Extensive Countrywide Molecular Identification and High Genetic Diversity of Haemonchus spp. in Domestic Ruminants in Greece.

The gastrointestinal nematode parasite Haemonchus spp. is one of the most pathogenic parasites of ruminants, due to its blood-sucking activity, which causes large economic losses in the ruminant industry. The latest epizootiological data recorded an increase in the infection, not only in Greece but also in other countries, mainly attributed to climatic changes. The study of the population structure and the investigation of the phylogenetic relationships of Haemonchus spp. are essential for the understanding of its biology and epizootiology to implement appropriate control and prevention strategies. In addition, the molecular approach allows the determination of evolutionary relationships between different species of this parasite, the diverse hosts they infect, as well as the different geographic compartments from which they originate. Therefore, the aim of the present study was to identify the species of the sympatric populations of the genus Haemonchus, a nematode parasite infecting ruminants (sheep, goats, cattle, and buffaloes) from different regions of Greece (continental and insular) using molecular methods. At the same time, an attempt was made to identify the possible subpopulations of Haemonchus spp. in Greece, to investigate their phylogenetic relationships, as well as to determine the genetic diversity of each population. A total of 288 worms of the genus Haemonchus were processed using molecular methods; of these, 96 were collected from sheep, 96 from goats, 48 from cattle, and finally, 48 from buffaloes. A fragment of 321 base pairs of the second internal transcribed spacer (ITS2) sequence of nuclear DNA was amplified for species identification, and, after basic local alignment search tool (Blast) analysis, it was revealed that they belonged to H. contortus. A fragment of 820 base pairs of subunit 4 of the nicotinamide dehydrogenase (ND4) gene of mitochondrial DNA was amplified for genetic diversity analysis. The Greek mitochondrial ND4 sequences of H. contortus were classified into 140 haplotypes, and the values of the average nucleotide and haplotype diversity were lower compared to the respective values derived from Italy, Malaysia, the USA, and China. The phylogenetic analysis of the ND4 gene revealed a clear grouping of the Greek haplotypes when compared with Asian ones, and, at the same time, there was no profound grouping of the same haplotypes with regard to their different hosts and geographical origin within different regions of Greece. The aforementioned findings confirmed that H. contortus prevails in our country and can infect all species of ruminants, without geographical boundaries, when the right conditions (i.e., common grazing) are created.

A genome-wide association study reveals novel SNP markers associated with resilience traits in two Mediterranean dairy sheep breeds.

Genetic selection for higher productivity increased dairy sheep susceptibility to diseases and environmental stressors, challenging their health and welfare status and production efficiency. Improving resilience to such stressors can enhance their ability to face these challenges without compromising productivity. Our objective was to estimate genomic heritability and perform genome-wide association studies (GWAS) to detect SNPs and candidate genes associated with three proxy traits for resilience (milk somatic cell count-SCC, lactation persistency-LP, body condition score-BCS) of Chios and Frizarta dairy ewes. We used genome-wide genotypes of 317 Chios and 346 Frizarta ewes. Individual records of milk yield and BCS, and milk samples were collected monthly for two consecutive milking periods; samples were analyzed to determine SCC. The LP was calculated as the regression coefficient of daily milk yield on days from lambing. Within breed, variance components analyses and GWAS were performed using genomic relatedness matrices in single-trait animal linear mixed models. Genomic-based heritability estimates were relatively high (BCS: h2 = 0.54 and 0.55, SCC: h2 = 0.25 and 0.38, LP: h2 = 0.43 and 0.45, for Chios and Frizarta ewes, respectively), compared to previous pedigree-based studies. The GWAS revealed 7 novel SNPs associated with the studied traits; one genome-wide and two suggestive significant SNPs for SCC (Frizarta: rs403061409, rs424064526 and rs428540973, on chromosomes 9, 1 and 12, respectively), one suggestive significant SNP for BCS (Chios: rs424834097 on chromosome 4) and three suggestive significant SNPs for LP (Frizarta: rs193632931 and rs412648955 on chromosomes 1 and 6, Chios: rs428128299 on chromosome 3). Nineteen candidate genes were detected: two for BCS (Chios: POT1, TMEM229A), thirteen for SCC (Frizarta: NTAQ1, ZHX1, ZHX2, LOC101109545, HAS2, DERL1, FAM83A, ATAD2, RBP7, FSTL1, CD80, HCLS1, GSK3B) and four for LP (Frizarta: GRID2, FAIM, CEP70-Chios: GRIP1). Present results show that resilience in the studied dairy sheep breeds is heritable and advance existing knowledge on the genomic background of SCC, LP, and BCS. Future research will quantify effects of different alleles of significant SNPs on the studied traits and search for possible correlations among traits to facilitate their effective incorporation in breeding programs aiming to improve resilience.

