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Prion diseases are progressive and fatal, neurodegenerative disorders described in humans and animals. According to the “protein-only” hypothesis, the normal host-encoded prion protein (PrPC) is converted into a pathological and infectious form (PrPSc) in these diseases. Transgenic knockout models have shown that PrPC is a prerequisite for the development of prion disease. In Norwegian dairy goats, a mutation (Ter) in the prion protein gene (PRNP) effectively blocks PrPC synthesis. We inoculated 12 goats (4 PRNP+/+, 4 PRNP+/Ter, and 4 PRNPTer/Ter) intracerebrally with goat scrapie prions. The mean incubation time until clinical signs of prion disease was 601 days post-inoculation (dpi) in PRNP+/+ goats and 773 dpi in PRNP+/Ter goats. PrPSc and vacuolation were similarly distributed in the central nervous system (CNS) of both groups and observed in all brain regions and segments of the spinal cord. Generally, accumulation of PrPSc was limited in peripheral organs, but all PRNP+/+ goats and 1 of 4 PRNP+/Ter goats were positive in head lymph nodes. The four PRNPTer/Ter goats remained healthy, without clinical signs of prion disease, and were euthanized 1260 dpi. As expected, no accumulation of PrPSc was observed in the CNS or peripheral tissues of this group, as assessed by immunohistochemistry, enzyme immunoassay, and real-time quaking-induced conversion. Our study shows for the first time that animals devoid of PrPC due to a natural mutation do not propagate prions and are resistant to scrapie. Clinical onset of disease is delayed in heterozygous goats expressing about 50% of PrPC levels.

Hemoglobin (Hb) constitutes an important protein in clinical diagnostics-both in humans and animals. Among the high number of sequence variants, some can cause severe diseases. Moreover, chemical modifications such as glycation and carbamylation serve as important biomarkers for conditions such as diabetes and kidney diseases. In clinical routine analysis of glycated Hb, sequence variants or other Hb proteoforms can cause interference, resulting in wrong quantification results. We present a versatile and flexible capillary zone electrophoresis-mass spectrometry screening method for Hb proteoforms including sequence variants and modified species extracted from dried blood spot (DBS) samples with virtually no sample preparation. High separation power was achieved by application of a 5-layers successive multiple ionic polymer layers-coated capillary, enabling separation of positional isomers of glycated α- and β-chains on the intact level. Quantification of glycated Hb was in good correlation with the results obtained in a clinical routine method. Identification and characterization of known and unknown proteoforms was performed by fragmentation of intact precursor ions. N-Terminal and lysine glycation could be identified on the α- and β-chain, respectively. The versatility of the method was demonstrated by application to dog and cat DBS samples. We discovered a putative new sequence variant of the β-chain in dog (T38 → A). The presented method enables separation, characterization, and quantification of intact proteoforms, including positional isomers of glycated species in a single run. Combined with the simple sample preparation, our method represents a valuable tool to be used for deeper characterization of clinical and veterinary samples.

International audience; Gallbladder mucocele (GBM) is a common extra-hepatic biliary syndrome in dogs with death rates ranging from 7 to 45%. Therefore, the aim of this study was to identify the association of survival with variables that could be utilized to improve clinical decisions. A total of 1194 dogs with a gross and histopathological diagnosis of GBM were included from 41 veterinary referral hospitals in this retrospective study. Dogs with GBM that demonstrated abnormal clinical signs had significantly greater odds of death than subclinical dogs in a univariable analysis (OR, 4.2; 95% CI, 2.14–8.23; P < 0.001). The multivariable model indicated that categorical variables including owner recognition of jaundice (OR, 2.12; 95% CI, 1.19–3.77; P = 0.011), concurrent hyperadrenocorticism (OR 1.94; 95% CI, 1.08–3.47; P = 0.026), and Pomeranian breed (OR, 2.46; 95% CI 1.10–5.50; P = 0.029) were associated with increased odds of death, and vomiting was associated with decreased odds of death (OR, 0.48; 95% CI, 0.30–0.72; P = 0.001). Continuous variables in the multivariable model, total serum/plasma bilirubin concentration (OR, 1.03; 95% CI, 1.01– 1.04; P < 0.001) and age (OR, 1.17; 95% CI, 1.08–1.26; P < 0.001), were associated with increased odds of death. The clinical utility of total serum/plasma bilirubin concentration as a biomarker to predict death was poor with a sensitivity of 0.61 (95% CI, 0.54–0.69) and a specificity of 0.63 (95% CI, 0.59–0.66). This study identified several prognostic variables in dogs with GBM including total serum/plasma bilirubin concentration, age, clinical signs, concurrent hyperadrenocorticism, and the Pomeranian breed. The presence of hypothyroidism or diabetes mellitus did not impact outcome in this study.

The International Agency for Research on Cancer has classified red meat as "probably carcinogenic to humans" (Group 2A). In mechanistic studies exploring the link between intake of red meat and CRC, heme iron, the pigment of red meat, is proposed to play a central role as a catalyzer of luminal lipid peroxidation and cytotoxicity. In the present work, the novel A/J Min/+ mouse was used to investigate the effects of dietary beef, pork, chicken, or salmon (40% muscle food (dry weight) and 60% powder diet) on Apc-driven intestinal carcinogenesis, from week 3-13 of age. Muscle food diets did not differentially affect carcinogenesis in the colon (flat ACF and tumors). In the small intestine, salmon intake resulted in a lower tumor size and load than did meat from terrestrial animals (beef, pork or chicken), while no differences were observed between the effects of white meat (chicken) and red meat (pork and beef). Additional results indicated that intestinal carcinogenesis was not related to dietary n-6 polyunsaturated fatty acids, intestinal formation of lipid peroxidation products (thiobarbituric acid reactive substances, TBARS), or cytotoxic effects of fecal water on Apc(-/+) cells. Notably, the amount of heme reaching the colon appeared to be relatively low in this study. The greatest tumor load was induced by the reference diet RM1, underlining the importance of the basic diets in experimental CRC. The present study in A/J Min/+ mice does not support the hypothesis of a role of red meat in intestinal carcinogenesis.

