Label‐free spectroscopic tissue characterization using fluorescence excitation‐scanning spectral imaging

Spectral imaging approaches provide new possibilities for measuring and discriminating fluorescent molecules in living cells and tissues. These approaches often employ tunable filters and robust image processing algorithms to identify many fluorescent labels in a single image set. Here, we present results from a novel spectral imaging technology that scans the fluorescence excitation spectrum, demonstrating that excitation‐scanning hyperspectral image data can discriminate among tissue types and estimate the molecular composition of tissues. This approach allows fast, accurate quantification of many fluorescent species from multivariate image data without the need of exogenous labels or dyes. We evaluated the ability of the excitation‐scanning approach to identify endogenous fluorescence signatures in multiple unlabeled tissue types. Signatures were screened using multi‐pass principal component analysis. Endmember extraction techniques revealed conserved autofluorescent signatures across multiple tissue types. We further examined the ability to detect known molecular signatures by constructing spectral libraries of common endogenous fluorophores and applying multiple spectral analysis techniques on test images from lung, liver and kidney. Spectral deconvolution revealed structure‐specific morphologic contrast generated from pure molecule signatures. These results demonstrate that excitation‐scanning spectral imaging, coupled with spectral imaging processing techniques, provides an approach for discriminating among tissue types and assessing the molecular composition of tissues. Additionally, excitation scanning offers the ability to rapidly screen molecular markers across a range of tissues without using fluorescent labels. This approach lays the groundwork for translation of excitation‐scanning technologies to clinical imaging platforms.     

Detección molecular de Toxoplasma gondii en carnes para consumo humano en Ibagué, Colombia

Introduction:

Toxoplasma gondii is a parasite with great zoonotic potential. It can infect a broad range of warm-blooded hosts (including livestock) and causes significant losses in the industry. In humans, it has been described as a pathogen in immunosuppressed people, it affects the fetus development in congenital infections, and is associated with various behavioral disorders in healthy people. Humans can acquire T. gondii by consuming undercooked, contaminated meat.

Objective:

To determine T. gondii positivity (currently unknown) in meat for human consumption (i.e., beef, chicken, and pork) in the city of Ibague, Colombia.

Materials and methods:

We used conventional nested PCR and the T. gondii B1 gene sequence as amplification target. We collected samples of meat (N=186) sold in the urban area of Ibagué (62 beef, 62 chicken, and 62 pork samples) and determined the T. gondii positivity percentage for each type of meat.

Results:

The study found an average of 18.8% positivity for all meat samples, pork having the highest percentage (22.5%; 14/62), followed by beef (19.3%; 12/62) and chicken (14.5%; 9/62). The best-amplified products were sequenced by macrogen and aligned with the B1 gene sequences in GenBank, thereby confirming their identity.

Conclusions:

This is the first study of T. gondii prevalence in meat for human consumption carried out in the city of Ibagué and the department of Tolima. All three types of meat sampled represent a risk for local human infection.     

Evolution of raw meat polarization-based properties by means of Mueller matrix imaging

The possibilities of using Mueller matrix (MM) imaging polarimetry to assess meat quality have not yet been sufficiently explored. In the current study, the fresh porcine muscles are imaged at room temperature with a wide-field MM imaging polarimeter over 26 hours to visualize dynamics of tissue optical properties through applying Lu-Chipman decomposition. The frequency distribution histograms (FDHs) and statistical analysis of the MM elements show prominent changes over time. The wavelength spectra of both total depolarization and scalar retardance have dips at 550 nm whereas their values continuously increase with time; the former is referred to the increase of number of scattering events and decrease of myoglobin absorption in the red part of visible spectra related to meat color and freshness, while the latter is associated with the increase in birefringence and meat tenderness. The obtained results are promising to develop a novel fast noncontact optical technique for monitoring of meat quality.     

Front Cover: Label‐free spectroscopic tissue characterization using fluorescence excitation‐scanning spectral imaging (J. Biophotonics 2/2020)

A false‐colored and merged image of fresh, ex vivo rat kidney acquired using an excitation‐scanning hyperspectral imaging system. The spectral image was acquired using excitation wavelengths from 360 to 550 nm. Colors represent principal components extracted from a spectral image cube featuring no added labels or markers. Further details can be found in the article by Peter F. Favreau, Joshua A. Deal, Bradley Harris, et al. ( e201900183 ).

