Carsten Krischek

Comparative Analysis and Limitations of Ethidium Monoazide and Propidium Monoazide Treatments for the Differentiation of Viable and Nonviable Campylobacter Cells

ABSTRACT The lack of differentiation between viable and nonviable bacterial cells limits the implementation of PCR-based methods for routine diagnostic approaches. Recently, the combination of a quantitative real-time PCR (qPCR) and ethidium monoazide (EMA) or propidium monoazide (PMA) pretreatment has been described to circumvent this disadvantage. In regard to the suitability of this approach for Campylobacter spp., conflicting results have been reported. Thus, we compared the suitabilities of EMA and PMA in various concentrations for a Campylobacter viability qPCR method. The presence of either intercalating dye, EMA or PMA, leads to concentration-dependent shifts toward higher threshold cycle (CT ) values, especially after EMA treatment. However, regression analysis resulted in high correlation coefficient (R 2) values of 0.99 (EMA) and 0.98 (PMA) between Campylobacter counts determined by qPCR and culture-based enumeration. EMA (10 μg/ml) and PMA (51.10 μg/ml) removed DNA selectively from nonviable cells in mixed samples at viable/nonviable ratios of up to 1:1,000. The optimized EMA protocol was successfully applied to 16 Campylobacter jejuni and Campylobacter coli field isolates from poultry and indicated the applicability for field isolates as well. EMA-qPCR and culture-based enumeration of Campylobacter spiked chicken leg quarters resulted in comparable bacterial cell counts. The correlation coefficient between the two analytical methods was 0.95. Nevertheless, larger amounts of nonviable cells (>104) resulted in an incomplete qPCR signal reduction, representing a serious methodological limitation, but double staining with EMA considerably improved the signal inhibition. Hence, the proposed Campylobacter viability EMA-qPCR provides a promising rapid method for diagnostic applications, but further research is needed to circumvent the limitation.

Color values and other meat quality characteristics of breast muscles collected from 3 broiler genetic lines slaughtered at 2 ages

Broilers from the lines Ross 308, Ross 708, and Cobb 700 were slaughtered at 28 and 41 d of age at a commercial abattoir. After slaughter, the carcass, breast, and leg weights as well as the breast and leg yields were determined. Further investigations analyzed the color [lightness (L*), redness (a*), and yellowness (b*)], pH at 24 h postmortem, electrical conductivity (EC), drip loss, grill loss, and shear force values as well as the muscle fiber cross-sectional areas of the breast muscles. The 41-d-old broilers had higher carcass, breast, and leg weights than the 28-d-old birds. The breast yield values were higher and the leg yields were lower in the 41-d-old broilers. The fiber cross-sectional area values were also higher in the older birds. Within the younger birds the slaughter characteristics were approximately comparable among the lines. The EC, L*, grill loss, and shear force values increased but the drip loss and a* values decreased with the age of the broiler. The genetic lines differed within the 28-d-old broilers with regard to EC, grill loss, and shear force values and within the 41-d-old broilers with regard to the EC, L*, grill loss, and shear force values. The pH correlated negatively with the EC, L*, b*, drip loss, and shear force values. During storage, L* and b* values of the breast muscles increased and a* values decreased in all genetic lines, whereas the L* values were generally higher in the older broilers and the a* and b* results were generally higher in the breast muscles of the younger broilers. In conclusion, the carcass and meat quality characteristics of broilers changed with age with positive (carcass and breast muscle weight, drip loss) but also negative (L*, a*, grill loss) effects. The effect of the genetic line was rather low. Despite the age-related changes of meat quality parameter, the pH values remained unchanged, indicating muscle structural influences on the muscle-to-meat-transition with increasing age of the broiler.

Adenine nucleotide concentrations and glycolytic enzyme activities in longissimus muscle samples of different pig genotypes collected before and after slaughter.

Longissimus muscle samples from the pig genotypes Duroc (Du), Pietrain (MHS homozygote negative (PiNN), positive (PiPP)) and a Duroc-Pietrain crossbreed (DuPi) were analyzed. The PiPP samples showed a faster pH drop and higher electrical conductivity, drip loss and lightness values. Before slaughter the concentrations of the adenine nucleotides were comparable between the genotypes, but 40 min after slaughter (p.m.) the ATP concentrations decreased and IMP increased, to a higher extent in the PiPP pigs. The nucleotide values of the 12 h p.m. samples were again comparable. Activities of glycogen phosporylase (GP), phosphofructokinase (PFK) and lactate dehydrogenase (LDH) were nearly similar before slaughter. Forty minutes after slaughter the LDH activities increased in all pigs and the PFK activities in all genotypes but not in the PiPP. GP results were rather inconsistent indicating an earlier activation of this enzyme. The study showed that the reduced meat quality in the PiPP pigs is accompanied with rapid ATP degradation and accelerated enzyme activation.