International audience; In the laboratory, prenatal stress produces alterations in the structure and function of corticolimbic neurons. Here we report changes in gene expression and corticolimbic dendritic spine morphology in the offspring of pregnant ewes subjected to aversive interactions with human handlers during the last five weeks of pregnancy (AVS) compared to control dams that received gentle handling (GEN). AVS Lambs had higher spine density on pyramidal neurons in area CA1 of the hippocampus and in medial prefrontal cortex compared to GEN lambs, as well as a lower ratio of mushroom spines to stubby and thin spines in area CA1. Expression of genes involved in brain development and spine morphogenesis was decreased in hippocampus and prefrontal cortex in AVS compared to GEN lambs. This study is the first demonstration that an ecologically relevant aversive experience in a field setting alters neuronal structure similarly to previous reports from laboratory settings and that even for animals domesticated over 12,000 years ago, an apparently mild stressor, resulting from human-animal interactions, can have similarly profound impacts on corticolimbic morphology. (C) 2012 Elsevier Ltd. All rights reserved.

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International audience; Humans and animals are exposed to PCBs and influences on developmental and endocrine processes are among the most pronounced effects. In the present study it was hypothesised that exposure to PCBs may interfere with sexually dimorphic behaviour. To test this hypothesis, behavioural studies in developmentally exposed sheep were conducted. Ewes were orally administered PCB 153 (98 mu g/kg bw day). PCB 118 (49 mu g/kg bw day) or corn oil from conception until delivery. However, because of accidental cross-contamination occurring twice causing a mixed exposure scenario in all three groups, the focus of this paper is to compare three distinct groups of lambs with different PCB levels (PCB 153 high-PCB 153h, PCB 118 high-PCB 118h, and low combined group-LC) rather than comparing animals exposed to single PCB congeners to those of a control group. Lambs were tested between 2 and 6 weeks of age. When LC males started the light/dark choice test in a dark box, they spent significantly more time in the dark part of the pen than LC females. This gender-related difference was not found in groups exposed to PCBs. A significant inhibitory effect on the activity level of males exposed to stress of confinement was found in the PCB 118h group. In a high stress situation females from PCB 118h and males from PCB 153h were less active than their gender counterparts. The results support the hypothesis that intrauterine exposure to PCBs can alter sexually dimorphic behaviour of offspring.

International audience; This study was conducted to evaluate the effect of early rearing conditions on physiological, haematological and immunological responses relevant to adaptation and long-term stress in white Leghorn hens with intact beaks housed in furnished cages (FC) or conventional cages (CC) during the laying period. Pullets were cage reared (CR) or litter floor reared (FR). From 16 to 76 weeks of age, hens were housed in FC (eight hens per cage) or in CC (three hens per cage). As measures of long-term stress at the end of the laying period, adrenal reactivity was quantified by assessing corticosterone responses to adrenocorticotropin challenge, and immune response was assessed by measuring antibody responses after immunization with sheep red blood cells (SRBC) and keyhole limpet haemocyanin (KLH). Heterophil to lymphocyte (H/L) ratio was employed as an indicator of stress. Rearing conditions significantly affected anti-SRBC titres (P < 0.0001) and tended to affect H/L ratios (P = 0.07), with the highest values found in FR hens. Layer housing affected H/L ratio (P < 0.01); the highest ratio was found in FR birds housed in FC during the laying period. This study shows that early rearing environment affects immunological indicators that are widely used to assess stress in laying hens. However, while results on H/L ratio indicated that FR birds experienced more stress particularly when they were housed in FC during the laying period, the immune responses to SRBC in FR hens was improved, indicating the opposite. This contradiction suggests that the effects on immune response may have been associated with pathogenic load due to environmental complexity in FR and FC hens rather than stress due to rearing system or housing system per se.

International audience; This paper is based on a workshop held in Oslo, Norway in November 2013, in which experts discussed how to reach consensus on the healthiness of red and processed meat. Recent nutritional recommendations include reducing intake of red and processed meat to reduce cancer risk, in particular colorectal cancer (CRC). Epidemiological and mechanistic data on associations between red and processed meat intake and CRC are inconsistent and underlying mechanisms are unclear. There is a need for further studies on differences between white and red meat, between processed and whole red meat and between different types of processed meats, as potential health risks may not be the same for all products. Better biomarkers of meat intake and of cancer occurrence and updated food composition databases are required for future studies. Modifying meat composition via animal feeding and breeding, improving meat processing by alternative methods such as adding phytochemicals and improving our diets in general are strategies that need to be followed up.

International audience; NKp46 has proven a reliable NK cell marker in both cattle and sheep. Four anti-bovine/ovine NKp46 mAbs reacted to caprine lymphocytes and specificity was confirmed as all mAbs reacted with caprine NKp46 transfectants. At young ages, goat NKp46+ cells were scarcer in circulation (~1% of PBMC) than in cattle and sheep, but the numbers increase with age. Similar to cattle and sheep, these cells are CD16+, CD8+, moderately perforin+, and CD2 marks a major negative and a minor positive subset. NKp46+ cells produce IFN-γ in response to cytokine stimulation, and following positive selection and culture they upregulate perforin and respond with NKp46-mediated cytotoxicity, thus confirming typical NK cell functions. NKp46 expression was non-overlapping with CD21+ B-cells, CD4+ T-cells and CD14+ monocytes. Most caprine-reactive gamma/delta TcR antibodies do not mark NKp46+ cells. However, in older goats, a significant proportion of NKp46+ cells express the TcR delta chain. This occurs at an age where the proportion of classical gamma-delta T-cells is in decline. NKp46+/TcRdelta+ cells were also present in cultures of IL-2 expanded caprine NKp46+ cells. Since NK cells in most species are CD3-/NKp46+, we stained permeabilized caprine PBMCs with a human cytoplasmatic CD3 epsilon mAb, which is cross-reactive in several species. Staining patterns corresponded well with expected T-cell populations, but this mAb also stained a subset of NKp46+ cells, the majority of which were also TcRdelta+. Thus, cytCD3e cannot be used straightforward for NK cell discrimination in goats, and these overlapping populations need closer investigation.

