Analysis of genome-wide DNA arrays reveals the genomic population structure and diversity in autochthonous Greek goat breeds.

Goats play an important role in the livestock sector in Greece. The national herd consists mainly of two indigenous breeds, the Eghoria and Skopelos. Here, we report the population structure and genomic profiles of these two native goat breeds using Illumina's Goat SNP50 BeadChip. Moreover, we present a panel of candidate markers acquired using different genetic models for breed discrimination. Quality control on the initial dataset resulted in 48,841 SNPs kept for downstream analysis. Principal component and admixture analyses were applied to assess population structure. The rate of inbreeding within breed was evaluated based on the distribution of runs of homozygosity in the genome and respective coefficients, the genomic relationship matrix, the patterns of linkage disequilibrium, and the historic effective population size. Results showed that both breeds exhibit high levels of genetic diversity. Level of inbreeding between the two breeds estimated by the Wright's fixation index FST was low (Fst = 0.04362), indicating the existence of a weak genetic differentiation between them. In addition, grouping of farms according to their geographical locations was observed. This study presents for the first time a genome-based analysis on the genetic structure of the two indigenous Greek goat breeds and identifies markers that can be potentially exploited in future selective breeding programs for traceability purposes, targeted genetic improvement schemes and conservation strategies.

Effect of herbal feed additives on performance parameters, intestinal microbiota, intestinal morphology and meat lipid oxidation of broiler chickens.

1. This feeding trial investigated the effects of herbal feed additives on performance of broiler chickens, jejunal and caecal microbiota, jejunal morphology, meat chemical composition and oxidative stability during refrigerated storage. 2. In a 42 days trial, 320 one-day-old broiler chickens were randomly allocated to 4 groups with 4 replicate pens each containing 20 chicks. The control group was fed maize-soybean-based diets. The diets of the other three groups were supplemented with herbal feed additives: HRB1 with StresomixTM (0.5 g/kg feed); HRB2 with AyuceeTM (1.0 g/kg feed); HRB3 with Salcochek ProTM (1.0 g/kg feed). The GC/MS analysis of the feed additives showed that the major components of HRB1 were ß-caryophyllene (14.4%) and menthol (9.8%); HRB2 were n-hexadecanoic acid (14.22%) and ß-caryophyllene (14.4%); and HRB3 were menthol (69.6%) and clavicol methyl ether (13.9%). 3. Intestinal samples were taken at 42 day to determine bacterial populations (total aerobe counts, Lactobacilli, and Escherichia coli) and perform gut morphology analysis. Meat samples were analysed for chemical composition and oxidative stability under storage. 4. The HRB1 group had improved (P < 0.05) body weight gain and tended to have improved (0.05 ≤ P < 0.10) feed conversion ratio, compared to the control group. Jejunum lactic acid bacteria counts were increased (P < 0.001) in groups HRB1 and HRB3, compared to the control group, whereas caecal lactic acid bacteria counts tended to increase (0.05 ≤ P < 0.10) in group HRB1, compared to the control group. Breast meat fat content tended to be lower (0.05 ≤ P < 0.10) in group HRB1. Meat oxidative stability was improved (P < 0.001), and jejunum villus height, crypt depth and goblet cells numbers were increased (P < 0.001) in all three herbal supplemented groups, compared to the control. 5. In conclusion, herbal feed additives may be able to improve both growth performance and antioxidant activity of broiler chickens, based on their phenolic compound content.

Genomic diversity and population structure of three autochthonous Greek sheep breeds assessed with genome-wide DNA arrays.