Investigating the role of genetic variation in vgll3 and six6 in the domestication of gilthead seabream (Sparus aurata Linnaeus) and European seabass (Dicentrarchus labrax Linnaeus).

Gene function conservation is crucial in molecular ecology, especially for key traits like growth and maturation in teleost fish. The vgll3 and six6 genes are known to influence age-at-maturity in Atlantic salmon, but their impact on other fish species is poorly understood. Here, we investigated the association of vgll3 and six6 in the domestication of gilthead seabream and European seabass, both undergoing selective breeding for growth-related traits in the Mediterranean. We analysed two different sets of samples using two different genotyping approaches. The first dataset comprised farmed and wild populations from Greece, genotyped for SNPs within the two genes ('gene-level genotyping'). The second dataset examined 300-600 k SNPs located in the chromosomes of the two genes, derived from a meta-analysis of a Pool-Seq experiment involving farmed and wild populations distributed widely across the Mediterranean ('chromosome-level genotyping'). The gene-level analysis revealed a statistically significant allele frequency differences between farmed and wild populations on both genes in each species. This finding was partially supported by the chromosome-level analysis, identifying highly differentiated regions may be involved in the domestication process at varying distances from the candidate genes. Noteworthy genomic features were found, such as a CpG island in gilthead seabream and novel candidate genes in European seabass, warranting further investigation. These findings support a putative role of vgll3 and six6 in the maturation and growth of gilthead seabream and European seabass, emphasizing the need for further research on their conserved function.

Genome sequencing of 2000 canids by the Dog10K consortium advances the understanding of demography, genome function and architecture.

BACKGROUND: The international Dog10K project aims to sequence and analyze several thousand canine genomes. Incorporating 20 × data from 1987 individuals, including 1611 dogs (321 breeds), 309 village dogs, 63 wolves, and four coyotes, we identify genomic variation across the canid family, setting the stage for detailed studies of domestication, behavior, morphology, disease susceptibility, and genome architecture and function. RESULTS: We report the analysis of > 48 M single-nucleotide, indel, and structural variants spanning the autosomes, X chromosome, and mitochondria. We discover more than 75% of variation for 239 sampled breeds. Allele sharing analysis indicates that 94.9% of breeds form monophyletic clusters and 25 major clades. German Shepherd Dogs and related breeds show the highest allele sharing with independent breeds from multiple clades. On average, each breed dog differs from the UU_Cfam_GSD_1.0 reference at 26,960 deletions and 14,034 insertions greater than 50 bp, with wolves having 14% more variants. Discovered variants include retrogene insertions from 926 parent genes. To aid functional prioritization, single-nucleotide variants were annotated with SnpEff and Zoonomia phyloP constraint scores. Constrained positions were negatively correlated with allele frequency. Finally, the utility of the Dog10K data as an imputation reference panel is assessed, generating high-confidence calls across varied genotyping platform densities including for breeds not included in the Dog10K collection. CONCLUSIONS: We have developed a dense dataset of 1987 sequenced canids that reveals patterns of allele sharing, identifies likely functional variants, informs breed structure, and enables accurate imputation. Dog10K data are publicly available.

Prevalence of SOD1 allele associated with degenerative myelopathy in canine population in Greece.

