A Review of Toxoplasma gondii in Animals in Greece: A FoodBorne Pathogen of Public Health Importance.

Toxoplasma gondii is a zoonotic protozoon with a complex life cycle and the second most important foodborne pathogen in Europe. Surveillance of toxoplasmosis is based on national considerations since there are no mandatory controls along the food chain in the European Union, and underreporting of meat is still a problem in many countries like Greece. The current review provides an overview of T. gondii prevalence, associated risk factors, and surveillance in animals in Greece, focusing on the transmission role of meat and highlighting the control measures that should be adopted by consumers. Sows, wild boars, hares, equines, and cats had lower, while sheep and goats generally had higher seroprevalence than their respective pooled European and global values. Seroprevalence in chickens was similar between Greece and Europe, while there was high variation in cattle studies, with no data regarding dairy products. Though a comprehensive meat safety assurance system is the most effective approach to control the principal biological hazards associated with meat, such as T. gondii, the prerequisite risk categorisation of farms and abattoirs based on EFSA's proposed harmonised epidemiological indicators has not materialised as yet in Greece. Therefore, comprehensive control strategies are still required to ensure food safety and safeguard public health.

Comparison of disk diffusion and broth microdilution methods for antimicrobial susceptibility testing of Campylobacter isolates of meat origin.

The aim of this study was to compare the disk diffusion (DD) and the broth microdilution (BMD) methods in determining the antimicrobial susceptibility of 36 Campylobacter isolates of meat-origin to six antibacterial drugs (erythromycin, ciprofloxacin, tetracycline, streptomycin, gentamicin and nalidixic acid). All the available zone diameter and minimum inhibitory concentration (MIC) breakpoints of C. jejuni and C. coli as recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) were utilized. In addition, the zone diameter breakpoints of Enterobacterales for nalidixic acid, gentamicin, and streptomycin, as recommended by the Clinical and Laboratory Standards Institute (CLSI), were applied. All Campylobacter isolates were categorised as susceptible to erythromycin and gentamicin by both methods indicating completely concordant classification results. The overall highest 'Very major error' (VME) and 'Major error' (ME) rates were detected for nalidixic acid (13.3%) and tetracycline (26.3%), respectively, whereas a 'Minor error' (mE) rate was detected only for ciprofloxacin (60.1%). However, the Cohen's kappa statistic indicated a substantial concordance between the DD and BMD classification results for tetracycline and streptomycin, and almost perfect agreement for nalidixic acid, with corresponding categorical agreement rates of over 86% and approximately up to 92%. The correlation between the complementary inhibition zones and MIC breakpoints was strong and statistically highly significant (p < 0.001) for ciprofloxacin, tetracycline, streptomycin, and nalidixic acid.

The Effects of Replacing Soybean Meal with Rapeseed Meal, Cottonseed Cake, and Fava Beans on the Milk Yield and Quality Traits in Milking Ewes.

The replacement of soybean meal (SBM) from intensively reared dairy sheep diets has emerged as a significant challenge for sustainable production. However, the effects of this replacement on milk production have not been sufficiently elucidated. The objective of this study was to prospectively assess the effects of replacing SBM with a mixture of alternative protein sources on the milk yield (MY) and the milk quality traits (MQT) in intensively reared dairy sheep. A total of 112 multiparous, purebred milking ewes of the Chios and Frizarta breeds, from two intensive dairy sheep farms, were involved in the study, postweaning, and were assigned to either the control (CR) or the experimental ration (ER) group. In the ER, 3/4 of the SBM was replaced by a mixture of rapeseed meal, cottonseed cake, and fava beans, producing a ration of a similar nutritional value. MY, MQT, and body condition scores were recorded for each individual ewe monthly for a period of 4 months during lactation. The experimental ration was associated with beneficial effects on daily and 100-day fat yields and on the electrical conductivity of milk as an improved udder health status indicator, with no adverse effects on any of the rest of the studied milk production traits.

Dry Fermented Sausages with Total Replacement of Fat by Extra Virgin Olive Oil Emulsion and Indigenous Lactic Acid Bacteria.