International audience; Robenacoxib and ketoprofen are acidic nonsteroidal anti-inflammatory drugs (NSAIDs). Both are licensed for once daily administration in the cat, despite having short blood half-lives. This study reports the pharmacokinetic/pharmacodynamic (PK/PD) modelling of each drug in a feline model of inflammation. Eight cats were enrolled in a randomized, controlled, three-period cross-over study. In each period, sterile inflammation was induced by the injection of carrageenan into a subcutaneously implanted tissue cage, immediately before the subcutaneous injection of robenacoxib (2 mg/kg), ketoprofen (2 mg/kg) or placebo. Blood samples were taken for the determination of drug and serum thromboxane (Tx)B2 concentrations (measuring COX-1 activity). Tissue cage exudate samples were obtained for drug and prostaglandin (PG)E2 concentrations (measuring COX-2 activity). Individual animal pharmacokinetic and pharmacodynamic parameters for COX-1 and COX-2 inhibition were generated by PK/PD modelling. S(+) ketoprofen clearance scaled by bioavailability (CL/F) was 0.114 L/kg/h (elimination half-life = 1.62 h). For robenacoxib, blood CL/F was 0.684 L/kg/h (elimination half-life = 1.13 h). Exudate elimination half-lives were 25.9 and 41.5 h for S(+) ketoprofen and robenacoxib, respectively. Both drugs reduced exudate PGE2 concentration significantly between 6 and 36 h. Ketoprofen significantly suppressed (>97%) serum TxB2 between 4 min and 24 h, whereas suppression was mild and transient with robenacoxib. In vivo IC50 COX-1/IC50 COX-2 ratios were 66.9:1 for robenacoxib and 1:107 for S(+) ketoprofen. The carboxylic acid nature of both drugs may contribute to the prolonged COX-2 inhibition in exudate, despite short half-lives in blood.

International audience; Background: Hibernation involves periods of severely depressed metabolism (torpor) and decreases in body temperature (Tb). Small arctic mammals ( < 5kg), in which Tb generally drop drastically, display leukopenia during hibernation. This raised the question of whether the decreased leukocyte counts in mammalian hibernators is due to torpor per se or is secondary to low Tb. The present study examined immune cell counts in brown bears (Ursus arctos), where torpor is only associated with shallow decreases in Tb. The results were compared across hibernator species for which immune and Tb data were available. Methods and Results: The white blood cell counts were determined by flow cytometry in 13 bears captured in the field both during summer and winter over 2 years time. Tb dropped from 39.6±0.8 to 33.5±1.1°C during hibernation. Blood neutrophils and monocytes were lower during hibernation than during the active period (47%, p= 0.001; 43%, p=0.039, respectively), whereas no change in lymphocyte counts was detected (p=0.599). Further, combining our data and those from 10 studies on 9 hibernating species suggested that the decline in Tb explained the decrease in innate immune cells (R2=0.83, p < 0.0001). Conclusions: Bears have fewer innate immune cells in circulation during hibernation, which may represent a suppressed innate immune system. Across species comparison suggests that, both in small and large hibernators, Tb is the main driver of immune function regulation during winter dormancy. The lack of a difference in lymphocyte counts in this context requires further investigations.

International audience; The objective of this study was to evaluate genome-enabled predictions of daughter yield deviations for clinical mastitis in Norwegian Red cows within and between environments according to mastitis pathogen status. Genome-based predictions of daughter yield deviations for clinical mastitis for 1,126 bulls within and between 5 environments were performed using Bayesian ridge regression. The environments were defined as herd-5-yr classes with the following prevalence of bacteriological milk samples found positive for contagious mastitis pathogens: 25% and

The current study presents a novel objective measure for characterizing sexually active groups (SAG 3-5) and relates this measure to other behaviors of lactating Holstein-Friesian cows. Cows in SAG 3-5 were required to participate in a minimum of 1 estrus behavior per 5 min while staying within 3 m (2 cow lengths) of its partner(s) for a minimum of 5 mm. Twenty Holstein-Friesian cows were video-monitored continuously through 1 complete estrous cycle. (22 d). Standing behavior, SAG 3-5, secondary estrus signs (SEC), and other social and agonistic behaviors were recorded continuously. The period of mounting estrus (MTE) was divided into the 3 parts: prestand, standing estrus (STE), and poststand. The mean durations of MTE, prestand, STE, and poststand period were 12.9 +/- 1.84, 4.0 +/- 1.93, 7.1 +/- 1.44, and 1.8 +/- 0.57 h (n = 13). The fractions of time spent in SAG 3-5 during MTE, prestand, STE, and poststand period were 13, 8, 19, and 1% (n = 11). During MTE, cows participated, on average, in 5.8 +/- 1.24 SAG 3-5 and initiated 9.5 +/- 2.99 mounts, with mean durations of 0.25 +/- 0.03 h and 4.00 +/- 0.36 s, respectively. The novel measure, SAG 3-5 was a sign of long duration not confined only to groups of STE cows. On one day when no cows were in estrus and during the periods 4 to 24 h before and after MTE, no SAG 3-5 behaviors were observed. Luteal-phase cows participated in SAG 3-5 only when the partner was a single cow in estrus. The time spent in SAG 3-5 increased between 1 and 3 h before MTE and the prestand period (3 vs. 8%) and reached a peak level during STE. From STE to poststand, time spent in SAG 3-5 decreased considerably (19 vs. 1%). The observed decrease in nonmutual agonistic behaviors 4 to 24 h before MTE is suggested as an early sign of pre-estrus. Changes in SAG 3-5, agonistic behaviors, and SEC are suggested as indicators of the specific stages of MTE. Increased SEC initiated and SAG 3-5 were indicators of late pre-estrus and early estrus (prestand). Peak levels of SAG 3-5, SEC, and social agonistic behaviors were indicators of STE. A sudden decrease in behaviors, preceded by frequent interactions, was indicative of late estrus (poststand). On the basis of the findings reported here, we propose that SAG 3-5, as well as proceptive and receptive patterns of SEC and agonistic behaviors, be included in estrus detection protocols. Updated knowledge of these behavioral interactions may assist when determining the stage of estrus and the optimal time to breed dairy cows.