Canine degenerative myelopathy (CDM) is a late-onset fatal disorder associated with a point mutation of the superoxide dismutase 1 (SOD1) gene (c.118G > A). The purpose of this study was to determine the genotype and allele frequencies of this mutation in 108 dogs, mainly in Belgian Malinois and German Shepherd dogs with (CDM-affected group) and without CDM clinical symptoms (control group) in Greece. Genotyping of the c.118G > A mutation was possible by Sanger sequencing and PCR-RFLP. The observed genotype frequencies for the control group were 89.4% for the homozygous (G/G), 9.6% for the heterozygous (A/G), and 0.96% for the homozygous mutant (A/A) allele. The mutant allele was not common in the Belgian Malinois dogs (allele frequency = 0.029), but quite common in the German Shepherd dogs (allele frequency = 0.138). In the CDM affected group, all 4 dogs were homozygous for the mutant allele. These frequencies were close to those expected, indicating no significant departure from Hardy-Weinberg equilibrium. A strong but not statistically significant association between the mutant allele and CDM was observed. A previously identified deletion upstream of the mutation of interest was found at a high frequency (0.361) in the population.

Genetic Identification and Traceability of Insect Meals.

Insects have been proposed as a rich alternative source of protein for the partial or total replacement of fishmeal in aquaculture. For maximum safety and effectiveness of insect meals, control of the quality composition of these products is considered mandatory. The aim of this study was the genetic analysis of the composition of commercially available insect meals at the species level. Commercially available Hermetia illucens, Tenebrio molitor and Musca domestica individuals, as well as nine insect meals produced from these species, were analyzed. The genetic identification of insects at the species level was based on a COI fragment, and analysis of the insect meals' composition was performed with the processes of cloning and colony PCR. Genetic analysis indicated that the commercially available larvae morphologically identified as Musca domestica belonged to the species Muscina stabulans. In the commercially available insect meals, no other animal species was identified beyond the expected one. However, in the insect meal produced for research purposes, fungal growth was detected. The used methodology, herein, allows for the qualitative genetic identification of insect meals and could be included in the methods of traceability of products containing insects and other animal species.

Indications, Safety, Efficacy and Survival Benefit of Intraperitoneal Chemotherapy in Patients With Advanced Gastric Cancer.

BACKGROUND/AIM: Advanced gastric cancer remains a significant concern for the medical community mostly due to the locoregional extension of the disease. Most commonly, stomach neoplasms are resectable, but not curable, due to the elevated percentage of peritoneal dissemination after gastrectomy and extensive lymph node dissection. Locoregional intraperitoneal chemotherapy plays a pivotal role in overall survival and prognosis of patients with advanced gastric cancer and shows a high probability of peritoneal dissemination after gastrectomy. In this review, we aimed to collect and present literature data concerning intraperitoneal chemotherapy in advanced stages of gastric cancer as well as evaluate the safety and survival benefit of the procedure. MATERIALS AND METHODS: We conducted a survey including all randomized controlled trials and clinical trials that were published in the last 30 years. The keywords used were: advanced gastric cancer, intraperitoneal chemotherapy and peritoneal carcinomatosis. We searched for clinical trials in Pubmed, Embase databases and the Cochrane library. Inclusion criteria were: patients with advanced gastric cancer with no macroscopical signs of peritoneal dissemination, who were treated with D2 gastrectomy and received one or more cycles of intraperitoneal chemotherapy. The final review included 20 articles. RESULTS: The safety of intraperitoneal chemotherapy, as well as the survival benefit of patients were evaluated. The majority of articles denoted that intraperitoneal chemotherapy is a safe procedure without severe or lethal complications. The majority of complications were hematological while non-hematologic complications were also noted. A survival benefit with statistically significant results (p<0.05) was observed in 6 out of 10 randomized controlled trials. CONCLUSION: Intraperitoneal chemotherapy for advanced gastric cancer is a safe procedure with promising results regarding survival benefit and prognosis. Further patient evaluation is required in order to standardize the type of chemotherapeutic agent and the sufficient dose and cycles for the most appropriate results.

Isolation of Listeria monocytogenes from poultry red mite (Dermanyssus gallinae) infesting a backyard chicken farm in Greece.

