Contents lists available at ScienceDirect Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth Note Identification of Campylobacter fetus by fluorescence in situ hybridization (FISH) Matti Karg a,1 , Hagen Frickmann b,c,1 , Helmut Hotzel d , Raimond Lugert a , Uwe Groß a , Ralf M. Hagen e , Herbert Tomaso d , Sven Poppert f,g , Andreas E. Zautner a, ⁎ a Institut für Medizinische Mikrobiologie, Universitätsmedizin Göttingen, Göttingen, Germany b Fachbereich Tropenmedizin am Bernhard-Nocht Institut, Bundeswehrkrankenhaus Hamburg, Hamburg, Germany c Institut für Medizinische Mikrobiologie, Virologie und Hygiene, Universitätsmedizin Rostock, Rostock, Germany d Institut für bakterielle Infektionen und Zoonosen, Friedrich-Loeffler-Institut Bundesforschungsinstitut für Tiergesundheit, Jena, Germany e Abteilung A – Lehre Gesundheitsversorgung, Sanitätsakademie der Bundeswehr, München, Germany f Schweizerisches Tropen- und Public Health-Institut (Swiss TPH), Basel, Switzerland g Switzerland & Medizinische Fakultät, Universität Basel, Basel, Switzerland ARTICLE INFO Keywords: Fluorescence in situ hybridization FISH Genital campylobacteriosis Bacteremia Campylobacter fetus Diagnostic microbiology ABSTRACT Two new DNA FISH-probes for Campylobacter fetus were designed, in silico checked for cross-reactions and successfully evaluated in a multi-centric approach with 41 Campylobacter fetus isolates including isolates of all three know subspecies: Campylobacter fetus ssp. fetus, Campylobacter fetus ssp. venerealis, and Campylobacter fetus ssp. testudinum and 40 strains of five non-target Campylobacter species. The bacterial species Campylobacter fetus is divided into three dif- ferent subspecies. C. fetus ssp. fetus is commonly associated with en- zootic abortion especially in sheep and bacteremia in humans (Abbass et al. 2011), whereas C. fetus ssp. venerealis typically causes bovine genital campylobacteriosis causing infertility with considerable eco- nomic losses (van Bergen et al. 2005; Wagenaar et al. 2014). A third subspecies – C. fetus ssp. testudinum – has been isolated from various reptile species, which is a colonizer in these hosts. C. fetus ssp. testu- dinum was also associated with bacteremia in humans (Dingle et al. 2010; Fitzgerald et al. 2014). Fluorescence in situ hybridization (FISH) is one of various methods established for molecular identification of Campylobacter spp. A FISH protocol using a hierarchical probe set for the rapid discrimination of isolates of thermotolerant Campylobacter spp. after growth in culture has been established for the diagnostic (Poppert et al. 2008). However, Campylobacter fetus could not be included due to a lack of well-char- acterized isolates as positive controls. To close this gap C. fetus specific FISH probes were designed and evaluated with a panel of well-char- acterized isolates in a multi-centric approach. A total of 20C. fetus spp. fetus, 14C. fetus spp. venerealis,7C. fetus spp. testudinum isolates, 10C. jejuni spp. jejuni,4C. jejuni ssp. doylei, 14C. coli, 10C. lari, and 2C. upsaliensis isolates were included for the probe evaluation. The C. fetus isolates originated from bull preputial washings (n = 16), cow vaginal mucus (n = 2), aborted calf fetuses (n = 4), aborted lamb fetus (n = 1), intestine of swine (n = 1), intestine of calf (n = 1), colonized reptiles (n = 3), and human blood samples (n = 11). Details are listed in Supplementary Tables 1–4. Two sulfoindocyanine-(Cy3-)labeled DNA probes for FISH targeting 16S rRNA of Campylobacter fetus (C_fetus_1: 5′- Cy3-GAG-ATT-AGT- TGG-ATA-TCA-AGC-CC-3′ and C_fetus_16s_1027: 5‘-Cy3-CCT-GTC-TCA- ACT-TTC-TAG-CAA-GC-3) were designed using the ARB software (Kumar et al. 2006; Ludwig et al. 2004). Afterwards, they were eval- uated in silico for cross-binding using the probeCheck software (http:// www.microbial-ecology.net/probecheck)(Loy et al. 2008) referring to the SILVA LSU reference database (Pruesse et al. 2007). Probes were synthesized by Sequence Laboratories (Seqlab) Göttingen GmbH, Göt- tingen, Germany. Optimal hybridization conditions for the new C. fetus probes were assessed by increasing the formamide concentration in the hybridiza- tion buffer at 46 °C from 10% to 50% as previously described (Frickmann et al. 2017; Moter and Göbel 2000). Campylobacter FISH was performed as described before (Poppert https://doi.org/10.1016/j.mimet.2018.05.020 Received 20 May 2018; Received in revised form 23 May 2018; Accepted 23 May 2018 ⁎ Corresponding author at: Institut für Medizinische Mikrobiologie, Universitätsmedizin Göttingen, Kreuzbergring 57, 37075 Göttingen, Germany. 1 Matti Karg and Hagen Frickmann contributed equally to this work. E-mail address: azautne@gwdg.de (A.E. Zautner). Journal of Microbiological Methods 151 (2018) 44–47 Available online 24 May 2018 0167-7012/ © 2018 Elsevier B.V. All rights reserved. T