Objective—To compare pharmacokinetics and clearances of creatinine and iohexol as estimates of glomerular filtration rate (GFR) in dogs with various degrees of renal function. Animals—50 Great Anglo-Francais Tricolor Hounds with various degrees of renal function. Procedures—Boluses of iohexol (40 mg/kg) and creatinine (647 mg/kg) were injected IV. Blood samples were collected before administration and 5 and 10 minutes and 1, 2, 4, 6, and 8 hours after administration. Plasma creatinine and iohexol concentrations were assayed via an enzymatic method and high-performance liquid chromatography, respectively. A noncompartmental approach was used for pharmacokinetic analysis. Pharmacokinetic variables were compared via a Bland-Altman plot and an ANOVA. Results—Compared with results for creatinine, iohexol had a significantly higher mean ± SD plasma clearance (3.4 ± 0.8 mL/min/kg vs 3.0 ± 0.7 mL/min/kg) and a significantly lower mean volume of distribution at steady state (250 ± 37 mL/kg vs 539 ± 73 mL/kg), mean residence time (80 ± 31 minutes vs 195 ± 73 minutes), and mean elimination half-life (74 ± 20 minutes vs 173 ± 53 minutes). Despite discrepancies between clearances, especially for high values, the difference was < 0.6 mL/min/kg for 34 (68%) dogs. Three dogs with a low GFR (< 2 mL/min/kg) were classified similarly by both methods. Conclusions and Clinical Relevance—Plasma iohexol and creatinine clearances can be used interchangeably for screening patients suspected of having chronic kidney disease (ie, low GFR), but large differences may exist for dogs with a GFR within or above the reference range.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:28:16Z No. of bitstreams: 0Bitstream added on 2014-05-27T14:32:16Z : No. of bitstreams: 1 2-s2.0-84873867256.pdf: 796974 bytes, checksum: 64f172274307a2abd3e5977743eb1671 (MD5) Made available in DSpace on 2014-05-27T11:28:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-01-30 Horses were domesticated from the Eurasian steppes 5,000-6,000 years ago. Since then, the use of horses for transportation, warfare, and agriculture, as well as selection for desired traits and fitness, has resulted in diverse populations distributed across the world, many of which have become or are in the process of becoming formally organized into closed, breeding populations (breeds). This report describes the use of a genome-wide set of autosomal SNPs and 814 horses from 36 breeds to provide the first detailed description of equine breed diversity. FST calculations, parsimony, and distance analysis demonstrated relationships among the breeds that largely reflect geographic origins and known breed histories. Low levels of population divergence were observed between breeds that are relatively early on in the process of breed development, and between those with high levels of within-breed diversity, whether due to large population size, ongoing outcrossing, or large within-breed phenotypic diversity. Populations with low within-breed diversity included those which have experienced population bottlenecks, have been under intense selective pressure, or are closed populations with long breed histories. These results provide new insights into the relationships among and the diversity within breeds of horses. In addition these results will facilitate future genome-wide association studies and investigations into genomic targets of selection. © 2013 Petersen et al. University of Minnesota College of Veterinary Medicine, St Paul, MN Texas AandM University College of Veterinary Medicine and Biomedical Science, College Station, TX Swedish University of Agricultural Sciences Department of Animal Breeding and Genetics, Uppsala University of Kentucky Department of Veterinary Science, Lexington, KY University of California Davis School of Veterinary Medicine, Davis, CA Equine Analysis, Midway, KY University Estadual Paulista Department of Veterinary Clinical Science, Botucatu-SP University College Dublin School of Veterinary Medicine, Dublin University of Azores Institute for Biotechnology and Bioengineering Biotechnology Centre of Azores, Angra do Heroísmo University of Veterinary Medicine Hannover Institute for Animal Breeding and Genetics, Hannover University of Perugia Faculty of Veterinary Medicine, Perugia Animal Health Trust, Lanwades Park, Newmarket, Suffolk French National Institute for Agricultural Research-Animal Genetics and Integrative Biology Unit, Jouy en Josas University of Sydney Veterinary Science, New South Wales Nihon Bioresource College, Koga, Ibaraki University of Helsinki Faculty of Veterinary Medicine, Helsinki University College Dublin College of Agriculture, Food Science and Veterinary Medicine, Belfield, Dublin University of Bern Institute of Genetics, Bern Institute of Cancer Research Breakthrough Breast Cancer Research Centre, London Royal Veterinary College Comparative Neuromuscular Diseases Laboratory, London Swiss National Stud Farm Agroscope Liebefeld-Posieux Research Station, Avenches Norwegian School of Veterinary Science Department of Basic Sciences and Aquatic Medicine, Oslo Animal DNA Diagnostics Ltd, Cambridge Laboratory of Racing Chemistry Department of Molecular Genetics, Utsunomiya, Tochigi

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The present study describes the occurrence of various gastrointestinal helminths, lungworms and liver flukes in Norwegian sheep and goats as assessed from faecal samples and post mortem examinations performed between 2007 and 2010. Faecal samples for gastrointestinal nematode egg counts were collected from 77 sheep flocks and 30 dairy goat flocks from three geographical regions in Norway. Additionally, thirty-two lambs and 16 adult goats were euthanized for necropsy examination and for identification of adult gastrointestinal nematodes and tapeworms, lungworms and liver flukes. The survey showed that there was a higher mean excretion of trichostrongyle eggs in sheep than in goats at the individual level (392 EPG vs. 154 EPG, p

An aerobic endospore-forming bacillus (NVH 391-98T) was isolated during a severe food poisoning outbreak in France in 1998, and four other similar strains have since been isolated, also mostly from food poisoning cases. Based on 16S rRNA gene sequence similarity, these strains were shown to belong to the Bacillus cereus Group (over 97 % similarity with the current Group species) and phylogenetic distance from other validly described species of the genus Bacillus was less than 95 %. Based on 16S rRNA gene sequence similarity and MLST data, these novel strains were shown to form a robust and well-separated cluster in the B. cereus Group, and constituted the most distant cluster from species of this Group. Major fatty acids (iso-C15 : 0, C16 : 0, iso-C17 : 0, anteiso-C15 : 0, iso-C16 : 0, iso-C13 : 0) supported the affiliation of these strains to the genus Bacillus , and more specifically to the B. cereus Group. NVH 391-98T taxon was more specifically characterized by an abundance of iso-C15 : 0 and low amounts of iso-C13 : 0 compared with other members of the B. cereus Group. Genome similarity together with DNA–DNA hybridization values and physiological and biochemical tests made it possible to genotypically and phenotypically differentiate NVH 391-98T taxon from the six current B. cereus Group species. NVH 391-98T therefore represents a novel species, for which the name Bacillus cytotoxicus sp. nov. is proposed, with the type strain NVH 391-98T ( = DSM 22905T = CIP 110041T).

