Prevalence and molecular characterization of Toxoplasma gondii in different types of poultry in Greece, associated risk factors and co-existence with Eimeria spp.

Toxoplasma gondii is a protozoan parasite of public health importance, infecting all warm-blooded animals, including chickens. Undercooked chicken meat or relevant products such as sausages could lead to human infections. In free-range, organic and slow-growth farming systems where the susceptibility period for chickens is extended, more knowledge about potential risk factors is essential. This study is the first seroepidemiological survey in different regions and types of chicken farms in Greece, using a major tachyzoite surface antigen-based ELISA (TgSAG1), combined with magnetic-capture PCR (mc-PCR) and bioassay for the isolation of strains from the chickens' tissues. Potential risk factors for T. gondii infection in these hosts were also investigated. Additionally, the co-existence of T. gondii and Eimeria spp. infections was assessed to elucidate epidemiological links between these two protozoan infections. Overall T. gondii seroprevalence was 9.5%. Of the backyard chickens sampled, 41.2% were seropositive and 70% of the organic and free-range layer farms had at least one T. gondii seropositive hen. No serologically positive broilers were found, although mc-PCR revealed a positive sample, highlighting the importance of accurate early-infection direct detection of T. gondii infections to ensure public health. T. gondii isolates obtained by mouse bioassay were genotyped. All belonged to type II (ToxoDB#3) as confirmed also by microsatellite typing. Production system, type of nutrition, and feeding system automation were identified as the most significant risk factors, while no association was found between the presence of cats and T. gondii seropositivity as calculated on both a farm level and per individual bird sampled.

Corrigendum: Overview of Cattle Diseases Listed Under Category C, D or E in the Animal Health Law for Which Control Programmes Are in Place Within Europe.

[This corrects the article DOI: 10.3389/fvets.2021.688078.].

The current situation and potential effects of climate change on the microbial load of marine bivalves of the Greek coastlines: an integrative review.

Global warming affects the aquatic ecosystems, accelerating pathogenic microorganisms' and toxic microalgae's growth and spread in marine habitats, and in bivalve molluscs. New parasite invasions are directly linked to oceanic warming. Consumption of pathogen-infected molluscs impacts human health at different rates, depending, inter alia, on the bacteria taxa. It is therefore necessary to monitor microbiological and chemical contamination of food. Many global cases of poisoning from bivalve consumption can be traced back to Mediterranean regions. This article aims to examine the marine bivalve's infestation rate within the scope of climate change, as well as to evaluate the risk posed by climate change to bivalve welfare and public health. Biological and climatic data literature review was performed from international scientific sources, Greek authorities and State organizations. Focusing on Greek aquaculture and bivalve fisheries, high-risk index pathogenic parasites and microalgae were observed during summer months, particularly in Thermaikos Gulf. Considering the climate models that predict further temperature increases, it seems that marine organisms will be subjected in the long term to higher temperatures. Due to the positive linkage between temperature and microbial load, the marine areas most affected by this phenomenon are characterized as 'high risk' for consumer health.

How Safe to Eat Are Raw Bivalves? Host Pathogenic and Public Health Concern Microbes within Mussels, Oysters, and Clams in Greek Markets.

Raw-bivalves consumption is a wide trend in Mediterranean countries. Despite the unambiguous nutritional value of seafood, raw consumption of bivalves may involve risks that could pose a significant threat to consumers' health. Their filter-feeding behavior is responsible for the potential hosting of a wide variety of microorganisms, either pathogenic for the bivalves or public health threats. Under this prism, the current study was conducted in an effort to evaluate the risk of eating raw bivalves originating from the two biggest seafood markets in Thessaloniki, the largest production area of bivalves in Greece. Both microbiological and molecular methodologies were applied in order to assess the presence of various harmful microbes, including noroviruses, Bonamia, Marteilia, Esherichia coli, Salmonella, and Vibrio. Results indicated the presence of several Vibrio strains in the analyzed samples, of which the halophilic Vibrio harveyi was verified by 16S rRNA sequencing; other than this, no enteropathogenic Vibrio spp. was detected. Furthermore, although Esherichia coli was detected in several samples, it was mostly below the European Union (EU) legislation thresholds. Interestingly, the non-target Photobacterium damselae was also detected, which is associated with both wound infections in human and aquatic animals. Regarding host pathogenic microorganisms, apart from Vibrio harveyi, the protozoan parasite Marteilia refrigens was identified in oysters, highlighting the continuous infection of this bivalve in Greece. In conclusion, bivalves can be generally characterized as a safe-to-eat raw food, hosting more bivalve pathogenic microbes than those of public health concern.