     

PCR detection of psychrophilic Clostridium spp. causing 'blown pack' spoilage of vacuum-packed chilled meats

AIMS: To develop a practical molecular procedure that directly, without isolation, and specifically detects the presence of clostridia which cause 'blown pack' spoilage of vacuum-packed meat. METHODS AND RESULTS: Primer sets and PCR amplification procedures were developed that detect the presence of 16S rDNA gene and/or 16S-23S rDNA internal transcribed spacer fragments of 'blown pack' causing clostridia in meat. The specificity of the developed procedures was evaluated with DNA obtained from close phylogenetic neighbours of 'blown pack' causing clostridia, food clostridia and common meat spoilage microorganisms. The sensitivity of detection was assessed in non-enriched and low-temperature-enriched beef mince inoculated with serially diluted pure cultures of Clostridium estertheticum DSMZ 8809T and Cl. gasigenes DB1AT. The efficacy of detection procedures was evaluated for naturally contaminated vacuum-packed meat samples. Three primer sets, 16SE, 16SDB and EISR, produced amplicons of the expected size with DNA templates from target clostridia, but failed to yield PCR products with DNAs from any other microorganisms tested. With 16SE and 16SDB primers, minimum levels of detection were 104 CFU g(-1) for non-enriched, and 102 CFU g(-1) for enriched meat samples. Based on the established specificity of these primers, as well as DNA sequencing of amplicons, Cl. gasigenes was confirmed as the causative agent of 'blown pack' spoilage in two packs, and Cl. estertheticum as the causative agent in the third. CONCLUSIONS: The developed method can be used for rapid detection of 'blown pack' causing clostridia in commercial blown packs, or following low temperature enrichment, for detection of these microorganisms in meat containing as few as 100 clostridial cells per gram. SIGNIFICANCE AND IMPACT OF THE STUDY: The paper reports practical procedures that can be used for rapid confirmation of the causative agents of clostridial 'blown pack' spoilage in commercial spoiled packs, or for detection of psychrophilic clostridia in epidemiological trace back of 'blown pack' spoilage incidents in meat processing plants.     

Sub millimetre flexible fibre probe for background and fluorescence free Raman spectroscopy

Using the shifted-excitation Raman difference spectroscopy technique and an optical fibre featuring a negative curvature excitation core and a coaxial ring of high numerical aperture collection cores, we have developed a portable, background and fluorescence free, endoscopic Raman probe. The probe consists of a single fibre with a diameter of less than 0.25 mm packaged in a sub-millimetre tubing, making it compatible with standard bronchoscopes. The Raman excitation light in the fibre is guided in air and therefore interacts little with silica, enabling an almost background free transmission of the excitation light. In addition, we used the shifted-excitation Raman difference spectroscopy technique and a tunable 785 nm laser to separate the fluorescence and the Raman spectrum from highly fluorescent samples, demonstrating the suitability of the probe for biomedical applications. Using this probe we also acquired fluorescence free human lung tissue data.     

Species identification in meat by using PCR-generated satellite probes

A convenient DNA-based identification system is described for testing the species origin of meat samples. Probes are generated by PCR with primers binding to species-specific satellite DNA and hybridized to DNA purified from meat. This method is more robust and versatile than methods based on oligonucleotide hybridization. With the exception of a slight cross-reaction of mutton and beef, each probe only recognized the species from which it was derived. Purifying the DNA with a DNA-binding resin improved the sensitivity. Admixtures of 0.1–0.5% can be detected in raw meat and 0.5–5% in autoclaved meat samples. The method can be adapted to detect any eukaryotic species for which species-specific DNA sequences are available. This method has proven its value in the routine inspection of meat samples by revealing more cases of deliberate or accidental species substitution and admixture than conventional techniques. Received 12 January 1998/ Accepted in revised form 15 June 1998     