The Bacillus cereus sensu lato complex has recently been divided into several phylogenetic groups with clear differences in growth temperature range. However, only a few studies have investigated the actual pathogenic potential of the psychrotolerant strains of the B. cereus group at low temperature, and little information is available concerning gene expression at low temperature. We found that vegetative cells of the psychrotolerant B. weihenstephanensis strain KBAB4 were pathogenic against the model insect Galleria mellonella at 15°C but not at 30°C. A similar temperature-dependent difference also was observed for the supernatant, which was cytotoxic to Vero epithelial cell lines and to murine macrophage J774 cells at 15°C but not at 30°C. We therefore determined the effect of low temperature on the production of various proteins putatively involved in virulence using two-dimensional protein gel electrophoresis, and we showed that the production of the Hbl enterotoxin and of two proteases, NprB and NprP2, was greater at a growth temperature of 15°C than at 30°C. The quantification of the mRNA levels for these virulence genes by real-time quantitative PCR at both temperatures showed that there was also more mRNA present at 15°C than at 30°C. We also found that at 15°C, hbl mRNA levels were maximal in the mid- to late exponential growth phase. In conclusion, we found that the higher virulence of the B. cereus KBAB4 strain at low temperature was accompanied by higher levels of the production of various known PlcR-controlled virulence factors and by a higher transcriptional activity of the corresponding genes.

In the period of 2008–2009, the efficacies of the benzimidazole (BZ) albendazole and the macrocyclic lactone (ML) ivermectin against gastrointestinal nematodes (GIN) of small ruminants were evaluated by means of the fecal egg count reduction (FECR) test and by post-treatment identification of surviving third stage (L3) larvae after coproculture. Sheep (n = 28) and goat (n = 28) flocks from three areas of Norway were randomly selected to assess the prevalence of anthelmintic resistance (AR), whereas only lambs from non-randomly selected sheep flocks (n = 32) with a farm management that could select for AR were investigated the second year. Only flocks with a mean excretion of nematode eggs per gram feces (EPG) ≥150 at time of treatment were included in the survey. In total, 48 (80%) and 13 (46.4%) of the selected sheep and goat flocks, respectively, fulfilled the inclusion criteria. The proportions of flocks classified as resistant (i.e., FECR Teladorsagia/Trichostrongylus in five flocks, Haemonchus in two flocks, and a mixture of these genera in the remaining two flocks. In the goat flocks, the pre-treatment infection levels of GIN were low compared to what was found in the sheep flocks. Still, in one flock, AR against BZ in Teladorsagia/Trichostrongylus was found. New strategies and recommendations to face the emerging AR situation in Rogaland County in order to limit the spread of resistant nematodes within and into other areas are urgently needed

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Chapitre 18; International audience

European eels are blocked at a prepubertal silver stage due to a deficient production of pituitary gonadotropins. We investigated the potential role of activin/follistatin system in the control of eel gonadotropins. Through the development of qPCR assays for European eel activin β B and follistatin, we first analyzed the tissue distribution of the expression of these two genes. Both activin β B and follistatin are expressed in the brain, pituitary and gonads. In addition, a striking expression of both transcripts was also found in the retina and in adipose tissue. The effects of recombinant human activins and follistatin on eel gonadotropin gene expression were studied using primary cultures of eel pituitary cells. Activins A and B strongly stimulated FSHβ subunit expression in a time- and dose-dependent manner. In contrast, activin reduced LHβ expression, an inhibitory effect which was highlighted in the presence of testosterone, a known activator of eel LHβ expression. No effect of activin was observed on other pituitary hormones. Follistatin antagonized both the stimulatory and inhibitory effects of activin on FSHβ and LHβ expression, respectively. Activin is the first major stimulator of FSH expression evidenced in the eel. These results in a basal teleost further support the ancient origin and strong conservation of the activin/follistatin system in the control of FSH in vertebrates. In contrast, the opposite regulation of FSH and LH may have emerged in the teleost lineage.

An equine SNP genotyping array was developed and evaluated on a panel of samples representing 14 domestic horse breeds and 18 evolutionarily related species. More than 54,000 polymorphic SNPs provided an average inter-SNP spacing of ∼43 kb. The mean minor allele frequency across domestic horse breeds was 0.23, and the number of polymorphic SNPs within breeds ranged from 43,287 to 52,085. Genome-wide linkage disequilibrium (LD) in most breeds declined rapidly over the first 50–100 kb and reached background levels within 1–2 Mb. The extent of LD and the level of inbreeding were highest in the Thoroughbred and lowest in the Mongolian and Quarter Horse. Multidimensional scaling (MDS) analyses demonstrated the tight grouping of individuals within most breeds, close proximity of related breeds, and less tight grouping in admixed breeds. The close relationship between the Przewalski's Horse and the domestic horse was demonstrated by pair-wise genetic distance and MDS. Genotyping of other Perissodactyla (zebras, asses, tapirs, and rhinoceros) was variably successful, with call rates and the number of polymorphic loci varying across taxa. Parsimony analysis placed the modern horse as sister taxa to Equus przewalski. The utility of the SNP array in genome-wide association was confirmed by mapping the known recessive chestnut coat color locus (MC1R) and defining a conserved haplotype of ∼750 kb across all breeds. These results demonstrate the high quality of this SNP genotyping resource, its usefulness in diverse genome analyses of the horse, and potential use in related species., Author Summary We utilized the previously generated horse genome sequence and a large SNP database to design an ∼54,000 SNP assay for use in the domestic horse and related species. The utility of this SNP array was demonstrated through genome-wide linkage disequilibrium, inbreeding and genetic distance measurements within breeds, as well as multidimensional scaling and parsimony analysis. Association mapping confirmed a large conserved segment containing the chestnut coat color locus in domestic horses. We also assess the utility of the SNP array in related species, including the Przewalski's Horse, zebras, asses, tapirs, and rhinoceros. This SNP genotyping tool will facilitate many genetics applications in equids, including identification of genes for health and performance traits, and compelling studies of the origins of the domestic horse, diversity within breeds, and evolutionary relationships among related species.