Overview of Cattle Diseases Listed Under Category C, D or E in the Animal Health Law for Which Control Programmes Are in Place Within Europe.

The COST action "Standardising output-based surveillance to control non-regulated diseases of cattle in the European Union (SOUND control)," aims to harmonise the results of surveillance and control programmes (CPs) for non-EU regulated cattle diseases to facilitate safe trade and improve overall control of cattle infectious diseases. In this paper we aimed to provide an overview on the diversity of control for these diseases in Europe. A non-EU regulated cattle disease was defined as an infectious disease of cattle with no or limited control at EU level, which is not included in the European Union Animal health law Categories A or B under Commission Implementing Regulation (EU) 2020/2002. A CP was defined as surveillance and/or intervention strategies designed to lower the incidence, prevalence, mortality or prove freedom from a specific disease in a region or country. Passive surveillance, and active surveillance of breeding bulls under Council Directive 88/407/EEC were not considered as CPs. A questionnaire was designed to obtain country-specific information about CPs for each disease. Animal health experts from 33 European countries completed the questionnaire. Overall, there are 23 diseases for which a CP exists in one or more of the countries studied. The diseases for which CPs exist in the highest number of countries are enzootic bovine leukosis, bluetongue, infectious bovine rhinotracheitis, bovine viral diarrhoea and anthrax (CPs reported by between 16 and 31 countries). Every participating country has on average, 6 CPs (min-max: 1-13) in place. Most programmes are implemented at a national level (86%) and are applied to both dairy and non-dairy cattle (75%). Approximately one-third of the CPs are voluntary, and the funding structure is divided between government and private resources. Countries that have eradicated diseases like enzootic bovine leukosis, bluetongue, infectious bovine rhinotracheitis and bovine viral diarrhoea have implemented CPs for other diseases to further improve the health status of cattle in their country. The control of non-EU regulated cattle diseases is very heterogenous in Europe. Therefore, the standardising of the outputs of these programmes to enable comparison represents a challenge.

Coxiella burnetii Shedding in Milk and Molecular Typing of Strains Infecting Dairy Cows in Greece.

Ruminants are considered the commonest animal reservoir for human infection of Coxiella burnetii, the Q fever causative agent. Considering the recently described importance of human Q fever in Greece, we aimed at providing the first comprehensive direct evidence of C. burnetii in dairy cows in Greece, including the genetic characterization of strains. The 462 examined dairy farms represented all geographical areas of Greece. One bulk tank milk sample was collected from every farm and tested for the presence of C. burnetii. Molecular genotyping of strains, performed directly on samples, revealed the existence of two separate clades characterized by single nucleotide polymorphism (SNP) genotypes of type 1 and type 2. The two clades were clearly distinguished in multiple locus variable-number tandem repeat analysis (MLVA) by two discriminative loci: MS30 and MS28. Whereas MLVA profiles of SNP-type 2 clade were closely related to strains described in other European cattle populations, the MLVA profile observed within the SNP type 1 clade highlighted a peculiar genetic signature for Greece, related to genotypes found in sheep and goats in Europe. The shedding of C. burnetii bearing this genotype might have yet undefined human epidemiological consequences. Surveillance of the genetic distribution of C. burnetii from different sources is needed to fully understand the epidemiology of Q fever in Greece.

Molecular detection of Crimean-Congo hemorrhagic fever virus in ticks, Greece, 2012-2014.

Crimean-Congo hemorrhagic fever virus (CCHFV) is transmitted to humans mainly through the bite of infected ticks. In Greece, only one clinical case has been observed, in 2008, but the seroprevalence in humans is relatively high (4.2%). To have a first insight into the circulation of CCHFV in Greece, 2000 ticks collected from livestock during 2012-2014 were tested. CCHFV was detected in 36 of the 1290 (2.8%) tick pools (1-5 ticks per pool). Two genetic lineages were identified: Europe 1 and Europe 2. Most Europe 1 sequences were obtained from Rhipicephalus sanguineus sensu lato ticks, while most Europe 2 sequences were recovered from Rhipicephalus bursa ticks. The number of collected Hyalomma marginatum ticks (the principal vector of CCHFV) was low (0.5% of ticks) and all were CCHFV negative. Since it is not known how efficient ticks of the Rhipicephalus genus are as vectors of the virus, laboratory studies will be required to explore the role of Rhipicephalus spp. ticks in CCHFV maintenance and transmission.