RESEARCH BACKGROUND: Formulations based on vegetable or fish oil and modifications in the production technology of dry fermented sausages have emerged in recent years aiming to achieve the desirable target of reducing the fat content of these meat products. However, previous efforts have confronted many difficulties, such as high mass loss and unacceptable appearance due to intensely wrinkled surfaces and case hardening. The objective of this study is to produce and evaluate dry fermented sausages by utilising a meat protein-olive oil emulsion as fat substitute and indigenous lactic acid bacteria (LAB) with probiotic properties isolated from traditional Greek meat products. EXPERIMENTAL APPROACH: A novel formulation with extra virgin olive oil and turkey protein was developed to totally replace the conventionally added pork fat. Probiotic and safety characteristics of autochthonous LAB isolates from spontaneously fermented sausages were evaluated and three LAB isolates were finally selected as starter cultures. Physicochemical, microbiological and sensory analyses were carried out in all treatments (control, Lactobacillus acidophilus, L. casei, L. sakei and Pediococcus pentosaceus) during fermentation. RESULTS AND CONCLUSIONS: Ready-to-eat sausages were found to be microbiologically stable. The olive oil-based formulation produced in this study generated a mosaic pattern visible in the sliced product simulating the fat in conventional fermented sausages and was regarded as an ideal fat substitute for the production of fermented sausages. An autochthonous isolate of Lactobacillus casei adapted the best to the final products as it was molecularly identified to be present in the highest counts among the LAB isolates used as starter cultures. NOVELTY AND SCIENTIFIC CONTRIBUTION: Α novel and high-quality dry fermented meat product was produced by replacing the added pork fat with a fat substitute based on a meat protein-olive oil emulsion. Autochthonous LAB with in vitro probiotic properties could have a potential use in large-scale novel dry fermented sausage production. Such isolates could be used as starters in an effort to standardise the production process and retain the typical organoleptic and sensory characteristics. Moreover, isolates like L. casei 62 that survived in high counts in the final products can increase the safety of fermented sausages by competing not only with pathogens but also with the indigenous microbiota and could have a potential functional value for the consumer.

Method-Dependent Implications in Foodborne Pathogen Quantification: The Case of Campylobacter coli Survival on Meat as Comparatively Assessed by Colony Count and Viability PCR.

The aim of the present study was to address method-dependent implications during the quantification of viable Campylobacter coli cells on meat over time. Traditional colony counting on selective and non-selective culture media along with an optimized viability real-time PCR utilizing propidium monoazide-quantitative PCR (PMA-qPCR), spheroplast formation and an internal sample process control (ISPC), were comparatively evaluated for monitoring the survival of C. coli on fresh lamb meat during refrigeration storage under normal atmospheric conditions. On day zero of three independent experiments, lamb meat pieces were artificially inoculated with C. coli and then stored under refrigeration for up to 8 days. Three meat samples were tested on different days and the mean counts were determined per quantification method. An overall reduction of the viable C. coli on lamb meat was observed regardless of the applied quantification scheme, but the rate of reduction followed a method-dependent pattern, the highest being observed for colony counting on modified charcoal cefoperazone deoxycholate agar (mCCDA). Univariate ANOVA indicated that the mean counts of viable C. coli using PMA-qPCR were significantly higher compared to Columbia blood agar (CBA) plating (0.32 log10 cell equivalents, p = 0.015) and significantly lower when mCCDA was compared to CBA plating (0.88 log10 CFU, p < 0.001), indicating that selective culture on mCCDA largely underestimated the number of culturable cells during the course of meat storage. PMA-qPCR outperformed the classical colony counting in terms of quantifying both the culturable and viable but non-culturable (VBNC) C. coli cells, which were generated over time on meat and are potentially infectious and equally important from a public health perspective as their culturable counterparts.

Plant-Derived Natural Antioxidants in Meat and Meat Products.

The global meat industry is constantly evolving due to changes in consumer preferences, concerns and lifestyles, as well as monetary, geographical, political, cultural and religious factors. Part of this evolution is the introduction of synthetic antioxidants to increase meat and meat products' shelf-life, and reduce meat spoilage due to lipid and protein oxidation. The public perception that natural compounds are safer and healthier per se has motivated the meat industry to replace synthetic antioxidants with plant-derived ones in meat systems. Despite several promising results from in vitro and in situ studies, the effectiveness of plant-derived antioxidants against lipid and protein oxidation has not been fully documented. Moreover, the utility, usability, marketability and potential health benefits of natural antioxidants are not yet fully proven. The present review aims to (i) describe the major chemical groups of plant-derived antioxidants and their courses of action; (ii) present the application of spices, herbs and fruits as antioxidants in meat systems; and (iii) discuss the legislative framework, future trends, challenges and limitations that are expected to shape their acceptance and mass exploitation by the meat industry.