Techno-economic modeling and assessment of cultivated meat: Impact of production bioreactor scale

Increases in global meat demands cannot be sustainably met with current methods of livestock farming, which has a substantial impact on greenhouse gas emissions, land use, water consumption, and farm animal welfare. Cultivated meat is a rapidly advancing technology that produces meat products by proliferating and differentiating animal stem cells in large bioreactors, avoiding conventional live-animal farming. While many companies are working in this area, there is a lack of existing infrastructure and experience at commercial scale, resulting in many technical bottlenecks such as scale-up of cell culture and media availability and costs. In this study, we evaluate theoretical cultivated beef production facilities with the goal of envisioning an industry with multiple facilities to produce in total 100,000,000 kg of cultured beef per year or ~0.14% of the annual global beef production. Using the computer-aided process design software, SuperPro Designer®, facilities are modeled to create a comprehensive analysis to highlight improvements that can lower the cost of such a production system and allow cultivated meat products to be competitive. Three facility scenarios are presented with different sized production reactors; ~42,000 L stirred tank bioreactor (STR) with a base case cost of goods sold (COGS) of $35/kg, ~211,000 L STR with a COGS of $25/kg, and ~262,000 L airlift reactor (ALR) with a COGS of $17/kg. This study outlines how advances in scaled up bioreactors, alternative bioreactor designs, and decreased media costs are necessary for commercialization of cultured meat products.     

Species authentication of commercial beef jerky based on PCR-RFLP analysis of the mitochondrial 12S rRNA gene

In this study,we determined species-specific variations by analyzing the mitochondrial 12S rRNA gene sequence variation(～440 bp) in 17 newly obtained sequences and 90 published cattle,yak,buffalo,goat,and pig sequences,which represent 62 breeds and 17 geographic regions.Based on the defined species-specific variations,two endonucleases,Alu I and Bfa I,were selected for species authentication using raw meat/tissue samples and the PCR-RFLP method.Goat and pig were identified using the Alu I enzyme,while cattle,yak,and buffalo were identified by digestion with Bfa I.Our approach had relatively high detection sensitivity of cattle DNA in mixed cattle and yak products,with the lowest detectable threshold equaling 20% of cattle DNA in a mixed cattle/yak sample.This method was successfully used to type commercial beef jerky products,which were produced by different companies utilizing various processing technologies.Our results show that several yak jerky products might be implicated in commercial fraud by using cattle meat instead of yak meat.     

Inside Cover

A novel 0.9 mm-diameter intravascular photoacoustic catheter with coaxial excitation and detection was developed to overcome the limitation of imaging range. A miniature ring-shaped ultrasound transducer with a 0.18 mm-diameter orifice in the center was successfully fabricated. The results demonstrated that the coaxial catheter exhibited much better photoacoustic/ultrasound imaging performance from the intima to the adventitia. Further details can be found in the article by Riqiang Lin, Qi Zhang, Shengmiao Lv, Jiaming Zhang, Xiatian Wang, Dongliang Shi, Xiaojing Gong, and Kwok-ho La ( e202200269 ).

     

The liver antioxidant status of fattening lambs is improved by naringin dietary supplementation at 0.15% rates but not meat quality

Twenty Assaf lambs fed barley straw plus a concentrate alone (CONTROL group) or enriched with naringin (1.5 g/kg DM, NARINGIN group) were used to assess the effect of this polyphenolic compound on meat quality attributes. Serum samples were collected for 7 weeks, then the animals were slaughtered and the livers and longissimus thoracis et lumborum muscles extracted for analysis. Triacylglycerol levels in the serum samples tended to show (P = 0.087) lower average values for the NARINGIN group when compared with the CONTROL, but no differences were observed when the meat was analysed for the intramuscular fat content. Lower thiobarbituric acid-reactive substances procedure (TBARS) values (P < 0.001) in the liver of the NARINGIN group were detected, probably as a consequence of naringenin accumulation in this organ. No significant differences were observed in the meat samples concerning TBARS or colour evolution during refrigerated storage, as not enough naringenin would have reached the muscle. Independent of naringin administration, the low levels of the most atherogenic oxysterols must be highlighted as the most important quality score in the lamb meat samples studied.     

