Friman et al. Acta Vet Scand (2019) 61:54 https://doi.org/10.1186/s13028-019-0488-7 Acta Veterinaria Scandinavica RESEARCH Open Access Description of two Serratia marcescens associated mastitis outbreaks in Finnish dairy farms and a review of literature Mari Johanna Friman1* , Marjut Hannele Eklund2, Anna Helena Pitkälä2, Päivi Johanna Rajala‑Schultz1 and Merja Hilma Johanna Rantala2 Abstract Background: Infection with Serratia spp. have been associated with mastitis outbreaks in dairy cattle herds. Environ‑ mental contamination or a point source, like a teat dip product, have often been observed to be potential sources of such outbreaks. We describe two Serratia marcescens associated mastitis outbreaks associated with a contaminated teat dip containing a tertiary alkyl amine, n,n‑bis (3‑aminopropyl) dodecylamine in two dairy cattle farms in Finland. S. marcescens strains isolated from milk and environmental samples were identifed by the MALDI‑TOF method. Results: Six specimens (n 19) on Herd 1 and all specimens (n 9) on Herd 2 were positive for S. marcescens. Positive specimens were from mastitis= milk and teat dip liquid and equipment.= Bacteria were not isolated from the unopened teat dip canister. The same clone of S. marcescens was isolated from milk samples and teat dip samples within the farms. Pulsed feld gel electrophoresis results to the S. marcescens isolates from these two diferent herds were tested with unweighted pair‑group method using arithmetic average clustering analysis. The isolates were not same clone in both herds, because similarity in that test was only 75% when cut‑of value to similarity is 85%. Conclusions: Our investigation showed that the post milking teat dip and/or temporary containers were contami‑ nated with S. marcescens and these were most likely the sources for new mastitis cases. The negative result from the unopened teat dip canister and positive results from refllable containers demonstrated that the product itself was not contaminated with S. marcescens at the production unit, but became contaminated at the farm level. Keywords: Bovine mastitis disinfectant, Outbreak, Serratia marcescens, Teat dip Background soil, diferent types of plants and insects [5], farm envi- Serratia species are rod-shaped Gram-negative bacte- ronment like bedding [6] and milking parlor [7] and from ria, which were recently classifed under the new family feces of dairy cows [3, 7]. Serratia spp. have also been of the order Enterobacterales: Yersiniaceae [1]. To date, detected in 1.3–2% of bulk tank milk samples [8, 9]. Ser- 20 diferent Serratia spp. have been described [2]. Ser- ratia spp. can form bioflm on inanimate surfaces [3] and ratia spp., in particular Serratia marcescens, and Serra- produce heat resistant enzymes, thus they are capable of tia liquefaciens are ubiquitous environmental bacteria causing spoilage at diferent points of milk processing capable of causing opportunistic infections in humans [10, 11]. However, certain subspecies like S. liquefaciens and many animal species [3] including mastitis in dairy can also be benefcial by contributing to ripening of raw cows [4]. Tese organisms have been isolated from water, milk cheese products due to their proteolytic activity [12]. *Correspondence: [email protected] Serratia marcescens and S. liquefaciens have been asso- 1 Department of Veterinary Production Animal Medicine, Faculty ciated with mastitis outbreaks in dairy cattle herds [6, 13, of Veterinary Medicine, University of Helsinki, Paroninkuja 20, 14]. Environmental contamination, or a point source that 04920 Saarentaus, Mäntsälä, Finland Full list of author information is available at the end of the article harbours the bacterium, has often been observed to be a © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Friman et al. Acta Vet Scand (2019) 61:54 Page 2 of 11 potential source of the outbreaks [15, 16], similar to out- (n = 1) and from the nozzle of the teat dip spray bot- breaks in human hospitals [17]. We describe two S. marc- tle (n = 1). Also, 5 mL samples of water from the bucket escens mastitis outbreaks associated with a contaminated (n = 2), where udder towels were kept during milking, teat dip in two dairy farms in Finland. In addition, the and from the teat dip product (n = 3) were collected into literature about Serratia spp. mastitis outbreaks in dairy the same kind of sterile plastic tubes as milk samples. Te cows is reviewed. specimens from the teat dip product were collected by spraying the teat dip product from a refllable spray bot- Methods tle, by pumping it through a reusable plastic pump that Farms and data collection was attached to the original opened canister, as well as by