Natural Phenolic Compounds for the Control of Oxidation, Bacterial Spoilage, and Foodborne Pathogens in Meat.

Alternative technologies for long-term preservation, quality assurance, and safety of meat are continuously pursued by the food industry to satisfy the demands of modern consumers for nutritious and healthy meat-based products. Naturally occurring phenolic compounds are considered promising substances by the meat industry for their antioxidant and antimicrobial properties, while consumers seem to embrace them for their claimed health benefits. Despite the numerous in vitro and in situ studies demonstrating their beneficial effects against meat oxidation, spoilage, and foodborne pathogens, wide application and commercialization has not been yet achieved. Major obstacles are still the scarcity of legislative framework, the large variety of meat-based products and targeted pathogens, the limited number of case-specific application protocols and the questionable universal efficiency of the applied ones. The objectives of the present review are i) to summarize the current knowledge about the applications of naturally occurring phenols in meat and meat-based products, emphasizing the mechanisms, determinants, and spectrum of their antioxidant and antimicrobial activity; ii) to present state-of-the-art technologies utilized for the application of phenolic compounds in meat systems; and iii) to discuss relevant regulation, limitations, perspectives, and future challenges for their mass industrial use.

Viability Quantitative PCR Utilizing Propidium Monoazide, Spheroplast Formation, and Campylobacter coli as a Bacterial Model.

A viability quantitative PCR (qPCR) utilizing propidium monoazide (PMA) is presented for rapid quantification of viable cells using the foodborne pathogen Campylobacter coli as a bacterial model. It includes optimized spheroplast formation via lysozyme and EDTA, induction of a mild osmotic shock for enhancing the selective penetration of PMA into dead cells, and exploitation of an internal sample process control (ISPC) involving cell inactivation to assess residual false-positive signals within each sample. Spheroplasting of bacteria in exponential phase did not permit PMA entrance into viable cells since a strong linear relationship was detected between simple qPCR and PMA-qPCR quantification, and no differences were observed regardless of whether spheroplasting was utilized. The PMA-qPCR signal suppression of dead cells was elevated using spheroplast formation. With regard to the ISPC, cell inactivation by hydrogen peroxide resulted in higher signal suppression during qPCR than heat inactivation did. Viability quantification of C. coli cells by optimized spheroplasting-PMA-qPCR with ISPC was successfully applied in an aging pure culture under aerobic conditions and artificially inoculated meat. The same method exhibited a high linear range of quantification (1.5 to 8.5 log10 viable cells ml-1), and results were highly correlated with culture-based enumeration. PMA-qPCR quantification of viable cells can be affected by their rigidity, age, culture media, and niches, but spheroplast formation along with osmotic shock and the use of a proper ISPC can address such variations. The developed methodology could detect cells in a viable-but-nonculturable state and might be utilized for the quantification of other Gram-negative bacteria.IMPORTANCE There is need for rapid and accurate methods to detect viable bacterial cells of foodborne pathogens. Conventional culture-based methods are time-consuming and unable to detect bacteria in a viable-but-nonculturable state. The high sensitivity and specificity of the quantitative PCR (qPCR) are negated by its inability to differentiate the DNAs from viable and dead cells. The combination of propidium monoazide (PMA), a DNA-intercalating dye, with qPCR assays is promising for detection of viable cells. Despite encouraging results, these assays still encounter various challenges, such as false-positive signals by dead cells and the lack of an internal control identifying these signals per sample. The significance of our research lies in enhancing the selective entrance of PMA into dead Campylobacter coli cells via spheroplasting and in developing an internal sample process control, thus delivering reliable results in pure cultures and meat samples, approaches that can be applicable to other Gram-negative pathogens.

Comparison of eleven methods for genomic DNA extraction suitable for large-scale whole-genome genotyping and long-term DNA banking using blood samples.