In the present study, genome-wide genotyping was applied to characterize the genetic diversity and population structure of three autochthonous Greek breeds: Boutsko, Karagouniko and Chios. Dairy sheep are among the most significant livestock species in Greece numbering approximately 9 million animals which are characterized by large phenotypic variation and reared under various farming systems. A total of 96 animals were genotyped with the Illumina's OvineSNP50K microarray beadchip, to study the population structure of the breeds and develop a specialized panel of single-nucleotide polymorphisms (SNPs), which could distinguish one breed from the others. Quality control on the dataset resulted in 46,125 SNPs, which were used to evaluate the genetic structure of the breeds. Population structure was assessed through principal component analysis (PCA) and admixture analysis, whereas inbreeding was estimated based on runs of homozygosity (ROHs) coefficients, genomic relationship matrix inbreeding coefficients (FGRM) and patterns of linkage disequilibrium (LD). Associations between SNPs and breeds were analyzed with different inheritance models, to identify SNPs that distinguish among the breeds. Results showed high levels of genetic heterogeneity in the three breeds. Genetic distances among breeds were modest, despite their different ancestries. Chios and Karagouniko breeds were more genetically related to each other compared to Boutsko. Analysis revealed 3802 candidate SNPs that can be used to identify two-breed crosses and purebred animals. The present study provides, for the first time, data on the genetic background of three Greek indigenous dairy sheep breeds as well as a specialized marker panel that can be applied for traceability purposes as well as targeted genetic improvement schemes and conservation programs.

The past, present and future in scaffold-based tendon treatments.

Tendon injuries represent a significant clinical burden on healthcare systems worldwide. As the human population ages and the life expectancy increases, tendon injuries will become more prevalent, especially among young individuals with long life ahead of them. Advancements in engineering, chemistry and biology have made available an array of three-dimensional scaffold-based intervention strategies, natural or synthetic in origin. Further, functionalisation strategies, based on biophysical, biochemical and biological cues, offer control over cellular functions; localisation and sustained release of therapeutics/biologics; and the ability to positively interact with the host to promote repair and regeneration. Herein, we critically discuss current therapies and emerging technologies that aim to transform tendon treatments in the years to come.

Chemical and microbiological characterization of artisan inoculants used for the fermentation of traditional dairy products in Epirus area (Greece).

The artisan production of a variety of dairy products is a historical tradition since antiquity, which is still practiced nowadays, in the area of Epirus, (northwestern Greece). The common denominator of these products, regardless of the final form of the end product, is the fermentation of raw milk by the use of dried artisan inoculants made of the dehydrated rumen of small ruminants, mainly lambs. The aim of this research project is to study the microbiological parameters as well as certain technological parameters (chymosin activity and lipolytic activity) of these inoculants. The results revealed a wide biodiversity of microorganisms such as Lactobacilli, Lactococci, Leuconostoc, Pediococci, Streptococci, Bifidobacteria, Enterococci, Clostridia and coliforms. Chymosin activity and lipolytic activity were found to be higher in artisan inoculants than in the commercial ones.

Hygienic quality and antibiotic resistance profile of sliced butchery.

In order to investigate the microbiological quality of different meat products on the Greek market, 200 samples were collected from the following preparations: boiled turkey (n=50), boiled pork ham (n=50), smoked turkey (n=50) and smoked pork ham (n=50). In all cold meat preparations Clostridium perfringens vegetative and spore forms, Staphylococcus aureus, Escherichia coli and other Clostridium sp lec(-), as well as Lactobacillus, Bacillus sp. and Salmonella sp. were recovered. For instance Bacillus cereus was present in 6% of the samples. L. monocytogenes, Campylobacter jejuni and Campylobacter coli were rarely present (1-4%) while Yersinia enterocolitica and Campylobacter lari were absent. Differences in the occurrence of S. aureus, Salmonella sp., E. coli and spore forms of C. perfringens in boiled and smoked samples, reflects either the differences in the processing of the foods or could be associated to the extensive handling by the personnel during the purchasing (storage, slicing, wrapping). Antibiotic resistance on specific antibiotics for each pathogen was also studied. A multiresistance antibiotic profile was effective for most bacterial strains, and pronounced resistance profiles were observed for the commonly used antibiotics as ampicillin, penicillin, cephalothin, streptomycin followed by ceftriaxone and gentamycin. Albeit this high observed resistance profile, the tested strains generally conserved their susceptibility to amikacin, aztreonam, chloramphenicol and tylosin conserved an almost absent resistance. Antibiotics commonly used for therapeutic purposes, as well as antibiotics added to feed stuff of animals for increasing animal flesh production should contribute to the extensive spreading of antibiotic resistance in food and the environment. Systematically monitoring of the microbiological quality of cold butchery preparations must be done, in order to preserve food quality, optimizing the processing and elaboration methods of the product and safeguard the public health.

