Øyvin Østensvik

Survival of Campylobacter on Frozen Broiler Carcasses as a Function of Time

In the Norwegian Action Plan against Campylobacter in broilers, carcasses from flocks identified as positive before slaughter are either heat treated or frozen for 5 weeks to reduce the number of Campylobacter. The objective of this study was to estimate the effect of freezing time and predict the number of Campylobacter on naturally infected or contaminated broiler carcasses following freezing for 2, 4, 6, 8, 10, 13, 21, 35, and 120 days by nonparametric and parametric linear statistical models. From each of the five flocks, 27 carcasses were sampled. Each carcass was cut in two pieces along the chest bone. Half was put into the freezer (-20 degrees C), whereas the other was deskinned and quantitative culturing was conducted from a 10-g sample of the skin. Fifteen frozen halves were selected at random at each time point following freezing from 2 to 120 days, and skin samples from these were cultured quantitatively and qualitatively. In regard to the log reduction of Campylobacter, almost similar results were obtained using three statistical methods; median regression on the change in Campylobacter counts, zero-inflated negative binomial regression, and a Bayesian Markov chain Monte Carlo (decay) model on original counts. Overall, a 2-log reduction of Campylobacter was obtained after 3 weeks of freezing. Only a marginal extra effect was observed when extending the freezing to 5 weeks. Although freezing appears to be an efficient way to reduce the level of Campylobacter on broiler carcasses, in 80% of the carcasses Campylobacter could still be detected using quantitative culturing following 120 days of freezing. Based on the high number of zeros, these data should be modeled by a zero-inflated model. The best statistical fit in regard to goodness-of-fit measures was the zero-inflated negative binomial log link model, closely followed by the Poisson model. Thus, in our continued search for a better way to describe the data, we used the Poisson distribution in the mixed Bayesian decay models.

SIMULTANEOUS DETERMINATION OF THE CYANOTOXINS ANATOXIN A, MICROCYSTIN DESMETHYL-3, LR, RR, AND YR IN FISH MUSCLE USING LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY

A liquid chromatographic-atmospheric pressure ionization ion spray method for the determination of anatoxin-a, microcystin desmethyl-3, LR, RR, and YR in fish muscle, is described. A salmon muscle sample was extracted with a mixture of methanol-water and acetone. The organic layer was evaporated and cleaned-up using LMS solid phase extraction columns. The method is simple, specific, and requires only small quantities of chemical reagents. The lower limits of quantification were 15, 2, 10, 1, and 10 ng/g for anatoksin-a, microcystin desmethyl-3, LR, RR, and YR, respectively.

Cytotoxic Bacillus spp. belonging to the B. cereus and B. subtilis groups in Norwegian surface waters

Aims:  To investigate the presence and numbers of Bacillus spp. spores in surface waters and examine isolates belonging to the B. cereus and B. subtilis groups for cytotoxicity, and to discuss the presence of cytotoxic Bacillus spp. in surface water as hazard identification in a risk assessment approach in the food industry.

Microcystin poisoning in roe deer (Capreolus capreolus)

Acute cyanobacterial hepatotoxicosis in a wild roe deer (Capreolus capreolus) from Norway is reported. The diagnosis was based upon the demonstration of typical liver lesions and high liver concentrations of microcystins. The liver was markedly enlarged and histopathological examination revealed diffuse hepatocellular dissociation, degeneration and necrosis and perisinusoidal haemorrhage. Liquid chromatography-mass spectrometry demonstrated the presence of 1361 ng microcystin-YR, -LR and -RR per gram liver (wet weight). This is believed to be the first report of cyanobacterial intoxication in wild mammalian species as confirmed by demonstration of high toxin levels in the animals tissues.

Determination of Flunixin and Tiamulin Hydrogen Fumarate in Meat and Toltrazuril and the Metabolite Toltrazurilsulfon in Meat and Eggs Using LC/MS

Abstract A liquid chromatographic‐atmospheric pressure ionization ion spray method is described for the determination of flunixin (FLU), tiamulin hydrogen fumarate (TIA) in meat, and toltrazuril (TOL) and the metabolite toltrazurilsulfon (TOLS) (Ponazuril) in meat and egg. The method can also be used for the determination of flunixin in milk. Samples were extracted with acetone–tetrahydrofurane, after which the organic layer was separated from water with dichloromethane and evaporated to dryness. The dry residue was diluted in methanol‐l‐heptane sulfonic acid and fat was extracted with hexane, filtered, and injected into the LC–MS. The lower limit of quantification for flunixin and toltrazuril were 5 ng/g and for tiamulin and toltrazurilsulfon in meat 2 ng/g.

DETERMINATION OF AMPROLIUM, ETHOPABATE, LASALOCID, MONENSIN, NARASIN, AND SALINOMYCIN IN FEED BY LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY

ABSTRACT A liquid chromatographic-atmospheric pressure ionization ion spray method for the determination of six coccidiostatics in feed is presented. Feed samples were homogenized with methanol–acetone–tetrahydrofuran. After addition of water, the samples were mixed and centrifuged. The compact bottom layer was re-extracted with methanol–water. After centrifugation, the combined supernatants were diluted and filtered through a Spin-X micro-centrifuge tube. Three different analytical columns were used. The calibration curves were linear in the investigated areas. The described assay offers a number of significant advantages compared to previously published methods for the detection and quantification of some coccidiostatics in feed. No derivatization is required.