Over the recent years, next generation sequencing and microarray technologies have revolutionized scientific research with their applications to high-throughput analysis of biological systems. Isolation of high quantities of pure, intact, double stranded, highly concentrated, not contaminated genomic DNA is prerequisite for successful and reliable large scale genotyping analysis. High quantities of pure DNA are also required for the creation of DNA-banks. In the present study, eleven different DNA extraction procedures, including phenol-chloroform, silica and magnetic beads based extractions, were examined to ascertain their relative effectiveness for extracting DNA from ovine blood samples. The quality and quantity of the differentially extracted DNA was subsequently assessed by spectrophotometric measurements, Qubit measurements, real-time PCR amplifications and gel electrophoresis. Processing time, intensity of labor and cost for each method were also evaluated. Results revealed significant differences among the eleven procedures and only four of the methods yielded satisfactory outputs. These four methods, comprising three modified silica based commercial kits (Modified Blood, Modified Tissue, Modified Dx kits) and an in-house developed magnetic beads based protocol, were most appropriate for extracting high quality and quantity DNA suitable for large-scale microarray genotyping and also for long-term DNA storage as demonstrated by their successful application to 600 individuals.

Campylobacter in small ruminants at slaughter: prevalence, pulsotypes and antibiotic resistance.

The present study aimed to address the prevalence, pulsotypes, and antimicrobial susceptibility patterns of Campylobacter species present in sheep and goat carcasses at slaughter. In total, 851 samples were collected (343 meat surfaces, 282 ileum contents, 226 liver surfaces) and 835 Campylobacter isolates were detected in 274 out of 343 carcasses (116 kids, 110 lambs, 63 goats and 54 sheep). The contamination rates per carcass category were 78.4% for kids, 94.5% for lambs, 63.5% for goats, and 72.2% for sheep. On average, 30% of the intestinal content samples and more than 70% of carcass and liver surfaces yielded the presence of campylobacters. Multiplex-PCR and RFLP analysis identified Campylobacter coli as the most prevalent species (76.2%) followed by Campylobacter jejuni (21.4%), albeit 2.4% of selected colonies yielded the concurrent presence of both these species. Macrorestriction profiling by pulsed-field gel electrophoresis (PFGE) was applied in order to characterise a subset of isolates. SmaI-PFGE successfully clustered 222 isolates in 82 SmaI-PFGE types indicating high heterogeneity among the campylobacter isolates (67 types among 174C. coli isolates and 15 types among 48C. jejuni isolates). No carcass-type (lamb, kid, sheep, and goat) specific PFGE clusters were recognised since there was a general overlapping of PFGE patterns regarding ovine and caprine isolates. Multiple pulsotypes were simultaneously present on single carcasses in the majority of tested animals. PFGE provided data regarding the potential routes of meat and liver contamination such as spillage of faecal material and cross-contamination during slaughter. Antimicrobial susceptibility patterns of Campylobacter isolates (n=240), determined by disk diffusion method, revealed resistance to tetracycline (47.9%) followed by streptomycin (22.9%) and ciprofloxacin along with nalidixic acid (18.3%). Isolates exhibited low resistance to erythromycin (2.5%) and were susceptible to gentamicin. The findings of the present study confirm the contamination of sheep and goats at slaughter with thermophilic campylobacters and underline their potential input in the epidemiology of human campylobacteriosis.

Diversity of Campylobacter in retail meat and liver of lambs and goat kids.

The presence, genetic diversity, and antimicrobial susceptibility profile of Campylobacter spp. in retail lamb and goat kid carcasses were assessed. A total of 200 samples consisting of 100 meat and 100 liver surface swabs were collected from 47 lamb and 53 goat kid carcasses at 23 retail markets in Northern Greece, and 125 Campylobacter isolates were recovered from 32 meat surfaces (32%) and 44 liver surfaces (44%). Multiplex polymerase chain reaction and restriction fragment length polymorphism analysis specified Campylobacter coli as the most frequently detected species (59.2%) followed by C. jejuni (40.8%). Pulsed-field gel electrophoresis (PFGE) was applied in order to typify a subset of randomly selected isolates (n=80). SmaI-PFGE successfully clustered the 80 isolates in 38 SmaI-PFGE types, indicating high heterogeneity among the analyzed Campylobacter isolates, and provided data regarding the dissemination of Camplobacter among carcasses stored in the same retail market. Antimicrobial susceptibility profiles of Campylobacter isolates, assessed by the disk-diffusion method, indicated that 31 isolates (24.8%) were multidrug resistant, and the most common profile was the concurrent resistance to tetracycline and streptomycin. Overall, 56.8% of isolates (n=71, multidrug-resistant isolates included) exhibited resistance to at least one antimicrobial (tetracycline 34.4%, quinolones 27.2%, and streptomycin 20.8%). However, all isolates were susceptible to erythromycin and gentamicin. The findings of this study verify the contamination of retail lamb and goat kid carcasses with a heterogeneous population of thermotolerant campylobacters. These data underscore the fact that retail meat and liver of small ruminants could serve as vehicles for consumer contamination with Campylobacter and that further investigation is necessary in order to evaluate the risk imposed by such products within the epidemiology of human campylobacteriosis cases.

