Clinical microbiology Clostridium novyi type B as a causative agent of bovine meat spoilage V. Eeckhaut a , F. Boyen a , F. Pasmans a , F.A. Uzal b , R. Ducatelle a , S. Van Hoorebeke c , J. Maris d , F. Haesebrouck a , F. Van Immerseel a, * a Ghent University, Faculty of Veterinary Medicine, Department of Pathology, Bacteriology and Avian Diseases, Salisburylaan 133, B-9820 Merelbeke, Belgium b California Animal Health and Food Safety Laboratory, University of California Davis,105 W Central Ave San Bernardino, CA 92408, USA c Quartes nv, Kapellestraat 70, B-9800 Deinze, Belgium d Animal Health Services Flanders (DGZ), Hagenbroeksesteenweg 167, B-2500 Lier, Belgium article info Article history: Received 30 January 2012 Received in revised form 28 February 2012 Accepted 22 March 2012 Available online 4 April 2012 Keywords: Clostridium novyi type B Bovine meat spoilage abstract A series of bovine meat spoilage cases in which meat from clinically healthy Belgian Blue cattle showed green discoloration are described. Histology of skeletal muscle revealed numerous spore-forming rods in the discolored areas of the meat. These organisms stained positively for Clostridium novyi by immuno- histochemistry. A combination of 16S rDNA and fliC gene sequencing of bacterial DNA, isolated from the spoiled meat samples, revealed the unique presence of C. novyi type B. Although this bacterium has been implicated in clinical necrotic hepatitis in cattle, the cases described here are the first implicating C. novyi type B as a cause of bovine meat spoilage. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction The perception of meat quality by consumers is highly influ- enced by sensory characteristics, including taste, tenderness, odor and color [1]. The bright red color of fresh bovine meat is caused by the oxygenated form of myoglobin, called oxymyoglobin. Prolonged exposure to air leads to metmyoglobin production, giving meat a brown-gray color [2]. Greening of meat in combination with rotten flavor caused by bacterial production of hydrogen sulphide and formation of sulfmyoglobin has been described in bovine carcasses [3,4]. In the current manuscript, we report a series of cases of greening of chilled bovine meat, in which Clostridium novyi type B was present in high numbers. C. novyi are anaerobic, spore- forming Gram-positive rods which can be divided into 4 types based on toxin production. The a-toxin is produced by types A and B and b-toxin by types B and D (also called Clostridium haemolyti- cum). Type C is non-toxigenic and therefore, avirulent. To the best of our knowledge, this is the first description of C. novyi causing spoilage of meat derived from clinically healthy animals. 2. Case report Between mid 2010 and mid 2011, multiple episodes of bovine meat spoilage were noticed at a cutting plant in Flanders, Belgium. The meat was derived from clinically healthy Belgian Blue male cattle from 3 fattening farms, all owned by members of the same family. Similar problems with bovine meat derived from other farms were not observed during that period. In the three farms, food waste (cooked carrots, beans and potatoes) derived from industrial kitchens was fed to the animals. At the farms, some of these food waste materials were stored on concrete floors at room temperature while others were in silos, until fed to the animals. No clinically sick animals were reported during the study period at any of the farms. No health problems were noticed during the pre- slaughter inspection of these animals at the slaughter house and no gross abnormalities of the carcasses were reported by the meat inspectors after slaughter. After chilling the carcasses at 7  C for several days, a strong rotten egg-like odor became apparent, and when cutting up the carcasses in the cutting plant, focal areas of green discoloration were noticeable in regions surrounding the joints of the hip (quadriceps muscle) and shoulder de (biceps femoris muscle), with the worst discoloration at sites closest to the joints (Fig. 1A). The areas of discoloration were sharply demarcated from the apparently normal surrounding tissues. On 10 February 2011, 28 April 2011 and 23 May 2011, samples from the spoiled meat were sent to the Faculty of Veterinary Medicine (Ghent University) for diagnostic work up. Samples of the meat were fixed by immersion in 10% buffered, pH 7 formalin for 24 h, embedded in paraffin and processed routinely for the production of hematoxylin and eosin (HE) sections; selected sections were also stained with Gram. Histologically, numerous large Gram-positive bacilli (w2 mm * Corresponding author. Tel.: þ32 9 264 77 48; fax: þ32 9 264 74 94. E-mail address: filip.vanimmerseel@UGent.be (F. Van Immerseel). Contents lists available at SciVerse ScienceDirect Anaerobe journal homepage: www.elsevier.com/locate/anaerobe 1075-9964/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2012.03.004 Anaerobe 18 (2012) 286e288