 days, which might be avoidable if sensitive PCR methods can be used. Since many matrices contain PCR inhibitors, the limit of detection (LOD) must be determined for each separate matrix. Another aim of this study was the determination of survival of Brucella abortus in the analyzed matrices. METHODS: The LOD for the detection of B. abortus in 14 matrices, relevant for human medicine, veterinary medicine and food and feed safety, was determined to evaluate the need of a pre-enrichment step prior to real-time PCR. The survival of B. abortus in the spiked matrices was tested by plate count in a 7-day interval for 132 days. RESULTS: The limit of detection for B. abortus in most matrices was in the range of 103-104 CFU/g for cultivation and 104-105 CFU/g for direct real-time PCR. The survival time of B. abortus was less than 21 days in apple purée and stomach content and 28 days in water while B. abortus remained viable at day 132 in milk, blood, spinach and minced meat. CONCLUSIONS: A direct PCR analysis without enrichment of bacteria saves at least 3 days. However, the limit of detection between direct PCR and plate count differs in a 10 fold range. We conclude that this lower sensitivity is acceptable in most cases especially if quick analysis are required.

A novel real-time PCR assay for specific detection of Brucella melitensis.

BACKGROUND: Brucellosis is a zoonosis that occurs worldwide. The disease has been completely eradicated in livestock in Sweden in 1994, and all cases of confirmed human brucellosis are imported into Sweden from other countries. However, due to an increase in the number of refugees and asylum seekers from the middle-east to Sweden, there is a need to improve the current diagnostic methodology for Brucella melitensis. Whilst culture of Brucella species can be used as a diagnostic tool, real-time PCR approaches provide a much faster result. The aim of this study was to set up a species-specific real-time PCR for the detection of all biovars of Brucella melitensis, which could be used routinely in diagnostic laboratories. METHODS: A Brucella melitensis real-time PCR assay was designed using all available genomes in the public database of Brucella (N = 96) including all complete genomes of Brucella melitensis (N = 17). The assay was validated with a collection of 37 Brucella species reference strains, 120 Brucella melitensis human clinical isolates, and 45 clinically relevant non-Brucella melitensis strains. RESULTS: In this study we developed a single real-time PCR for the specific detection of all biovars of Brucella melitensis. CONCLUSIONS: This new real-time PCR method shows a high specificity (100%) and a high sensitivity (1.25 GE/µl) and has been implemented in the laboratories of four governmental authorities across Sweden.

Brucellosis outbreak in a Swedish kennel in 2013: determination of genetic markers for source tracing.

Brucellosis is a highly infectious zoonotic disease but rare in Sweden. Nonetheless, an outbreak of canine brucellosis caused by an infected dog imported to Sweden was verified in 2013. In total 25 dogs were tested at least duplicated by the following approaches: real-time PCR for the detection of Brucella canis, a Brucella genus-specific real-time PCR, selective cultivation, and microscopic examination. The whole genome of B. canis strain SVA13 was analysed regarding genetic markers for epidemiological examination. The genome of an intact prophage of Roseobacter was detected in B. canis strain SVA13 with whole genome sequence prophage analysis (WGS-PA). It was shown that the prophage gene content in the American, African and European isolates differs remarkably from the Asian strains. The prophage sequences in Brucella may therefore serve of use as genetic markers in epidemiological investigations. Phage DNA fragments were also detected in clustered, regularly interspaced short palindromic repeats (CRISPR) in the genome of strain SVA13. In addition to the recommendations for genetic markers in Brucella outbreak tracing, our paper reports a validated two-step stand-alone real-time PCR for the detection of B. canis and its first successful use in an outbreak investigation.

Draft Genome Sequences of Brucella suis Biovar 4 Strain NCTC 10385, Brucella ceti Strain NCTC 12891T, Brucella inopinata Strain CAMP 6436T, and Brucella neotomae Strain ATCC 23459T.

With the aim of developing quantitative PCR methods for the detection and differentiation of Brucella species, the genomes of Brucella ceti, Brucella inopinata, Brucella netotomae, and Brucella suis biovar 4 were sequenced and analyzed.

Whole-Genome Sequence of Brucella canis Strain SVA13, Isolated from an Infected Dog.

An outbreak of canine brucellosis in Sweden was confirmed by the National Veterinary Institute (SVA) in August 2013. The whole genome of the causative agent was sequenced, assembled, and analyzed.