Occurrence of Clostridium perfringens from different cultivated soils.

The occurrence of Clostridium perfringens was estimated in 750 samples originated from a variety of soils bearing various bulb crops: Brawnica oderacea (vegetable), Olea europaea, Daucus carota (carote), Solanum tuberosum (potato), Phaseolus vulgaris (green haricot), Beta vulgaris var. rapaceum (beetroot), Cucurbita pepo (squash), Allium cepa (onion), Cucumis sativus (cucumber) and Capsicum annum (pepper). All isolated strains were tested for their antimicrobial activities to amoxicillin, penicillin G, kanamycin, tetracycline, streptomycin, erythromycin, chloramphenicol and metronidazole. When considering the type of the bulb production, it was observed increased number of C. perfringens spore densities in the most undersurface bulb soils. Moreover, C. perfringens spore are likely to occur in particularly large numbers in soil contaminated by fecal matter. Additionally, there is a close relationship between the spore amount and nature of organic content. Presence of C. perfringens was associated with acidic soil. Most of our strains showed resistance to the studied antibiotics applied usually for human and veterinary care. A systematic monitoring of the cultivated soil ecosystems must include bacteriological parameters together with chemical indices of organic pollution in order to obtain information adequate for assessing their overall quality.

Isolation of microbial pathogens of subclinical mastitis from raw sheep's milk of Epirus (Greece) and their role in its hygiene.

The natural raw milk microflora is a factor that expresses its sensorial characteristics. The microbial charge into the mammary gland of healthy animal is low and the application of right and healthy conditions during milking and cheese making procedure, prevents from contaminating as well as maintains the natural microflora in order to lend the particular characteristics of milk. The purpose of the present project was the study of the Total Viable Count (T.V.C.) and the count of total psychrotropic bacteria of raw sheep milk from Boutsiko and Karamaniko breeds, collected from healthy animals, as well as the isolation, identification and enumeration of pathogenic bacteria related with the hygiene and the quality of raw sheep milk (with a particular interest in bacteria that may cause human infection). During the experiment we examined two hundred forty (240) samples of raw sheep milk. In these samples a) Staphylococcus aureus, Salmonella sp., Escherichia coli, Clostridium perfringens (vegetative cells and spores) and Bacillus sp. were isolated and identified b) the Total Viable Count and the total number of psychrotropic bacteria were also specified. The sampling, the preparation of samples and decimal dilutions were based on international methods. The Total viable count was determined using the standard methods of the American Public Health Association, 2002. The total number of psychrotropic bacteria was determined using APHA 1976, 1978 rules. The identification of the bacteria was carried out according to the Bergey's manual. Microscopic examination of Gram stained cells, catalase, oxidase and biochemical tests were performed when necessary to further identify. From the 240 milk samples tested, only 5% were E. coli positive, with mean counts ranged from 2 × 10(3) to 2.4 × 10(4) cfu/ml. S. aureus was isolated from 24% of the samples and the mean count per ml was ranged from <10 to 3.4 × 10(2). Meanwhile, Bacillus spp. was also detected in 29% samples. Vegetative forms and spores of C. perfringens were detected in 13% and 63% of the samples respectively. However, microbiological analyses revealed the presence of a small number of selected pathogens in milk samples such as Salmonella, which was only detected in 5% of the samples. Listeria sp., Pseudomonas sp. and Vibrio cholerae were never found. From the experimental results, the Total Viable Count from raw sheep milk samples, fulfils the microbiological criteria of EU Legislation in a percentage of approximately 97%.

Antibacterial activity of different honeys against pathogenic bacteria.

To study the antimicrobial activity of honey, 60 samples of various botanical origin were evaluated for their antimicrobial activities against 16 clinical pathogens and their respective reference strains. The microbiological quality of honeys and the antibiotic susceptibility of the various isolates were also examined. The bioassay applied for determining the antimicrobial effect employs the well-agar diffusion method and the estimation of minimum active dilution which produces a 1mm diameter inhibition zone. All honey samples, despite their origin (coniferous, citrus, thyme or polyfloral), showed antibacterial activity against the pathogenic and their respective reference strains at variable levels. Coniferous and thyme honeys showed the highest activity with an average minimum dilution of 17.4 and 19.2% (w/v) followed by citrus and polyfloral honeys with 20.8 and 23.8% respectively. Clinical isolates of Staphylococcus aureus subsp. aureus, Escherichia coli, Salmonella enterica subsp. Enterica, Streptococcus pyogenes, Bacillus cereus and Bacillus subtilis were proven to be up to 60% more resistant than their equal reference strains thus emphasizing the variability in the antibacterial effect of honey and the need for further research.