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International audience; Following the recent discovery of new Brucella strains from different animal species and from the environment, ten Brucella species are nowadays included in the genus Brucella. Although the intracellular trafficking of Brucella is well described, the strategies developed by Brucella to survive and multiply in phagocytic and non-phagocytic cells, particularly to access nutriments during its intracellular journey, are still largely unknown. Metabolism and virulence of Brucella are now considered to be two sides of the same coin. Mechanisms presiding to the colonization of the pregnant uterus in different animal species are not known. Vaccination is the cornerstone of control programs in livestock and although the 519, RB51 (both in cattle) and Rev 1 (in sheep and goats) vaccines have been successfully used worldwide, they have drawbacks and thus the ideal brucellosis vaccine is still very much awaited. There is no vaccine available for pigs and wildlife. Animal brucellosis control strategies differ in the developed and the developing world. Most emphasis is put on eradication and on risk analysis to avoid the re-introduction of Brucella in the developed world. Information related to the prevalence of brucellosis is still scarce in the developing world and control programs are rarely implemented. Since there is no vaccine available for humans, prevention of human brucellosis relies on its control in the animal reservoir. Brucella is also considered to be an agent to be used in bio- and agroterrorism attacks. At the animal/ecosystem/human interface it is critical to reduce opportunities for Brucella to jump host species as already seen in livestock, wildlife and humans. This task is a challenge for the future in terms of veterinary public health, as for wildlife and ecosystem managers and will need a "One Health" approach to be successful.

International audience; In mammals, sex specialization is reflected by differences in brain anatomy and function. Measurable differences are documented in reproductive behavior, cognition, and emotion. We hypothesized that gonadotropin-releasing hormone (GnRH) plays a crucial role in controlling the extent of the brain's sex specificity and that changes in GnRH action during critical periods of brain development, such as puberty, will result in altered sex-specific behavioral and physiological patterns. We blocked puberty in half of the 48 same-sex Scottish mule Texel cross sheep twins with GnRH analog (GnRHa) goserelin acetate every 3 weeks, beginning just before puberty. To determine the effects of GnRHa treatment on sex-specific behavior and emotion regulation in different social contexts, we employed the food acquisition task (FAT) and measurement of heart rate variability (HRV). ANOVA revealed significant sex and sex x treatment interaction effects, suggesting that treated males were more likely to leave their companions to acquire food than untreated, while the opposite effect was observed in females. Concordant results were seen in HRV; treated males displayed higher HRV than untreated, while the reverse pattern was found in females, as shown by significant sex and sex x treatment interaction effects. We conclude that long-term prepubertal GnRHa treatment significantly affected sex-specific brain development, which impacted emotion and behavior regulation in sheep. These results suggest that GnRH is a modulator of cognitive function in the developing brain and that the sexes are differentially affected by GnRH modulation.

The closely related bacterial species Bacillus cereus and Bacillus weihenstephanensis are adapted to the mesophilic and the psychrotrophic temperature range, respectively. While B. cereus strains are associated with foodborne diseases, B. weihenstephanensis strains are so far not, although similar virulence genes are found in both species. Our investigations show that both species were virulent in the insect model, Galleria mellonella, following infection via oral and haemocoel routes. However, virulence of B. weihenstephanensis was much higher at 15 degrees C than at 37 degrees C. Furthermore, a temperature-dependent difference between the species was seen in a cell culture cytotoxicity assay. In summary, our results demonstrate for the first time virulence of B. weihenstephanensis strains in an in vivo model. In addition, we found that G. mellonella is a useful model for studies of the psychrotolerant species of the B. cereus group, suggesting that insects might be an ecological growth niche for several members of this bacterial group.

International audience; The chiral pharmacokinetics and pharmacodynamics of ketoprofen were investigated in a placebo-controlled study in piglets after intramuscular administration of 6 mg⁄ kg racemic ketoprofen. The absorption half-lives of both enantiomers were short, and S-ketoprofen predominated over R-ketoprofen in plasma. A kaolin-induced inflammation model was used to evaluate the antiinflammatory,antipyretic and analgesic effects of ketoprofen. Skin temperatures increased after the kaolin injection, but the effect of ketoprofen was small. No significant antipyretic effects could be detected, but body temperatures tended tobe lower in the ketoprofen-treated piglets. Mechanical nociceptive threshold testing was used to evaluate the analgesic effects. The piglets in the ketoprofentreatedgroup had significantly higher mechanical nociceptive thresholds compared to the piglets in the placebo group for 12–24 h following the treatment. Pharmacokinetic ⁄ pharmacodynamic modelling of the results from the mechanical nociceptive threshold testing gave a median IC50 for S-ketoprofen of 26.7 lg ⁄ mL and an IC50 for R-ketoprofen of 1.6 lg ⁄ mL. This indicates that Rketoprofenis a more potent analgesic than S-ketoprofen in piglets. Estimated ED50 for racemic ketoprofen was 2.5 mg⁄ kg.

Background Gene expression profiling studies of mastitis in ruminants have provided key but fragmented knowledge for the understanding of the disease. A systematic combination of different expression profiling studies via meta-analysis techniques has the potential to test the extensibility of conclusions based on single studies. Using the program Pointillist, we performed meta-analysis of transcription-profiling data from six independent studies of infections with mammary gland pathogens, including samples from cattle challenged in vivo with S. aureus, E. coli, and S. uberis, samples from goats challenged in vivo with S. aureus, as well as cattle macrophages and ovine dendritic cells infected in vitro with S. aureus. We combined different time points from those studies, testing different responses to mastitis infection: overall (common signature), early stage, late stage, and cattle-specific. Results Ingenuity Pathway Analysis of affected genes showed that the four meta-analysis combinations share biological functions and pathways (e.g. protein ubiquitination and polyamine regulation) which are intrinsic to the general disease response. In the overall response, pathways related to immune response and inflammation, as well as biological functions related to lipid metabolism were altered. This latter observation is consistent with the milk fat content depression commonly observed during mastitis infection. Complementarities between early and late stage responses were found, with a prominence of metabolic and stress signals in the early stage and of the immune response related to the lipid metabolism in the late stage; both mechanisms apparently modulated by few genes, including XBP1 and SREBF1. The cattle-specific response was characterized by alteration of the immune response and by modification of lipid metabolism. Comparison of E. coli and S. aureus infections in cattle in vivo revealed that affected genes showing opposite regulation had the same altered biological functions and provided evidence that E. coli caused a stronger host response. Conclusions This meta-analysis approach reinforces previous findings but also reveals several novel themes, including the involvement of genes, biological functions, and pathways that were not identified in individual studies. As such, it provides an interesting proof of principle for future studies combining information from diverse heterogeneous sources.

Chantier qualité GA; International audience; Atlantic salmon Salmo salar microsatellite markers from a large database were analysed and selected with technical, economic and genetic criteria to provide an optimized set of polymorphic DNA markers for the analysis of the genetic diversity and the structure of anadromous Atlantic salmon populations. A set of 37 microsatellite markers was identified that are easy to use and provide a high level of differentiation power.