Owners of two dairy farms, located over 300 km apart, taking the product straight from an opened canister. In contacted the Production Animal Hospital of the Faculty addition, fve litres of drill well water was collected into of Veterinary Medicine, University of Helsinki, in 2016 a sterile can for bacteriological investigation. In total, the due to several Serratia mastitis cases that had occurred number of environmental samples was 13. on the farms during the past years. Te frst case of S. Samples (n = 9) from Herd 2 were collected by the marcescens from mastitis milk sample in Herd 1 was in farmer. Tese consisted of quarter milk samples from two August 2011, and in Herd 2 in September 2016. After the cows, each with one clinical mastitis quarter, and seven farmer contacts, an outbreak investigation was initiated teat dip samples. Te teat dip product from an opened to fnd a possible source of the organism and to control teat dip canister (n = 1), and all teat dip spray bottles the outbreaks. (n = 6) were sampled as described for Herd 1. Information on farm type, herd size, annual milk yield, In addition, a new, unopened 20 L canister of the teat milking and hygienic practices, including the use of dis- dip product (Viri-Dip Plus, Oy Teollisuushankinta TH infectants, and laboratory reports of tested mastitis milk Ab, Kokkola, Finland) was ordered straight from the samples (from 2014 to 2016) were obtained from the importer of the product to our laboratory to test the bac- farmers. Milk samples had been tested in a milk process- terial quality of the product. ing company’s laboratory (Valio Ltd, Lapinlahti, Finland) using multiplex real-time PCR method (PathoProof™ Microbiological methods Mastitis Complete-16 Kit, A Termo Scientifc™, Van- Culturing and identifcation taa, Finland). Data about udder health in the herds were All samples were cultured immediately after arrival to retrieved from the national health care recording system, the Laboratory of Production Animal Hospital (Univer- ETT Naseva [18], per the owners’ permission. sity of Helsinki, Mäntsälä, Finland). Culture swabs and 10 µL of fuid samples (milk, teat dip and bucket water) Sample collection were streaked onto 5% sheep blood agar plates (Tammer- During the farm visit (08/2016) to Herd 1 to investigate Tutkan Maljat Oy, Tampere, Finland), and incubated in the outbreak, all lactating cows (n = 33) were tested with ambient air at 37 °C for up to 48 h. Preliminary identifca- the California Mastitis Test (CMT). Quarter milk sam- tion of the species was made according to the guidelines ples for bacteriological culture were collected by the of NMC [20]. investigating veterinarian from all quarters that showed Well water specimen (5 L) was fltered through a com- CMT score > 2 (n = 5) on a 1–5 scale [19]. Milk sam- mercial membrane flter with a pore size of 0.45 µm (Mil- ples were collected into 10 mL plastic tubes (Linkoputki lipore Corporation, MA, USA), after which the flter was 16 × 100 mm Plastone, Mekalasi, Helsinki, Finland). One placed aseptically on a nonselective chromogenic agar of the fve samples was from an acute mastitis case and (UriSelect, Bio-rad Finland Oy, Helsinki, Finland), and the rest were from subclinical mastitis. In addition, one incubated at 35 °C for up to 48 h. frozen quarter milk sample from the most recent acute Identifcation of the isolates, that were tentatively mastitis case that had occurred within a week prior to the identifed as Serratia spp. based on colony and bac- herd visit was available for testing. Tus, six mastitis milk terium morphology, were further confrmed in the samples were cultured from Herd 1. Clinical Microbiology Laboratory of the Faculty of Vet- To fnd the potential source of S. marcescens in the erinary Medicine (University of Helsinki, Finland), by environment, surface samples for bacteriological culture matrix-assisted laser desorption ionization-time of fight were taken using sterile cotton swabs (M40, Transystem mass spectrometry using MALDI Biotyper Microfex Amies Agar Gel without charcoal, Copan Diagnostics, LT (Bruker Daltonics GmbH, Bremen, Germany) and CA, USA) from a water faucet (n = 1), milking unit lin- MALDI Biotype MSP Identifcation Standard Method ers (n = 2), washing centres of the milking units (n = 2), 1.1.; using score ≥ 2.00 for species level identifcation. In water cups of the cows (approximately 5 × 5 cm area) addition, 16S rRNA gene sequence based identifcation Friman et al. Acta Vet Scand (2019) 61:54 Page 3 of 11 was performed for seven isolates. Bacterial superna- for the search were ‘cow’ AND ‘mastitis’ AND ‘Serratia’ tant in InstaGene Matrix (Bio-Rad Laboratories Inc., AND as subject-specifc terms.