The effects of chronically increased intra-abdominal pressure on the rabbit diaphragm.

BACKGROUND: Diaphragmatic muscular remodeling is caused by various conditions and was mainly studied in pulmonary pathologies and chronic alterations of intra-thoracic pressure. We investigate the effect of the chronically increased intra-abdominal pressure (IAP) on the diaphragm by morphological and biochemical analysis. METHODS: Thirty rabbits were divided into control and study groups. IAP was increased in group B to 12 mmHg for 2 months. The left hemidiaphragm underwent morphological, while the right underwent biochemical analysis. RESULTS: In H&E, all fibers were normal. ATPase analysis demonstrated that type I fibers show no differences between groups. Type ΙΙ(Α) were decreased (p = 0.016) while type ΙΙ(Β/X) fibers were increased (p = 0.025) in group B. Fibers with resistance to fatigue were decreased in group B (p = 0.024). In group B, biochemical activity for glutathione reductase (p = 0.004), glutathione peroxidase (p = 0.021), protein carbonylation (0.029), lipid peroxidation (p = 0.005), and balance of preoxidative-antioxidative factors (p = 0.006) was increased. CONCLUSIONS: Chronically increased IAP induces alterations to the rabbit diaphragm. Adaptation, equivalent to strenuous contraction, transforms the diaphragm to be functionally more efficient toward workload but makes it vulnerable against oxidative stress.

Can chronic intra-abdominal hypertension cause oxidative stress to the abdominal wall muscles? An experimental study.

BACKGROUND: The aim of this study was to test the hypothesis that intra-abdominal hypertension alone could trigger such changes to the rectus abdominis muscle that would lead to an imbalance between oxidant production and antioxidant protection. MATERIALS AND METHODS: Forty-five New Zealand white rabbits were divided into three groups and a rubber bag was implanted into their peritoneal cavity. In group A (n = 15), the bag was empty. In group B (n = 15), it was filled with normal saline to achieve an intra-abdominal pressure of over 12 mm Hg. In group C (n = 15), it was filled with lead equiponderant to the mean weight of the normal saline injected in group B. After 8 weeks, we measured in rectus abdominis muscle biopsies the lipid peroxidation products, the protein carbonyl content, the total glutathione and superoxide dismutase (SOD) concentration, the activity of glutathione reductase and glutathione peroxidase, and the pro-oxidant-antioxidant balance. RESULTS: The lipid peroxidation products were significantly higher in group B compared with both group A (P = 0.026) and group C (P < 0.001). The total protein carbonyl content was significantly higher in group B compared with both group A (P = 0.006) and group C (P < 0.001). No difference was found between the three groups in total glutathione (P = 0.735) and SOD (P = 0.410) concentration. Glutathione peroxidase activity was higher in groups B and C compared with group A (P = 0.05 and P = 0.003, respectively). Glutathione reductase activity was higher in group B compared with group A (P = 0.005) and group C (P = 0.001). The pro-oxidant antioxidant balance was higher in group B compared with the group A (P = 0.012). CONCLUSIONS: Maintaining the IP over 12 mm Hg for 8 wk caused increased oxidative damage to both lipids and proteins with an increased pro-oxidant-antioxidant balance. In an attempt to compensate for this damage the muscle fibers increased their glutathione reductase and glutathione peroxidase activity.

The effect of chronically increased intra-abdominal pressure on rectus abdominis muscle histology an experimental study on rabbits.