International audience; Human BDCA3+ dendritic cells (DCs) were suggested to be homologous to mouse CD8alpha+ DCs. We demonstrate that human BDCA3+ DCs are more efficient than their BDCA1+ counterparts or plasmacytoid DCs (pDCs) in cross-presenting antigen and activating CD8+ T cells, which is similar to mouse CD8alpha+ DCs as compared with CD11b+ DCs or pDCs, although with more moderate differences between human DC subsets. Yet, no specific marker was known to be shared between homologous DC subsets across species. We found that XC chemokine receptor 1 (XCR1) is specifically expressed and active in mouse CD8alpha+, human BDCA3+, and sheep CD26+ DCs and is conserved across species. The mRNA encoding the XCR1 ligand chemokine (C motif) ligand 1 (XCL1) is selectively expressed in natural killer (NK) and CD8+ T lymphocytes at steady-state and is enhanced upon activation. Moreover, the Xcl1 mRNA is selectively expressed at high levels in central memory compared with naive CD8+ T lymphocytes. Finally, XCR1-/- mice have decreased early CD8+ T cell responses to Listeria monocytogenes infection, which is associated with higher bacterial loads early in infection. Therefore, XCR1 constitutes the first conserved specific marker for cell subsets homologous to mouse CD8alpha+ DCs in higher vertebrates and promotes their ability to activate early CD8+ T cell defenses against an intracellular pathogenic bacteria.

Contributeurs Inra de l'European Cattle Genetic Diversity Consortium pour cet article : A. Oulmouden, H. Levéziel, Inra, UMR 1061, Limoges, France; The introduction of livestock species in Europe has been followed by various genetic events, which created a complex spatial pattern of genetic differentiation. Spatial principal component (sPCA) analysis and spatial metric multidimensional scaling (sMDS) incorporate geography in multivariate analysis. This method was applied to three microsatellite data sets for 45 goat breeds, 46 sheep breeds, and 101 cattle breeds from Europe, Southwest Asia, and India. The first two sPCA coordinates for goat and cattle, and the first sPCA coordinate of sheep, correspond to the coordinates of ordinary PCA analysis. However, higher sPCA coordinates suggest, for all three species, additional spatial structuring. The goat is the most geographically structured species, followed by cattle. For all three species, the main genetic cline is from southeast to northwest, but other geographic patterns depend on the species. We propose sPCA and sMDS to be useful tools for describing the correlation of genetic variation with geography.

Background Classification of bacteria within the genus Brucella has been difficult due in part to considerable genomic homogeneity between the different species and biovars, in spite of clear differences in phenotypes. Therefore, many different methods have been used to assess Brucella taxonomy. In the current work, we examine 32 sequenced genomes from genus Brucella representing the six classical species, as well as more recently described species, using bioinformatical methods. Comparisons were made at the level of genomic DNA using oligonucleotide based methods (Markov chain based genomic signatures, genomic codon and amino acid frequencies based comparisons) and proteomes (all-against-all BLAST protein comparisons and pan-genomic analyses). Results We found that the oligonucleotide based methods gave different results compared to that of the proteome based methods. Differences were also found between the oligonucleotide based methods used. Whilst the Markov chain based genomic signatures grouped the different species in genus Brucella according to host preference, the codon and amino acid frequencies based methods reflected small differences between the Brucella species. Only minor differences could be detected between all genera included in this study using the codon and amino acid frequencies based methods. Proteome comparisons were found to be in strong accordance with current Brucella taxonomy indicating a remarkable association between gene gain or loss on one hand and mutations in marker genes on the other. The proteome based methods found greater similarity between Brucella species and Ochrobactrum species than between species within genus Agrobacterium compared to each other. In other words, proteome comparisons of species within genus Agrobacterium were found to be more diverse than proteome comparisons between species in genus Brucella and genus Ochrobactrum. Pan-genomic analyses indicated that uptake of DNA from outside genus Brucella appears to be limited. Conclusions While both the proteome based methods and the Markov chain based genomic signatures were able to reflect environmental diversity between the different species and strains of genus Brucella, the genomic codon and amino acid frequencies based comparisons were not found adequate for such comparisons. The proteome comparison based phylogenies of the species in genus Brucella showed a surprising consistency with current Brucella taxonomy.

The domestication of livestock represented a crucial step in human history. By using endogenous retroviruses as genetic markers, we found that sheep differentiated on the basis of their "retrotype" and morphological traits dispersed across Eurasia and Africa via separate migratory episodes. Relicts of the first migrations include the Mouflon, as well as breeds previously recognized as "primitive" on the basis of their morphology, such as the Orkney, Soay, and the Nordic short-tailed sheep now confined to the periphery of northwest Europe. A later migratory episode, involving sheep with improved production traits, shaped the great majority of present-day breeds. The ability to differentiate genetically primitive sheep from more modern breeds provides valuable insights into the history of sheep domestication.

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The aim of the present study was to study the effects of social instability (regrouping) during the second trimester (7 weeks) of pregnancy on aggression, cortisol concentrations and growth in goats and its consequences for survival, growth and some aspects of behavioural development in the kids. Six weeks after mating, 32 goats were distributed into eight groups. In four of the groups, randomly chosen pairs of goats were rotated between groups every Monday morning (around 08:30 h) for 7 weeks (unstable groups), starting 6 weeks into gestation (second trimester). The remaining four groups were kept stable throughout the entire pregnancy (stable groups). The adult goats were video recorded for 6 h twice a week for the first, second, fourth and seventh regrouping and for the 2 last weeks before expected birth. Blood samples of the adult goats were collected in a period from I week before the start of the first regrouping and until I week before expected birth. Blood samples from a maximum of two kids from each litter were collected at 3 weeks of age. Two kids from each litter were subjected to two types of behavioural tests: a 'social test' at the age of 1 and 7 weeks and a 'novel object test' at the age of 5 weeks. Except for the higher aggression level in the unstable groups, social instability did not have any other significant effects on factors such as growth, cortisol level or kid production in the goats. There were only minor effects on the behavioural development in the goat kids. However, kids from the unstable groups showed more escape attempts in the first trial of a,social test', spent more time in contact with a novel object and unfamiliar kids, and showed less fear (escape attempts) after they were given one exposure to the social test situation. Furthermore, kids from unstable groups had a lower basal cortisol level than kids from stable groups. (C) 2008 Elsevier B.V. All rights reserved.