Microbiological quality of grey-mullet roe.

The Greek grey-mullet roe is produced from the fully developed gonads of the female mullet (Mugil cephalus) couth in lagoons during their reproductive migration. The traditional processing method of the roe includes, air drying, salting, shape formation and covering with multiple layers of natural beeswax for preservation and distribution. Fish Roe brands have been a staple in local diet and is increasingly becoming popular in the international market. As a ready-to-eat food it's microbial quality should be of concern for the protection of consumers health. In this study, 48 samples of fish roe, just before waxing, were collected from various local processors for microbiological examination by using selective media and incubated under aerobic and anaerobic conditions. The identification of the bacteria was carried out according to the Bergey's manual. Microscopic examination of Gram stained cells, catalase, oxidase and biochemical tests were performed when necessary to further identify. V. parahaemolyticus, Vibrio spp., Salmonella spp., and Aeromonas hydrophila were detected in one sample (2%). Shigella spp., and Flavobacterium spp. in two samples (4%), Clotriduim perfringens (vegetative forms), E. coli, and spores of Bacillus spp., were detected in three samples (6%), Staphylococcus aureus in four samples (8%). Various Micrococcus spp., and spores of C. perfringens in 16% and 35% of the samples respectively. From the Listeria genus, only the species Listeria innocua, Listeria welshimeri, Listeria seeligeri Listeria ivanovii and Listeria grayi were recovered from 2 to 10% of the samples. Microbiological analyses revealed the presence of a small number of pathogens in grey-mullet roe samples which are in accordance with the findings of similar studies. Traditional processing of the fish roe, seems inadequate to ensure the food safety and even waxing isn't expected to fully protect them against facultative anaerobes with salt tolerance. Therefore, additional measures should be taken during processing and marketing of fish roe to minimize potential health risks for the consumers.

Microbial ecology of fish species ongrowing in Greek sea farms and their watery environment.

The present study focuses on the bacteriological profile of both watery ecosystem and fishes from different North and Central West Greek fish-farms ongrowing euryhaline fish species. The natural microflora of the fish and the water of their ongrowing units in selected farms were studied for a period of 20 months. The analyzed samples were mainly sea bream (Sparus aurata) 61.3% and sea bass (Dicentrarchus labrax) 24%. In most of the watery ecosystems coming from the different sampling areas, total and fecal coliforms as well as total and fecal streptococci were abundant in all water samples. Enterococcus, Escherichia coli and Pseudomonas were present at a level of 3 logs cfu/100 ml. The anaerobic Clostridium perfringens was found in vegetative (21.3%) and spore forms (13.3%). It is of interest to note that pathogens as Pasteurella piscicida and Vibrio anguillarum were isolated only in a small number of samples. Staphylococcus aureus was detected in 4% of the samples, other Staphylococcus sp. in 29.3%, E. coli in 30.7%, Salmonella sp. in 1.3%, Pseudomonas sp. in 13.3%, Clostridia lec(-) in 49.3%, Bacillus sp. in 38.7%, Vibrio sp. in 18.7%, Lactobacillus and Lactococcus sp. in 36% και 29.3% respectively. Vegetative forms of C. perfringens were detected in 22.7%. Although, our results showed no significant correlations between the sea water and fish microflora, more focus on this bipolar interacting system should be necessary in order to avoid any possible disturbance in the balance of the healthy farming ecosystem with the host organisms.

Transmission of Mannheimia haemolytica from the tonsils of lambs to the teat of ewes during sucking.