Low temperature cooking of pork meat - Physicochemical and sensory aspects.

Low-temperature cooking is increasingly used in the food sector. This study compared three different low temperature heating methods and one conventional cooking procedure of pork meat in a combi steamer with special emphasis on sensory parameters. Low temperature, long time (LTLT) treatments over 20h at 53°C or 58°C (LTLT 53°C or 58°C) showed considerable effects on meat tenderization. Heating to a core temperature of 60°C (low temperature method=LT) at 60°C oven temperature resulted in less tender but clearly juicier meat. LTLT 53°C and LT were evaluated as being equally acceptable by the panelists. The tenderest meat (LTLT 58°C) was mainly rejected because of a crumbly and dry mouth feeling. Conventional heating to a core temperature of 80°C at 180°C oven temperature resulted in low eating quality due to high toughness and low juiciness.

Influence of intermittent administration of parathyroid hormone on muscle tissue and bone healing in orchiectomized rats or controls.

Influence of human parathyroid hormone (hPTH 1-34) on muscle and bone healing was studied in either orchiectomized (Orx at 8 months of age) or sham-operated male rats. Eleven-month-old Sprague-Dawley rats underwent bilateral transverse metaphyseal osteotomy of tibia and were divided into four groups (n=12): 1) sham-vehicle, 2) sham group-PTH everyday, 3) Orx-vehicle, 4) Orx-PTH everyday, and 5) Orx-PTH every other day. PTH dosage was 40  μg/kg body weight. After 5 weeks, fiber cross-sectional area, capillary density, and enzyme activity (lactate dehydrogenase, citrate synthase, and complex I) were measured in soleus (MS), gastrocnemius (MG), and longissimus (ML) muscles; tibiae were analyzed by computed tomographical, histological, and gene expression analyses. The effect of PTH in all rats was increased serum osteocalcin, cortical and callus densities and callus area. In sham rats capillary density was increased in limb muscles (MS: 1.3-1.7, MG: 1.2-1.4 capillaries/fiber), and rate of osseous bridging of osteotomy was enhanced (67-100%). In Orx rats serum creatine kinase was decreased (6670-2847 U/l), and bone genes (Igf-1, osteoprotegerin, and receptor activator of nuclear factor kB ligand) were up-regulated. Cross-sectional area, enzyme activity, food intake, weight of body, visceral organs, adipose tissue, MG, and MS were not affected by PTH. PTH had a favorable effect on muscle capillary density and improved bone healing being more effective in sham rats and having no adverse systemic effect. The effect was less if PTH was applied every other day. The findings may show up trends for therapeutic treatment of male patients.

High pressure as an alternative processing step for ham production

As high pressure processing (HPP) is becoming more and more important in the food industry, this study examined the application of HPP (500 and 600MPa) as a manufacturing step during simulated ham production. By replacing conventional heating with HPP steps, ham-like texture or color attributes could not be achieved. HPP products showed a less pale, less red appearance, softer texture and higher yields. However, a combination of mild temperature (53°C) and 500MPa resulted in parameters more comparable to cooked ham. We conclude that HPP can be used for novel food development, providing novel textures and colors. However, when it comes to ham production, a heating step seems to be unavoidable to obtain characteristic ham properties.

Muscle Transcriptional Profile Based on Muscle Fiber, Mitochondrial Respiratory Activity, and Metabolic Enzymes

Skeletal muscle is a highly metabolically active tissue that both stores and consumes energy. Important biological pathways that affect energy metabolism and metabolic fiber type in muscle cells may be identified through transcriptomic profiling of the muscle, especially ante mortem. Here, gene expression was investigated in malignant hyperthermia syndrome (MHS)-negative Duroc and Pietrian (PiNN) pigs significantly differing for the muscle fiber types slow-twitch-oxidative fiber (STO) and fast-twitch-oxidative fiber (FTO) as well as mitochondrial activity (succinate-dependent state 3 respiration rate). Longissimus muscle samples were obtained 24 h before slaughter and profiled using cDNA microarrays. Differential gene expression between Duroc and PiNN muscle samples were associated with protein ubiquitination, stem cell pluripotency, amyloid processing, and 3-phosphoinositide biosynthesis and degradation pathways. In addition, weighted gene co-expression network analysis within both breeds identified several co-expression modules that were associated with the proportion of different fiber types, mitochondrial respiratory activity, and ATP metabolism. In particular, Duroc results revealed strong correlations between mitochondrion-associated co-expression modules and STO (r = 0.78), fast-twitch glycolytic fiber (r = -0.98), complex I (r=0.72) and COX activity (r = 0.86). Other pathways in the protein-kinase-activity enriched module were positively correlated with STO (r=0.93), while negatively correlated with FTO (r = -0.72). In contrast to PiNN, co-expression modules enriched in macromolecule catabolic process, actin cytoskeleton, and transcription activator activity were associated with fiber types, mitochondrial respiratory activity, and metabolic enzyme activities. Our results highlight the importance of mitochondria for the oxidative capacity of porcine muscle and for breed-dependent molecular pathways in muscle cell fibers.