The poultry red mite (PRM), Dermanyssus gallinae, is arguably the most harmful, ubiquitous haematophagous ectoparasite infesting egg-laying hens. PRM is a vector of various microorganisms, with some being important for food microbiology and public health. The present study aimed to investigate the presence of specific pathogens, including Escherichia coli, Salmonella spp. and Listeria spp., carried by PRM infesting a chicken farm in Greece. Mites were caught using cardboard traps (Avivet), and 100 unwashed PRM were homogenized and used for microbiological cultures. Microbiological cultures were carried out on general and selective substrates to detect the above-mentioned bacteria. Specifically for Listeria spp., DNA was extracted from bacteria grown in Tryptone Soya Yeast Extract Agar using a commercial kit. The hly gene encoding the Listeriolysin O protein was amplified by PCR. Mites were identified as D. gallinae using morphological keys as well as by COI DNA barcoding. Microbiological cultures and PCR assays were positive for Listeria monocytogenes. No other bacteria were detected. The current study constitutes the first molecular isolation of L. monocytogenes from D. gallinae, confirming that PRM can carry this food-borne pathogen. PRM control measures and hygiene practices should be applied to minimize any possible contamination risk of poultry products with L. monocytogenes and safeguard public health.

Bulgarian Medicinal Extracts as Natural Inhibitors with Antiviral and Antibacterial Activity.

BACKGROUND: Bulgaria is a country with a wide range of medicinal plants, with uses in traditional medicine dating back for centuries. METHODS: Disc diffusion assay was used to evaluate the antimicrobial activity of the plant extracts. A cytopathic effect inhibition test was used for the assessment of the antiviral activity of the extracts. The virucidal activity of the extracts, their influence on the stage of viral adsorption, and their protective effect on uninfected cells were reported using the end-point dilution method, and Δlgs was determined as compared to the untreated controls. RESULTS: The results of the study reveal that the antibacterial potential of G. glabra and H. perforatum extracts in Gram-positive bacteria is more effective than in Gram-negative bacteria. When applied during the replication of HSV-1 and HCov-OC-43, only some of the extracts showed weak activity, with SI between 2 to 8.5. Almost all tested extracts inhibited the extracellular virions of the studied enveloped viruses (HSV-1 and HCov-OC-43) to a greater extent than of the non-enveloped viruses (PV-1 and HAdV-5). They inhibited the stage of viral adsorption (HSV-1) in the host cell (MDBK) to varying degrees and showed a protective effect on healthy cells (MDBK) before they were subjected to viral invasion (HSV-1). CONCLUSION: The antipathogenic potential of extracts of H. perforatum and G. glabra suggests their effectiveness as antimicrobial agents. All 13 extracts of the Bulgarian medicinal plants studied can be used to reduce viral yield in a wide range of viral infections.

DNA barcoding identification of Greek freshwater fishes.

Biodiversity is a key factor for the functioning and efficiency of an ecosystem. Greece, though covering a relatively small surface area, hosts a great deal of species diversity. This is especially true for freshwater fishes. In recent years, the traditional methods of species identification have been supplemented by the use of molecular markers. The present study therefore aims to extensively produce DNA barcodes for Greek freshwater fish species and investigate thoroughly if the presently accepted species classification is in agreement with molecular data. A 624-bases long fragment of the COI gene was sequenced, from 406 freshwater fish specimens belonging to 24 genera and originating from 18 lake and river sites. These sequences were used along with 596 sequences from the same genera, recovered from BOLD, for the construction of phylogenetic trees and the estimation of genetic distances between individuals. In total, 1002 sequences belonging to 72 species were analyzed. The method was found to be effective for 55 of 72 studied species. 17 closely related species with low interspecific genetic distances were observed, for which further study is proposed. It should also be noted that, in four cases, cryptic diversity was observed, where groups originally identified as one species exhibited genetic distance great enough to be separated into discrete species. Region specific haplotypes were also detected within populations of 14 species, giving the possibility to identify even the geographic origin of a species. Our findings are discussed in the light of the rich history of the Balkan peninsula and provide a significant steppingstone for the further study of Greek and European freshwater fish biodiversity.

A comprehensive genome-wide scan detects genomic regions related to local adaptation and climate resilience in Mediterranean domestic sheep.