Despite their key role in a wide range of fields relating to animal and public health, there is currently a lack of veterinary pathologists in Europe. In 1999, to help address the problem, the European College of Veterinary Pathologists (ECVP) and the European Society of Veterinary Pathology (ESVP) established a joint Education Committee. In this Special Article, Professor Anja Kipar and colleagues, all members of the committee, describe the ECVP/ESVP Summer Schools in Veterinary Pathology programme, which aims to provide high-quality research training for veterinary pathologists from all over Europe and beyond.

International audience; An accurate screening of blood donors would constitute a major improvement for transfusion safety from vCJD infection. Several companies already claim positive results with new ante mortem tests for prion in blood but no standardized samples are available to allow comparative evaluations. In the context of the NeuroPrion BloodDiag project, we have constructed a library of blood fraction samples from sheep for the evaluation of these new tests. Sheep were experimentally infected with scrapie and, in parallel with healthy controls, have been sampled regularly since 2004. Blood was fractionated by the French Transfusion Center of Lille (France) using a protocol similar to the one used for humans. With complete information about the animals (genotype, source of contamination, days post-contamination and presence of clinical signs), this library comprises now more than 8000 aliquots from over 110 different blood samples. Assessment of infectivity of the different blood fractions (plasma, platelets, red cells and buffy-coat) has been performed by inoculation of transgenic mice overexpressing ovine PrP gene (tg338). Successful transmissions were observed with the buffy coat of several animals. Now, two years post-infection, some positive results are also being observed with plasma samples inoculated by intraperitoneal route (a still ongoing study). This bank constitutes a powerful tool for the evaluation of blood tests under development for use as pre-clinical diagnostic screens for TSE infection.

The Neolithic introduction of domestic cattle into Europe was followed by differential adaptation, selection, migration and genetic isolation, leading ultimately to the emergence of specialized breeds. We have studied the differentiation of European cattle by amplified fragment length polymorphism (AFLP) fingerprinting. Combining AFLP data sets from two laboratories yielded 81 biallelic polymorphic markers scored in 19-22 individual animals from 51 breeds. Model-based clustering differentiated Podolian cattle as well as French and Alpine breeds from other European cattle. AFLP genetic distances correlated well with microsatellite-based genetic distances calculated for the same breeds. However, the AFLP data emphasized the divergence of taurine and indicine cattle relative to the variation among European breeds and indicated an Eastern influence on Italian and Hungarian Podolian breeds. This probably reflects import from the East after the original introduction of domestic cattle into Europe. Our data suggest that Italian cattle breeds are relatively diverse at the DNA sequence level.

Background Three enterotoxins are implicated in diarrhoeal food poisoning due to Bacillus cereus: Haemolysin BL (Hbl), Non-haemolytic enterotoxin (Nhe), and Cytotoxin K (CytK). Toxin gene profiling and assays for detection of toxin-producing stains have been used in attempts to evaluate the enterotoxic potential of B. cereus group strains. B. cereus strain NVH 391/98, isolated from a case of fatal enteritis, was genetically remote from other B. cereus group strains. This strain lacked the genes encoding Hbl and Nhe, but contains CytK-1. The high virulence of this strain is thought to be due to the greater cytotoxic activity of CytK-1 compared to CytK-2, and to a high level of cytK expression. To date, only three strains containing cytK-1 have been identified; B. cereus strains NVH 391/98, NVH 883/00, and INRA AF2. Results A novel gene variant encoding Nhe was identified in these three strains, which had an average of 80% identity in protein sequence with previously identified Nhe toxins. While culture supernatants containing CytK and Nhe from NVH 391/98 and INRA AF2 were highly cytotoxic, NVH 883/00 expressed little or no CytK and Nhe and was non-cytotoxic. Comparative sequence and expression studies indicated that neither the PlcR/PapR quorum sensing system, nor theYvrGH and YvfTU two-component systems, were responsible for the observed difference in toxin production. Additionally, phylogenetic analysis of 13 genes showed that NVH 391/98, NVH 883/00, and INRA AF2 comprise a novel cluster of strains genetically distant from other B. cereus group strains. Conclusion Due to its divergent sequence, the novel nhe operon had previously not been detected in NVH 391/98 using PCR and several monoclonal antibodies. Thus, toxigenic profiling based on the original nhe sequence will fail to detect the toxin in this group of strains. The observation that strain NVH 883/00 carries cytK-1 but is non-cytotoxic indicates that the detection of this gene variant is not a sufficient criterion for identification of highly cytotoxic strains. The presence of the novel nhe operon and the cytK-1 gene variant in this cluster of strains reflect their phylogenetically remote relationship towards other B. cereus group strains.

International audience; Haemolysin BL (HBL) and nonhaemolytic enterotoxin (Nhe), each consisting of three components, represent the major enterotoxins produced by Bacillus cereus. To evaluate the expression of these toxins, a set of 100 B. cereus strains was examined. Molecular biological characterization showed that 42% of the strains harboured the genes for HBL and 99% for Nhe. The production of all Nhe and HBL components were analyzed using specific antibodies and, in culture supernatants, detectable levels of HBL and Nhe were found for 100% of hbl-positive and 96% of nhe-positive strains. The concentrations of the HBL–L2 and NheB component ranged from 0.02 to 5.6 mgmL 1 and from 0.03 to 14.2 mgmL 1, respectively. Comparison of the amount of NheB produced by food poisoning and food/environmental strains revealed that the median value for all food poisoning strains was significantly higher than for the food/environmental isolates. The data presented in this study provide evidence that specific and quantitative determination of the enterotoxins is necessary to evaluate the toxic potential of B. cereus. In particular, the level of Nhe seems to explain most of the cytotoxic activity of B. cereus isolates and may indicate a highly diarrheic potential.

International audience; Bacillus cereus is the causative agent of gastrointestinal diarrhoea. At least three known enterotoxins may be involved in this syndrome: nonhaemolytic (Nhe) enterotoxin, Hbl enterotoxin and cytotoxin K. Two different forms were recently described for cytotoxin K, encoded by cytK-1 and cytK-2 genes. The CytK-1 toxin appeared to carry a high toxicity, but there is currently no method available to rapidly detect and discriminate the B. cereus strains able to produce this CytK-1 form. In this study, a duplex PCR assay was developed and validated on 162 known cytotoxin-containing strains. This PCR method is the first molecular tool to provide rapid detection and discrimination of cytK-1- and cytK-2-carrying B. cereus strains.

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