Animal performance and meat characteristics in steers reared in intensive conditions fed with different vegetable oils

Enhancing the quality of beef meat is an important goal in terms of improving both the nutritional value for the consumer and the commercial value for producers. The aim of this work was to study the effects of different vegetable oil supplements on growth performance, carcass quality and meat quality in beef steers reared under intensive conditions. A total of 240 Blonde D’ Aquitaine steers (average BW=293.7±38.88 kg) were grouped into 24 batches (10 steers/batch) and were randomly assigned to one of the three dietary treatments (eight batches per treatment), each supplemented with either 4% hydrogenated palm oil (PALM) or fatty acids (FAs) from olive oil (OLI) or soybean oil (SOY). No differences in growth performance or carcass quality were observed. For the meat quality analysis, a steer was randomly selected from each batch and the 6th rib on the left half of the carcass was dissected. PALM meat had the highest percentage of 16:0 (P<0.05) and the lowest n-6/n-3 polyunsaturated fatty acids (PUFA) ratio (P<0.05), OLI had the highest content of t11-18:1 (P<0.01) and c9,t11-18:2 (P<0.05) and SOY showed the lowest value of monounsaturated fatty acids (MUFA) (P<0.001), the highest percentage of PUFA (P<0.01) and a lower index of atherogenicity (P=0.07) than PALM. No significant differences in the sensory characteristics of the meat were noted. However, the results of the principal component analysis of meat characteristics enabled meat from those steers that consumed fatty acids from olive oil to be differentiated from that of steers that consumed soybean oil.     

Effects of cloned-cattle meat on reproductive physiology in rats

The influence of the cloned-cattle meat diets upon reproduction in mammals was rarely studied. This study was performed to analyze the effects of the diets containing cloned-cattle (Korean native beef, Hanwoo) meat on the reproductive physiology in rats. The male and female rats were fed with the diets containing 5% or 10% of normal- (N-5 or N-10) or cloned- (C-5 or C-10) cattle meat during test periods. The rats fed with commercial pellets were used as control. Lower food consumption in normal- and cloned-cattle meat diet groups is detected in both male and female rats compared with that of control (P < 0.05, 0.01 and 0.001). No signs of cloned-cattle meat diets on male reproductive parameters are found in all groups, except for lower sperm deformity in C-5 group (P < 0.05) and higher testosterone concentration in C-10 group (P < 0.05), respectively. There are no significant test substance-related differences of Caesarean section and delivery in dams and external examination and physiological development test in neonate compared with control and normal meat groups. Based on these results, it can be postulated that there are no obvious negative effects on the reproductive physiology in rats fed with cloned-cattle meat diets compared to their comparators.     

Investigation of arcobacters in meat and faecal samples of clinically healthy cattle in Turkey

AIMS: To investigate the presence of Arcobacter spp. in minced beef meat (n = 97) and rectal faecal samples (n = 200) collected from cattle immediately after slaughter at a local abattoir in Turkey. METHODS AND RESULTS: Meat samples were examined using three different isolation procedures (CAT-supplemented media, de Boer arcobacter isolation method and membrane filtration method), but only one method (CAT-supplemented media) was employed for faecal samples. The isolated Arcobacter strains were identified by genus- and species-(multiplex) specific PCR assays. Arcobacter spp. were isolated from 5 and 9.5% of meat and faecal samples respectively. Although the only Arcobacter sp. found in meat samples was Arcobacter butzleri, all three pathogenic species--A. butzleri, A. cryaerophilus and A. skirrowii--were detected in the rectal swabs. No Arcobacter was isolated when the de Boer method was used for minced meat samples but the same five meat samples were found positive for arcobacters when CAT-supplemented media and membrane filtration method were used. CONCLUSIONS: The membrane filtration method was found to be superior to the CAT-supplemented media, because it led to a reduction in competing microflora. However, the necessity for one filter and medium for each sample makes this method somewhat expensive. The multiplex-PCR (m-PCR) assay shortened significantly the time required for the identification of Arcobacter spp. and also removed the possibility of false positive results due to other campylobacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports the isolation of Arcobacter spp. in cattle for the first time in Turkey. The m-PCR assay enables the identification and differentiation of all arcobacters simultaneously in one-step PCR.     