BACKGROUND: The aim of this study was to specify the histologic response of the rectus abdominis muscle of the rabbit, to the chronically increased intra-abdominal pressure. MATERIALS AND METHODS: Forty-five New Zealand white rabbits were divided into three groups. In all groups, a rubber bag was implanted into the peritoneal cavity. In group A (n=15) the bags were kept empty. In group B (n=15) the bags were filled with normal saline in order to achieve an intra-abdominal pressure of over 12 mmHg. This pressure was kept at this level for 8 wk. In group C (n=15) the intra-abdominal rubber bags were filled with lead covered by silicone, equiponderant to the mean weight of the normal saline insufflated in group B. After 8 wk we took biopsies of the rectus abdominis muscle and counted the proportion of the different types of muscular fibers (type I, IIA, and IIB/X). RESULTS: Significant difference was found in the proportion of the three types of muscle fibers. Intra-abdominal hypertension led to an increase in type I fibers (P=0.008). No difference was noticed between groups A and C. CONCLUSIONS: The histologic response to the increased intra-abdominal pressure was an increase in type I muscle fibers. Charging with lead did not cause any significant change in the proportion of muscular fibers.

Diaphragmatic adaptation following intra-abdominal weight changing.

BACKGROUND: The diaphragm, the major respiratory muscle, contains three types of muscular fibers in dynamic balance between them. The fiber ratios vary in time in function of conditions, such as aging, hypoproteinemia, exercise, and chronic respiratory load. The diaphragmatic adaptation following abdominal conditions remains an unexplored field. This experimental study aims to identify the changes of the diaphragm due to chronic abdominal weight load. This may find application in conditions such as pregnancy, ascites, visceromegaly, large masses, and morbid obesity. METHODS: Thirty rabbits were divided into control (A) and study (B) groups. Group B was loaded with weight for 2 months. The left costal hemidiaphragm were stained with H&E and ATPase (fiber typing), while the right underwent biochemical analysis (prooxidative-antioxidative balance, lipid peroxidation, superoxide dismutase, glutathione reductase and peroxidase activities, total glutathione, and protein carbonylation). RESULTS: In H&E, all fibers were within normal range. ATPase analysis demonstrated reduction of type I (p = 0.019) and an increase of the type ΙΙ(Α) fibers ratio (p < 0.001) in group B, while the type ΙΙ(Β/X) fibers ratio remained stable. The above suggest remodeling of type I fibers into type II(A). Concerning biochemical analysis, difference was observed in glutathione peroxidase activity (p < 0.001). CONCLUSIONS: Chronically loaded abdomen leads to morphological adaptations of the costal diaphragm, but with minor oxidative stress. These diaphragmatic morphological changes are equivalent to heart failure or severe COPD, showing that this remodeling makes the muscle more efficient towards work load, but more vulnerable to fatigue.

Chronically increased intra-abdominal pressure: validating a model.

BACKGROUND: Pregnancy is the only physiologic condition in which we encounter chronically elevated intra-abdominal pressure (IAP), while pathologically several pathologies, such as ascites and morbid obesity, are affected by this phenomenon. This paper introduces and validates a new model that is able to create and maintain chronically increased IAP, facilitating the study of phenomena related to chronically elevated IAP, i.e., obesity. METHODS: An experimental device was implanted in 15 rabbits, which consisted of an intra-abdominal balloon (IAB), an external control valve, and a connecting tube. A Foley catheter was inserted in their urinary bladders. IAPs were measured simultaneously transvesically and via the device. During the acute phase, IAB was gradually inflated to 16 cmH(2)O, and IAPs were consecutively measured. During the chronic phase, residual IAPs were measured in a weekly rate for 8 weeks. Statistical significances, mean bias, and precisions were calculated. RESULTS: During the acute phase, the saline in the IAB efficiently increases IAP to 16 cmH(2)O. IAPs measured both through the urinary bladder and the device correlate well with small bias and high precision. Our model maintains sufficiently chronically increased IAP for at least 8 weeks. No mortality was observed. CONCLUSIONS: A rabbit model establishing and maintaining chronically increased IAP was successfully created and proved to be simple, effective, and repeatable. This model established chronically increased IAP permitting this way the study of its effect on organs and systems.