Quantification of Bacillus cereus Emetic Toxin (Cereulide) in Figs Using LC/MS

Abstract An LC/MS method is described for the quantification of cereulide, the emetic toxin of Bacillus cereus in figs. The method can also be used for the determination of cereulide in rice. The sample was extracted with a mixture of acetone–tetrahydrofurane, methanol, and water, after which the organic layer was separated from water with chloroform and evaporated to dryness. The dry residue was diluted in chloroform–hexane and purified using a silica solid phase extraction column. The detection limit was 1 ng/g.

Effect of water treatment on the growth potential of Vibrio cholerae and Vibrio parahaemolyticus in seawater.

In laboratory experiments we added Vibrio cholerae and Vibrio parahaemolyticus to bottles with seawater previously treated by filtration, UV, chlorine or ozone. The purpose was to investigate the influence of different treatment techniques on the growth potential of these bacteria in simulated ballast water tanks. Residual oxidants were removed before inoculation, and the bottles were incubated at 21 ± 1 °C. The growth potential of the vibrios was investigated in two different experimental setups, i.e. in presence and absence of added natural microorganisms. In general, V. cholerae and V. parahaemolyticus rapidly lost their culturability after inoculation and storage in untreated seawater, but showed increased survival or growth in the treated water. Highest growth was observed in water previously exposed to high concentrations of ozone. Addition of natural microorganisms reduced the growth of V. cholerae and V. parahaemolyticus.

Impact of rainfall on the hygienic quality of blue mussels and water in urban areas in the Inner Oslofjord, Norway.

The effects of precipitation on the hygienic quality of water and blue mussels collected from five different localities in the urban areas in the Inner Oslofjord were investigated, with samples analysed for Escherichia coli, Salmonella spp., pathogenic Vibrio spp., Norovirus, Sapovirus, Cryptosporidium spp. and Giardia duodenalis. The sampling sites were located at varying distances from the outlet of combined sewer overflows (CSO)-impacted rivers/streams. In general, 1-3 log₁₀ increases in fecal indicator bacteria and human pathogens were observed after heavy rainfalls. Blue mussels appeared to be a useful indicator of the impact of sewage at these sites, and generally a good correlation was identified between concentrations of E. coli and other human pathogens in the mussels. Provision of general advice to the public of avoiding areas near the outlets of CSO-impacted rivers after heavy rainfall may reduce the risk of gastroenteritis by bathers and others that may swallow water during recreational activities.

Effects of hygienic treatments during slaughtering on microbial dynamics and contamination of sheep meat.

The aims of this study were to investigate bacterial dynamics in the sheep meat chain, from fleece to meat trimmings, using both quantitative and qualitative analyses, and to study the effects on microbial load associated with the hygienic interventions of: i) shearing sheep immediately before slaughter, ii) manual steam vacuum pasteurisation, iii) hot water pasteurisation of carcasses, followed by iv) chilling. A further aim was to provide evidence to determine whether or not unshorn sheep should be handled in a processing line separate from that of shorn sheep in Norwegian abattoirs. A total of 176 surface swab samples were collected from three sites along the value chain: i) on fleeces, ii) on carcasses at the end of the slaughter line, and iii) on carcasses after chilling for 24h, and 32 samples were collected from meat trimmings. The results showed that Aerobic Plate Counts (APC) were lower for the shorn group compared to the unshorn group, both on carcasses before chilling and after chilling (difference of 0.8 and 0.9logCFU/1000cm(2) (p≤0.05), respectively) and in meat trimmings (difference of 0.5logCFU/g (p≤0.05)). Hygienic treatments were used on carcasses derived from unshorn sheep, and steam vacuum treatment reduced Escherichia coli, Enterobacteriaceae, and APC before chilling by 1.2, 1.0, and 0.6logCFU/1000cm(2) (p≤0.05), respectively, and hot water pasteurisation, in addition to chilling, reduced E. coli, Enterobacteriaceae, and APC by 0.7, 1.0, and 0.9logCFU/1000cm(2) (p≤0.05), respectively, compared with untreated carcasses. The effect of chilling was shown by the significant reduction of number of carcasses where E. coli were detected; from 65% (13/20) of the shorn group before chilling to 35% (7/20) after chilling, and from 90% (36/40) to 45% (9/20) of the unshorn group. Sequencing of the 16S rRNA gene derived from 316 colonies of Enterobacteriaceae showed a tendency for the relative proportion of the genus Escherichia/Shigella, compared with other genera within Enterobacteriaceae, to be greater for unshorn, untreated sheep than from the other groups at the sampling locations along the meat chain. The study showed that steam vacuum and hot water pasteurisation reduced the contamination of carcasses derived from unshorn sheep, down to the level of the shorn group, and thus can replace the separate processing line for unshorn sheep. Indeed, the low microbial contamination in meat trimmings for all groups indicates that the separate processing line is unnecessary.