Genetic parameters of meat technological quality traits in a grand-parental commercial line of turkey

Genetic parameters for meat quality traits and their relationships with body weight and breast development were estimated for a total of 420 male turkeys using REML. The birds were slaughtered in a commercial plant and the traits measured included pH at 20 min (pH₂₀) and 24 h post-mortem (pHu) and colour of the breast and thigh meat. The heritabilities of the rate and the extent of the pH fall in the breast muscle were estimated at h² = 0.21 ± 0.04 and h² = 0.16 ± 0.04, respectively. Heritabilities ranging from 0.10 to 0.32 were obtained for the colour indicators in the breast muscle. A marked negative genetic correlation (r_g = -0.80 ± 0.10) was found between pH₂₀ and lightness (L*) of breast meat, both traits corresponding to PSE indicators. The pH₂₀ in the thigh muscle had a moderate heritability (h² = 0.20 ± 0.07) and was partially genetically related to pH₂₀ in the breast muscle (r_g = 0.45 ± 0.17). Body weight and breast yield were positively correlated with both initial and ultimate pH and negatively with the lightness of breast meat.     

Small animal irradiator dose distribution verification using radioluminescence imaging

The main objective of this work was the development of a novel 2D dosimetry approach for small animal external radiotherapy using radioluminescence imaging (RLI) with a commercial complementary metal oxide semiconductor detector. Measurements of RLI were performed on the small animal image‐guided platform SmART, RLI data were corrected for perspective distortion using Matlab. Four irradiation fields were tested and the planar 2D dose distributions and dose profiles were compared against dose calculations performed with a Monte Carlo based treatment planning system and gafchromic film. System linearity and RLI image noise against dose were also measured. The maximum difference between beam size measured with RLI and nominal beam size was less than 8% for all the tested beams. The image correction procedure was able to reduce perspective distortion. A novel RLI approach for quality assurance of a small animal irradiator was presented and tested. Results are in agreement with MC dose calculations and gafchromic film measurements.     

Association of halothane sensitivity with growth and meat quality in pigs

Previous reports have indicated that a proportion of pigs, homozygous normal for the skeletal muscle ryanodine receptor gene (RYR1), was halothane sensitive, and this was associated with poor meat quality when pigs were handled aggressively. This study was conducted to evaluate halothane sensitivity in RYR1-normal pigs, managed under simulated commercial conditions, to ascertain the association of halothane sensitivity with growth rate and meat quality. A total of 363 pigs across four farrowing groups, from seven Landrace sires and 38 Yorkshire–Landrace F1 dams, were tested at 8 weeks of age for halothane sensitivity using a closed system that delivered 5% halothane at 2 l/min for 3 (group 1) or 2 (groups 2 to 4) min. After 1 min, limb rigidity, limb tremors and abdominal discoloration were evaluated on a binomial scale with 0 indicating no reaction and 1 indicating reaction. Testing was repeated 2 days later. At 10 weeks of age, pigs were moved to finishing pens and not moved again until marketing. Within farrowing group, pigs were harvested in one of two groups, and at marketing were moved a distance of 91 m, weighed, tattooed, loaded and transported a distance of 550 km to a commercial harvest plant. After overnight rest, pigs were harvested and the pH of the loin muscle was measured at 45 min (pH45) after stunning. After an 18-h chill, loin muscle pH (pHu), International Commission on Illumination (CIE) L*, a*, b*, color (1 to 6) and marbling (1 to 10) scores and fluid loss percent were collected. Generalized linear mixed models were used to estimate repeatabilities for response to halothane challenge. Repeatabilities for limb rigidity for the front right and left legs were 0.24 and 0.31, respectively, whereas rear right and left leg repeatabilities were 0.19 and 0.17, respectively. Repeatabilities for front right and left leg tremors were 0.16 and 0.20, respectively. Growth rate was not influenced by any measure of halothane sensitivity. Carcasses from pigs exhibiting limb rigidity tended to have lower pH45 (5.88 v. 5.97; P = 0.06), similar pHu (5.47 v. 5.49; P = 0.32), less pH decline from 45 min to 18 h (−0.40 v. −0.50; P = 0.04) and a tendency for greater fluid loss percent (5.01 v. 4.55; P = 0.08) than carcasses from pigs that did not exhibit limb rigidity during halothane challenge. A proportion of pigs normal for RYR1 did exhibit limb rigidity during halothane gas challenge, and subsequently tended to have lower 45 min pH and greater longissimus muscle fluid loss post harvest.     