BACKGROUND: The management of farm animal genetic resources and the adaptation of animals to climate change will probably have major effects on the long-term sustainability of the livestock sector. Genomic data harbour useful relevant information that needs to be harnessed for effectively managing genetic resources. In this paper, we report the genome characterization of the highly productive Mediterranean Chios dairy sheep and focus on genetic diversity measures related with local adaptation and selection and the genetic architecture of animal resilience to weather fluctuations as a novel adaptative trait linked to climate change. RESULTS: We detected runs of homozygosity (ROH) and heterozygosity (ROHet) that revealed multiple highly homozygous and heterozygous hotspots across the Chios sheep genome. A particularly highly homozygous region was identified on chromosome 13 as a candidate of directional genetic selection associated with milk traits, which includes annotated genes that were previously shown to be linked to local adaptation to harsh environmental conditions. Favourable heterozygosity related with a potentially protective role against livestock diseases and enhanced overall fitness was revealed in heterozygous-rich regions on sheep chromosomes 3, 10, 13 and 19. Furthermore, genomic analyses were conducted on sheep resilience phenotypes that display changes in milk production in response to weather variation. Sheep resilience to heat stress was a significantly heritable trait (h2 = 0.26) and genetically antagonistic to milk production. Genome-wide association and regional heritability mapping analyses revealed novel genomic markers and regions on chromosome 5 that were significantly associated with sheep resilience to climate change. Subsequently, an annotation analysis detected a set of genes on chromosome 5 that were associated with olfactory receptor complexes that could participate in heat stress mitigation through changes in respiration rate and respiratory evaporation. Other genes were grouped in previously reported biological processes relevant to livestock heat dissipation, including stress and immune response. CONCLUSIONS: Our results may contribute to the optimal management of sheep genetic resources and inform modern selective breeding programmes that aim at mitigating future environmental challenges towards sustainable farming, while better balancing animal adaptation and productivity. Our results are directly relevant to the studied breed and the respective environmental conditions; however, the methodology may be extended to other livestock species of interest.

An unusual case of obstructive uropathy.

Although inguinal bladder hernia associated with obstructive uropathy is an extremely rare entity, it should be suspected in elderly patients with bladder outlet obstruction presented with inguinal hernia and lower urinary tract symptoms.

Case of Human Infestation with Dermanyssus gallinae (Poultry Red Mite) from Swallows (Hirundinidae).

Dermanyssus gallinae (the poultry red mite, PRM) is an important ectoparasite in the laying hen industry. PRM can also infest humans, causing gamasoidosis, which is manifested as skin lesions characterized by rash and itching. Recently, there has been an increase in the reported number of human infestation cases with D. gallinae, mostly associated with the proliferation of pigeons in cities where they build their nests. The human form of the disease has not been linked to swallows (Hirundinidae) before. In this report, we describe an incident of human gamasoidosis linked to a nest of swallows built on the window ledge of an apartment in the island of Kefalonia, Greece. Mites were identified as D. gallinae using morphological keys and amplifying the Cytochrome C oxidase subunit I (COI) gene by PCR. Bayesian phylogenetic analysis and median-joining network supported the identification of three PRM haplogroups and the haplotype isolated from swallows was identical to three PRM sequences isolated from hens in Portugal. The patient was treated with topical corticosteroids, while the house was sprayed with deltamethrin. After one week, the mites disappeared and clinical symptoms subsided. The current study is the first report of human gamasoidosis from PRM found in swallows' nest.

Understanding the seasonality of performance resilience to climate volatility in Mediterranean dairy sheep.