First Identification and Molecular Characterization of Avian metapneumovirus Subtype B from Chickens in Greece.

Avian metapneumovirus (aMPV) is considered a major pathogen for turkeys but its impact on chicken production is still partially neglected, even though it is fully acknowledged as a primary pathogen in chickens as well. The lack of structured diagnostic surveys does not allow a pervasive understanding of aMPV epidemiology. Being that aMPV is almost an everyday challenge for farmers and veterinarians, a more accurate report of its presence should be detailed, posing the basis for a deep and global epidemiologic analysis. With these premises, the present work aims to report the first detection and molecular characterization of aMPV subtype B field strains from unvaccinated chickens in Greece. The Greek strains appear to be phylogenetically related among each other and with other recent Mediterranean strains while being distant from the currently applied vaccines, thus stressing once more the necessity to evaluate aMPV diffusion and evolution.

Combination of RT-PCR and proteomics for the identification of Crimean-Congo hemorrhagic fever virus in ticks.

Crimean-Congo hemorrhagic fever (CCHF) is an emerging tick-borne zoonotic disease caused by the CCHF virus (CCHFV). In this study, an experimental approach combining RT-PCR and proteomics was used for the identification and characterization of CCHFV in 106 ticks from 7 species that were collected from small ruminants in Greece. The methodological approach included an initial screening for CCHFV by RT-PCR followed by proteomics analysis of positive and control negative tick samples. This novel approach allowed the identification of CCHFV-positive ticks and provided additional information to corroborate the RT-PCR findings using a different approach. Two ticks, Dermacentor marginatus and Haemaphysalis parva collected from a goat and a sheep, respectively were positive for CCHFV. The sequences for CCHFV RNA segments S and L were characterized by RT-PCR and proteomics analysis of tick samples, respectively. These results showed the possibility of combining analyses at the RNA and protein levels using RT-PCR and proteomics for the characterization of CCHFV in ticks. The results supported that the CCHFV identified in ticks are genetic variants of the AP92 strain. Although the AP92-like strains probably do not represent a high risk of CCHF to the population, the circulation of genetically diverse CCHFV strains could potentially result in the appearance of novel viral genotypes with increased pathogenicity and fitness.

Molecular characterization of Echinococcus granulosus isolates from Bulgarian human cystic echinococcosis patients.

Although cystic echinococcosis (CE) is highly endemic in Bulgaria, there is still scarce information about species and/or genotypes of the Echinococcus granulosus complex that infect humans. Our study tackled the genetic diversity of E. granulosus complex in a cohort of 30 Bulgarian CE patients. Ten animal E. granulosus isolates from neighboring Greece were additionally included. Specimens were comparatively analyzed for partial sequences of five mitochondrial (mt) (cox I, nad I, rrnS, rrnL, and atp6) and three nuclear (nc) genes (act II, hbx 2, and ef-1α) using a PCR-sequencing approach. All 30 Bulgarian isolates were identified as E. granulosus sensu stricto (s.s.) and were showing identical sequences for each of the three examined partial nc gene markers. Based upon concatenated sequences from partial mtDNA markers, we detected 10 haplotypes: 6 haplotypes (H1-H6) clustering with E. granulosus s.s. (G1) and 4 haplotypes (H9-H13) grouping with E. granulosus s.s. (G3), with H1 and H10 being the most frequent in Bulgarian patients. The haplotypes H1, H4, and H11 were also present in Greek hydatid cyst samples of animal origin. In conclusion, E. granulosus s.s. (G1 and G3 genotypes) is the only causative agent found so far to cause human CE in Bulgaria. However, further studies including larger sample sizes and other additional geographic regions in Bulgaria will have to be performed to confirm our results.

Rickettsia species in human-parasitizing ticks in Greece.