A new validated HPLC-FLD method for detecting ochratoxin A in dry-cured meat and in blue cheese

In the present study, a fast and sensitive method for the quantification of ochratoxin A in two lipidicproteic food matrices has been developed. In particular, the sample preparation procedure has been optimized for dry-cured meat products and blue cheeses and tested for several validation parameters (LOD, LOQ, recovery, repeatability and within-laboratory precision). The procedure has been then applied to several dry-cured meat products and blue cheeses from the market. Ochratoxin A has been occasionally found in dry-cured and smoked ham from the market and the contamination occurred both in the outer and in the inner part of the products. Concerning the blue cheese, the occurrence of ochratoxin A is reported for the first time: OTA was occasionally found at low levels (0.1–3 μg/kg) in commercial samples of Roquefort from France and Gorgonzola from Italy, opening a new issue for risk assessment and quality control. Presented at the 29^th Mykotoxin-Workshop, Fellbach, Germany, May 14–16, 2007 Financial support: This work was partially granted by Emilia-Romagna region (projects: SIQUAL and “Safety and quality of typical pork meat production chain”)     

Association of corticotropin-releasing hormone gene variation with performance and meat quality traits in commercial pig lines

The porcine corticotropin-releasing hormone(CRH) gene is a functional-positional candidate for quantitative tract loci on porcine chromosome 4 with major effects on growth and carcass composition. In addition, the central role of CRH in the neuroendocrine response to stress implicates the CRH gene as a functional candidate for meat quality. Association of a single nucleotide polymorphism (SNP) in the promoter region of the porcine CRH gene (g.233C > T) with several growth, carcass and meat quality traits was examined using more than 2000 individuals from four commercial lines: German Landrace (LR), Pietrain (Pi), German Large White x German Landrace (F1) and the German commercial fattening pig cross of Pietrain x F1 (PiF1). Significant association of the CRH SNP was found with feed conversion ratio in the PiF1 line, with carcass length in the LR line and with lean content in the F1, LR and Pi lines. Moreover, significant association with meat colour was found in the Pi and LR lines; however, the effects were in opposite directions. The presented results indicate that sequence variation in the porcine CRH gene has no major effect on growth and carcass composition in commercial pig lines, although it may significantly contribute to variation in meat quality. The g.233C>T SNP may be in incomplete linkage disequilibrium with causal mutations and/or exhibit effects in the context of DNA variation at other interacting loci.     

Identification of contemporary selection signatures using composite log likelihood and their associations with marbling score in Korean cattle

Positive selection not only increases beneficial allele frequency but also causes augmentation of allele frequencies of sequence variants in close proximity. Signals for positive selection were detected by the statistical differences in subsequent allele frequencies. To identify selection signatures in Korean cattle, we applied a composite log‐likelihood (CLL)‐based method, which calculates a composite likelihood of the allelic frequencies observed across sliding windows of five adjacent loci and compares the value with the critical statistic estimated by 50 000 permutations. Data for a total of 11 799 nucleotide polymorphisms were used with 71 Korean cattle and 209 foreign beef cattle. As a result, 147 signals were identified for Korean cattle based on CLL estimates (P < 0.01). The signals might be candidate genetic factors for meat quality by which the Korean cattle have been selected. Further genetic association analysis with 41 intragenic variants in the selection signatures with the greatest CLL for each chromosome revealed that marbling score was associated with five variants. Intensive association studies with all the selection signatures identified in this study are required to exclude signals associated with other phenotypes or signals falsely detected and thus to identify genetic markers for meat quality.