As future climate challenges become increasingly evident, enhancing performance resilience of farm animals may contribute to mitigation against adverse weather and seasonal variation, and underpin livestock farming sustainability. In the present study, we develop novel seasonal resilience phenotypes reflecting milk production changes to fluctuating weather. We evaluate the impact of calendar season (autumn, winter and spring) on animal performance resilience by analysing 420,534 milk records of 36,908 milking ewes of the Chios breed together with relevant meteorological data from eastern Mediterranean. We reveal substantial seasonal effects on resilience and significant heritable trait variation (h2 = 0.03-0.17). Resilience to cold weather (10 °C) of animals that start producing milk in spring was under different genetic control compared to autumn and winter as exemplified by negative genetic correlations (- 0.09 to - 0.27). Animal resilience to hot weather (25 °C) was partially under the same genetic control with genetic correlations between seasons ranging from 0.43 to 0.86. We report both favourable and antagonistic associations between animal resilience and lifetime milk production, depending on calendar season and the desirable direction of genetic selection. Concluding, we emphasise on seasonal adaptation of animals to climate and the need to incorporate the novel seasonal traits in future selective breeding programmes.

Genome-wide analysis clarifies the population genetic structure of wild gilthead sea bream (Sparus aurata).

Gilthead sea bream is an important target for both recreational and commercial fishing in Europe, where it is also one of the most important cultured fish. Its distribution ranges from the Mediterranean to the African and European coasts of the North-East Atlantic. Until now, the population genetic structure of this species in the wild has largely been studied using microsatellite DNA markers, with minimal genetic differentiation being detected. In this geographically widespread study, 958 wild gilthead sea bream from 23 locations within the Mediterranean Sea and Atlantic Ocean were genotyped at 1159 genome-wide SNP markers by RAD sequencing. Outlier analyses identified 18 loci potentially under selection. Neutral marker analyses identified weak subdivision into three genetic clusters: Atlantic, West, and East Mediterranean. The latter group could be further subdivided into an Ionian/Adriatic and an Aegean group using the outlier markers alone. Seascape analysis suggested that this differentiation was mainly due to difference in salinity, this being also supported by preliminary genomic functional analysis. These results are of fundamental importance for the development of proper management of this species in the wild and are a first step toward the study of the potential genetic impact of the sea bream aquaculture industry.

The genus Diaphanosoma (Diplostraca: Sididae) in Greece: morphological and molecular assessment.

For the genus Diaphanosoma Fischer (Cladocera) the species name D. brachyurum has been widely used for many decades to identify other species belonging to this genus. To clarify the diversity of the genus in Greek lakes in the present study, we morphologically and genetically identified the Diaphanosoma species occurring in eight lakes. Three hundred twenty-nine Diaphanosoma individuals were morphologically examined, while for the genetic analyses the mtDNA COI gene was sequenced in 48 individuals. Combining the morphological and genetic results, we verified the occurrence of D. mongolianum, D. orghidani and D. macedonicum in our study area. We could not confirm prior records of D. brachyurum and D. lacustris while we provide the molecular identity of D. macedonicum. Furthermore, we highlight the need to check whether the European D. mongolianum populations are characterised of mitochondrial discordance and hybridization as the individuals from the Asian type locality of the species. Our results support the importance of combining both approaches to correctly identify taxonomic species, despite the extra effort and cost during the sample analysis.

Population Genetic Structure and Connectivity of the European Lobster Homarus gammarus in the Adriatic and Mediterranean Seas.

Highly selective fishing has the potential to permanently change the characteristics within a population and could drive the decline of genetic diversity. European lobster is an intensively fished crustacean species in the Adriatic Sea which reaches high market value. Since knowledge of population structure and dynamics is important for effective fisheries management, in this study, we used 14 neutral microsatellites loci and partial mitochondrial COI region sequencing to explore population connectivity and genetic structure by comparing samples from the Adriatic Sea and the adjacent basins of the Mediterranean Sea. The obtained results suggest that neutral genetic diversity has not been significantly affected by decrease in population size due to overfishing, habitat degradation and other anthropogenic activities. Global genetic differentiation across all populations was low (F ST = 0.0062). Populations from the Adriatic Sea were panmictic, while genetic differentiation was found among populations from different Mediterranean basins. Observed gene flow for European lobster suggest that populations in the north eastern Adriatic act as a source for surrounding areas, emphasizing the need to protect these populations by establishing interconnected MPAs that will be beneficial for both fisheries and conservation management.

Mystery of the lost gallstone-Part 2.

If gallbladder perforation occurs during cholecystectomy, every spilled gallstone should be retrieved to minimize possible late gallstone-related complications.