BACKGROUND: Ticks serve as vectors and reservoirs for a variety of bacterial, viral and protozoan pathogens affecting humans and animals. Unusual increased tick aggressiveness was observed in 2008-2009 in northeastern Greece. The aim of the study was to check ticks removed from persons during 2009 for infection with Rickettsia species. METHODS: A total of 159 ticks were removed from 147 persons who sought medical advice in a hospital. Tick identification was performed morphologically using taxonomic keys. DNA was extracted from each individual tick and a PCR assay targeting the rickettsial outer membrane protein A gene of Rickettsia spp. was applied. RESULTS: Most of the adult ticks (132/153, 86.3%) were Rhipicephalus sanguineus. Rickettsiae were detected in 23 of the 153 (15.0%) adult ticks. Five Rickettsiae species were identified: R. aeschlimannii, R. africae (n=6), R. massilae (4), R. monacensis (1), and Candidatus R. barbariae (1). To our knowledge, this is the first report of R. africae, R. monacensis, and Candidatus R. barbariae in Greece. CONCLUSIONS: Several Rickettsia species were identified in ticks removed from humans in Greece, including those that are prevalent in northern and southern latitudes.

Novel phleboviruses detected in ticks, Greece.

Since 2009, when severe fever with thrombocytopenia syndrome virus and Heartland virus have been identified and associated with disease in humans, the interest on tick-borne phleboviruses is increasing rapidly. The aim of the present study was to investigate the presence of tick-borne phleboviruses in Greece and compare them with respective ones detected worldwide. Ticks collected from goats and sheep in 60 sites of 13 regional units of Greece were grouped in pools (1-3 ticks per pool) and tested for the presence of phleboviral RNA. Six of 210 pools were positive; they consisted of Rhipicephalus sanguineus ticks collected from sheep in 3 regional units of Greece: Pella (2/30, 6.7%), Imathia (2/21, 9.5%), and Ioannina (2/28, 7.1%). The overall tick minimum infection rate was 2.1%. The sequences of the Greek phlebovirus (provisionally named Antigone virus) form a distinct clade in the tick-borne phleboviruses, differing by >40% from the currently known phleboviruses. Any probable implication of these viruses to public health remains to be elucidated.

A novel AP92-like Crimean-Congo hemorrhagic fever virus strain, Greece.

Ticks were collected from various regions of northern Greece and tested for the presence of Crimean-Congo hemorrhagic fever virus (CCHFV) RNA. Human and animal sera were collected in the regions where CCHFV-positive ticks were detected, and they were tested for the presence of IgG antibodies against the virus. A CCHFV strain was detected in Rhipicephalus bursa ticks collected from sheep in Kastoria regional unit, differing by 9.7% at the nucleotide level from the AP92 strain, which was isolated in 1975 in another region of Greece. Up to date, CCHF cases have not been reported in these regions. The human seroprevalence in the area was estimated at 6%, while IgG-positive sheep was detected in two of the four neighboring farms tested. The circulation of this specific CCHFV lineage in Greece, especially in a region where the seroprevalence is high, together with the lack of human CCHF cases, suggests a probable antigenic, but non- or low-pathogenic character of this lineage. Further studies on these strains will increase our knowledge about the role of AP92-like strains in the CCHF epidemiology, which might be useful for drug and vaccine design.

Anisakidae infection in fish of the Aegean Sea.

Nematode worms of the family Anisakidae are the causative agents of infections in humans when fish is consumed raw and of serious allergies up to the death, when fish is consumed raw or cooked by previously sensitized people. From April until November 2009, 462 fish belonging to 26 species, fished in three areas of the Aegean Sea were tested for Anisakidae larvae. Anisakidae larvae were detected in 87 (18.83%) fish of 13 species. These larvae were identified by morphology as the third-stage larvae of the genera Hysterothylacium sp. or Anisakis. Larvae of the genus Anisakis were identified by PCR-RFLP as belonging to A. simplex s.str., A. pegreffii, or as hybrids between A. simplex s.str and A. pegreffii.

Ticks parasitizing humans in Greece.

In summer 2008, two fatal cases were observed in Northeastern Greece: a Crimean-Congo hemorrhagic fever (CCHF) case (first report in Greece) and a Mediterranean spotted fever case. In total, 537 ticks removed from humans who referred for this reason to the two hospitals of the region during June-September 2008 were identified. The vast majority of them (81.5%) were Rhipicephalus sanguineus, which is the main vector of Rickettsia conorii, while Hyalomma marginatum, the main vector of CCHF virus, accounted for 5.2%. The increased aggressiveness of R. sanguineus might be related to the weather conditions occurred during 2007-2008, while a variety of factors, including climate, might play a role in CCHF emergence.