Objective of the work was to study whether Mannheimia haemolytica may be transmitted from the mouth of the lambs into the teat of the dam during sucking. We compared bacterial populations within the teat duct and milk of ewes immediately before and immediately after sucking by the lambs. Tonsils of lambs of the ewes were swabbed. M. haemolytica strain DAG21T recovered from a teat duct of a ewe was compared to strain DAG21R recovered from the tonsils of her lamb by using 16s rRNA sequencing. We used those two isolates and another one of known pathogenicity, for challenging ewes: (i) 2 mm deep into healthy teats, (ii) 2 mm deep into teats with chapping lesions or (iii) into the cistern of healthy mammary glands. Of samples collected before suckling, 20/792 were bacteriologically positive, and of those after, 50/792 were bacteriologically positive (P<0.001); in 37 cases, a negative sample became positive. One M. haemolytica (DAG21T) was recovered after suckling from a teat duct of a ewe. The organism was isolated from 57/90 tonsillar swabs from lambs. Risk of infection of ewe' teats was 0.004 throughout lactation, being greatest (0.021) during the 3rd week of lactation. The 16s rRNA sequences of strains DAG21T and DAG21R were identical over 1450 nucleotides. Phylogenetic analysis showed that the two isolates clustered together with isolates of M. haemolytica. Organism deposition into healthy teats caused subclinical mastitis; deposition into teats with lesions or directly into mammary gland caused clinical mastitis. When results of inoculation of the three strains were compared between them, statistical significance was always P>0.9. Results provide clear evidence that suckling by lambs can lead to transmission of M. haemolytica into the teats of the ewes; the bacteria have the potential to cause mastitis if circumstances are favourable.

Post-partum involution of the canine uterus - gross anatomical and histological features.

We aimed to study the normal puerperium in the bitch. Ovariohysterectomy was performed in nine bitches, each at a different day after normal whelping; their genital tract was subject to gross anatomical examination, as well as to histological examination and electron microscopy scanning. Corpora albicans were evenly distributed in the left and right ovaries and placental sites were evenly distributed among left and right uterine horns. Placental sites were initially of dark green to grey colour, later becoming dark brown; their length and height progressively decreased. Height of the myometrium and diameter of the uterine glands progressively decreased. Trophoblast-like cells were consistently observed at the placental sites and on the surface of the interplacental areas, at all time points where hysterectomy had been performed. It is suggested that involution of the canine genital tract can last up to 3 months and is slow. Continuous (up to D84 post-partum) presence of prominent placental sites should be considered a normal feature of canine uterine post-partum involution.

Effects of lamb sucking on the bacterial flora of teat duct and mammary gland of ewes.

We aimed (i) to determine differences in bacterial flora of teat duct and mammary gland of ewes before and after suckling, (ii) to evaluate factors potentially affecting those. We collected samples of teat duct material and mammary secretion from 11 ewes immediately before and after sucking by lambs, as well as 120 min later. We processed samples bacteriologically and compared changes in infection by the Sign Test. We isolated bacteria from 3.5% duct and 1.5% secretion samples before suckling. Respective figures post-suckling were 10.6% and 2.0%, and 120 min later 6.8% and 1.5%. We recorded differences in infection of duct samples before and after suckling in 40 cases; bacteria were isolated before suckling from six samples, whereas after it from 34 (p < 0.001). Also, we recorded differences in samples collected after suckling and 120 min later in 12 cases; bacteria were isolated immediately post-suckling from eight samples, whereas 120 min later from four (p = 0.375). No significant changes were seen for secretion. We found neither difference between ewes with single or twin lambs, nor among stages of lactation. Mostly, we isolated staphylococci: 70% of isolates before suckling, 80% of isolates after it, 91% of isolates 120 min later. After suckling we also isolated two Mannheimia haemolytica strains. Suckling predisposes to entrance of bacteria into the teat; however, increased teat infections did not result in mammary infections. Isolation of M. haemolytica post-suckling indicates that lambs act as source of infection for this pathogen.

Aerobic and anaerobic microbiology of the immersion chilling procedure during poultry processing.

