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.

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.

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.

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.

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.

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.

Frequency of Resistance to Benzimidazoles of Haemonchus contortus Helminths from Dairy Sheep, Goats, Cattle and Buffaloes in Greece.

The study investigated the presence of resistance to benzimidazoles in Haemonchus contortus helminths from ruminant species in Greece through the detection of the Phe/Tyr polymorphism in the amino acid at position 200 of the ß-tubulin protein. In total, 288 adult female H. contortus helminths collected from the abomasum of various ruminant animals in Greece were tested. Of these, 96 were collected from sheep, 96 from goats, 48 from cattle, and 48 from buffaloes. The frequencies of the homozygous and heterozygous resistant genotypes at the position 200 of the ß-tubulin gene of helminths recovered from sheep were 96.9% and 3.1%, respectively. The frequencies of the homozygous and heterozygous resistant genotypes, respectively, were 100.0% and 0.0% in helminths from goats, 25.0% and 75.0% in helminths from cattle and 8.3% and 91.7% in helminths from buffaloes. In all parasitic populations, no homozygous susceptible genotypes were detected. The present study highlighted, for the first time, the emergence of benzimidazole-resistant H. contortus in goats, cattle, and buffaloes in Greece, using an allele-specific PCR. It is postulated that benzimidazole-resistant alleles were transferred from sheep or goats to cattle and buffaloes at the commonly grazing pastures in Greece.

Genetic analyses of brown hare (Lepus europaeus) support limited migration and translocation of Greek populations.

Numerous studies have shown that the phylogeography of many species, including European brown hare, has been affected by the climatic oscillations of the Pleistocene. During this period the Balkans acted as a major refugium offering habitable conditions for many species. However, few studies have focused on the specific role of the Greek peninsula in the phylogeographic history of species in this southernmost margin of Balkans. We, therefore analyzed a 528 bp fragment of the D-loop region of mitochondrial DNA in 154 wild brown hare individuals from unsampled areas from both mainland and island Greece and compared it to 310 available brown hare sequences (including 110 Greek samples). Newly identified haplotypes show characteristic distribution in specific Greek areas reinforcing the theory that Greece can be considered as a subrefuge within Balkans for a number of species, with several "refugia within refugia" spots, holding significant genetic diversity. No haplotypes from wild Greek individuals clustered with the Central and Western Europe group revealing a minimal contribution of this area to the colonization of central Europe. One hundred and ten reared brown hares were also analyzed to elucidate the impact of introductions on local populations. Most of these samples presented close genetic affinity with haplotypes from Central and Western Europe indicating that farms in Greece use breeders from those areas. Therefore, despite human translocation of individuals, the genetic structure of brown hare has mostly been influenced by paleoclimatic conditions and minimally by human actions.