Effects of a higher incubation temperature between embryonic day 9 and 12 on growth and meat quality characteristics of turkeys

1. The study investigated the influence of manipulating incubation temperature for a short period on the post-hatch development up to week 16 in male and female BUT Big 6 turkeys. 2. Eggs were incubated at a control temperature of 37·5°C and 55% RH until d 18 when transferred to a hatcher at 37·5°C and 85% RH. For a 4 d period between embryonic day 9 (ED 9) and 12, eggs were incubated at 38·5°C and 55% RH (HT). 3. Birds were slaughtered at 16 weeks of age to analyse meat quality parameters of the Musculus pectoralis superficialis (MPS). 4. Across both incubation treatments, the turkey males had significantly higher live and breast weights, but lower breast yields than the females. The sex of the animals only influenced the yellowness of the MPS with lower values in the males. 5. Temperature manipulation resulted in significantly decreased live weights of HT birds compared with the control animals across all ages in both sexes. No impact of incubation treatment on meat quality characteristics was found. 6. The results indicate a negative effect of higher incubation temperature on the post-hatch growth, possibly by influencing the mechanisms that regulate the hypertrophic growth of the muscle fibres.

Altered incubation temperatures between embryonic Days 7 and 13 influence the weights and the mitochondrial respiratory and enzyme activities in breast and leg muscles of broiler embryos.

Altering incubation temperature during embryogenesis has an impact on chicken embryo growth, but the underlying molecular mechanisms are not understood; the present study was performed to address these changes. Broiler eggs were incubated at low (36.8°C), control (37.8°C), and high (38.8°C) temperatures between Embryonic Day (ED) 7 and 10 or ED 10 and 13, which cover critical periods of embryonic myogenesis. The embryos were then dissected immediately after treatment on ED 10 or 13 to assess body, liver, and heart weights as well as to analyze breast and leg muscle fibers for their mitochondrial respiratory activity (MRA). Breast muscle samples were additionally used to evaluate the activity of enzymes involved in energy metabolism and cell‐cycle progression. ED‐10 embryos incubated at 38.8°C showed elevated weights (body, liver, and heart), MRA, and activities of lactate dehydrogenase and cytochrome oxidase compared to the ED‐10 embryos incubated at 36.8°C. Similarly, the ED‐13 embryos incubated at 38.8°C showed elevated body weight, MRA, and activities of glycogen phosphorylase, phosphofructokinase, and cytochrome oxidase compared to their 36.8°C counterparts. Embryos incubated at the normal temperature (37.8°C), however, showed variable differences from those incubated at 38.8°C versus 36.8°C. Cell‐cycle enzyme activities were not impacted by the different temperature treatments. Thus, an increase or decrease in the incubation temperature during embryonic broiler myogenesis results in altered embryo activity, muscle energy metabolism, and activity‐dependent muscle growth. Mol. Reprod. Dev. 83: 71–78, 2016.

Influencing factors and applicability of the viability EMA-qPCR for a detection and quantification of Campylobacter cells from water samples.

In recent years, increasing numbers of human campylobacteriosis cases caused by contaminated water have been reported. As the culture-based detection of Campylobacter is time consuming and can yield false-negative results, the suitability of a quantitative real-time PCR method in combination with an ethidium monoazide pretreatment of samples (EMA-qPCR) for the rapid, quantitative detection of viable Campylobacter cells from water samples was investigated. EMA-qPCR has been shown to be a promising rapid method for the detection of viable Campylobacter spp. from food samples. Application of membrane filtration and centrifugation, two methods frequently used for the isolation of bacteria from water, revealed a mean loss of up to 1.08 log10 cells/ml from spiked samples. Both methods used alone lead to a loss of dead bacteria and accumulation of viable bacteria in the sample as shown by fluorescence microscopy. After filtration of samples, no significant differences could be detected in subsequent qPCR experiments with and without EMA pretreatment compared to culture-based enumeration. High correlations (R2 = 0.942 without EMA, R2 = 0.893 with EMA) were obtained. After centrifugation of samples, qPCR results overestimated Campylobacter counts, whereas results from both EMA-qPCR and the reference method were comparable. As up to 81.59% of nonviable cells were detected in pond water, EMA-qPCR failed to detect correct quantities of viable cells. However, analyses of spiked tap water samples revealed a high correlation (R2 = 0.863) between results from EMA-qPCR and the reference method. After membrane filtration, EMA-qPCR was successfully applied to Campylobacter field isolates, and results indicated an advantage over qPCR by analysing defined mixtures of viable and nonviable cells. In conclusion, EMA-qPCR is a suitable method to detect viable Campylobacter from water samples, but the isolation technique and the type/quality of the water sample impact the results.