The development of treatments to reduce bacterial numbers on poultry carcasses is important for the overall hygienic quality of birds. The important washing effect of the immersion chilling procedure is discussed. Systematic monitoring of fecal bacterial indicators as well as some classic pathogens was performed at selected critical points in a water chiller ecosystem. Clostridium perfringens, fecal coliforms, Enterococcus sp., and Streptococcus sp. were found in all water chiller samples. The temperature of the chiller ecosystem varied according to location: Escherichia coli and Salmonella sp. were found at 16 degrees C, compared with the 4 degrees C location, where these species were found in lower numbers. Moreover, the psychrotrophic bacterium Pseudomonas was found only at this last location. The temperature of the water during the immersion chilling procedure was unfavorable for the growth of Campylobacter sp., whose presence was always strictly associated with a pH close to 6. Spore forms of C. perfringens were persistent in all locations and seemed to be a reliable indicator of contamination of the water chiller ecosystem.

Effects of machine-milking on the bacterial flora of teat duct and mammary gland of ewes.

In total, 308 paired-samples of teat duct material and milk, were collected before and 50-70 min after machine-milking, from 30 ewes. Samples were processed bacteriologically. For analysis of results, we compared changes in bacterial isolation following milking, for duct and milk samples; statistical significance was assessed by the Sign Test. Bacteria were isolated from 18 (6%) duct and 19 (6%) milk samples collected before the milking procedure; respective figures after it, were 81 (26%) and 33 (11%). In 77 (25%) cases, bacteriological findings in the two duct samples of each pair were different; in seven cases bacteria were isolated only before, whilst in 70 cases bacteria were isolated only after milking (P < 0.005); respective results for milk samples were 26 (8%): 6 and 20 cases (P = 0.693). The majority of bacterial isolates were staphylococci, accounting for 63% of 99 isolates. The milking procedure predisposes to entrance of bacteria into the teat duct; however, increased bacterial isolation from the teat did not result to increased mammary infections, likely as a consequence of defence mechanisms present in healthy teats.

Bacteriological and epidemiological findings during examination of the uterine content of ewes with retention of fetal membranes.

We included 92 pairs of ewes with or without retention of fetal membranes in a cohort study of 25 flocks in Southern Greece. We obtained two uterine content samples under aseptic conditions, by introducing a swab into the uterus of these ewes, on the 2nd-4th and the 5th-9th day after lambing. We used conventional bacteriological techniques to isolate and identify bacteria and to carry out antimicrobial agents susceptibility testing. The prevalence of bacterial intrauterine contamination among ewes with retention was 24% on the first and 46% on the second sampling (P < 0.0001) and that among ewes without retention was 8 and 2% (P > 0.05), respectively. Clinical signs accompanying the retention of fetal membranes were more frequently observed among ewes with intrauterine contamination than among those without (P = 0.0007). The odds of an ewe having an intrauterine contamination increased multiplicatively by 1.06 when the median duration of retention in the flock increased by 6 h. The principal bacteria isolated from the ewes with retention were Arcanobacterium pyogenes and Escherichia coli; 21% of 73 isolates tested were found resistant to at least one antimicrobial agent.

The effects of inoculation of Listeria monocytogenes into the ovine mammary gland.

In each of two experiments, the effects of inoculation of Listeria monocytogenes into the ovine mammary gland were studied. In the first experiment, ewes were challenged with one or other of five different Listeria spp. isolates to study differences in their pathogenicity. In the second, ewes were challenged with L. monocytogenes serotype 1/2a to study the sequential features of the infection. The reaction of the mammary glands was assessed by bacteriological, cytological and histological methods. No distinct variation in the pathogenicity of L. monocytogenes isolates was evident: all produced subclinical mastitis, independently of their origin or serotype; a L. innocua isolate caused only a transient increase of milk somatic cell counts. After challenge, L. monocytogenes was isolated for 88 days from the milk of inoculated glands, whose milk somatic cell counts were greater than 1.0 x 10(6) cells ml-1. The organism was also isolated from the mammary lymph nodes, but not from any internal organ of any inoculated ewe. In early stages of the infection neutrophilic infiltration was the predominant histological feature, but hyperaemia, and degeneration of alveolar epithelial cells were also recorded. Later, chronic inflammatory features predominated, with lymphocytes as the principal cell types, destruction of alveoli and fibrous tissue proliferation. In the final stage of the experiment, fibrosis was the salient finding. It is concluded that L. monocytogenes can cause subclinical mastitis after intramammary inoculation into ewes.