Detection of Antibodies Against Toxoplasma gondii in Filter Paper-Dried Blood Dot Spots Compared with Serum in Pigs and Assessment of Variation Associated with Packed Cell Volume.

The aim of this study was to assess the agreement between anti-Toxoplasma gondii IgG antibody detection in serum and filter paper (FP) blood spots using the indirect immunofluorescence antibody assay (IFA) and to evaluate the potential impact of the packed cell volume (PCV) on antibody detection in FPs. A pair of a serum and an FP sample was collected from 96 sows at various farms in Greece, with previously identified high seropositivity and/or risk factors associated with high seropositivity against T. gondii. The PCV value was determined using the microhematocrit method. IFA was used for the detection of antibodies against T. gondii. T. gondii-specific antibodies were detected in 45.8% serum samples and 41.6% FP samples showing almost perfect agreement. Detection in FP samples presented high sensitivity (87.1-92.8%) and excellent specificity (100%) when compared with detection in serum, regardless of the PCV values. The findings of this study support the reliability of FPs for the evaluation of the serological status of swine against T. gondii. FPs could be a good alternative sample type compared with serum for large-scale epidemiological studies.

Evaluation of an Indirect Immunofluorescence Assay for the Detection of Anaplasma phagocytophilum Antigen in Ovine Buffy Coat Smears.

Diagnosis of anaplasmosis is challenging considering the great variation in clinical signs and the limitations of the available diagnostic assays, while the detection of carrier animals that play a significant role in disease epidemiology as reservoirs is of great significance. In this study, we evaluated the diagnostic accuracy of a newly developed indirect immunofluorescent assay (Ag-IFAT) for the detection of A. phagocytophilum antigens in buffy coat specimens, alone and in combination with cytology, using PCR as a reference. Blood samples were collected from 138 sheep of the Chios breed from six farms in Greece. A buffy coat was extruded from the centrifuged blood. Buffy coat smears were used for cytological examination and the Ag-IFAT assay. The Ag-IFAT assay presented excellent specificity (100%) and high sensitivity (85.4%) for the detection of A. phagocytophilum antigens in buffy coats, and it has an almost perfect agreement with PCR and cytology (κ value = 0.88 and 0.85, respectively). A. phagocytophilum antigens are likely to be detected using Ag-IFAT in a PCR-positive animal, as indicated by the good performance of the assay. Overall, this assay presents high diagnostic accuracy, and it could be used for the detection of animals during the early stage of infection.

Wild Rabbit Exposure to Leishmania infantum, Toxoplasma gondii, Anaplasma phagocytophilum and Babesia caballi Evidenced by Serum and Aqueous Humor Antibody Detection.

Wild rabbits (Oryctolagus cuniculus) can be important sentinel species for the presence of zoonotic pathogens. Therefore, we collected blood samples from wild rabbits harvested by hunters during the hunting season 2019-2020 on the island of Lemnos, to determine exposure of wild rabbits to the zoonotic pathogens Leishmania infantum, Toxoplasma gondii, Anaplasma phagocytophilum and Babesia caballi, as well as aqueous humor to assess its diagnostic performance in terms of sensitivity, specificity, positive and negative likelihood ratios. Antibodies against these pathogens were detected by Indirect Immunofluorescence Antibody (IFA) assay. Out of the 72 wild rabbits included in the study, 4.2%, 5.5%, 18% and 9.7% were seropositive to L. infantum, T. gondii, A. phagocytophilum and B. caballi, respectively. Although less frequently, antibodies were also detected in aqueous humor of wild rabbits. The antibody detection in aqueous humor presented 100% specificity but decreased sensitivity compared to serum suggesting that aqueous humor could be successfully used in epidemiological studies to confirm exposure at the population level but has little diagnostic value at the individual level. This is the first report on the seropositivity of wild rabbits to A. phagocytophilum and B. caballi and the detection of antibodies against A. phagocytopylum, L. infantum, T. gondii and B. caballi in the aqueous humor.

Quantitative and Morphological Blood Cell Findings Associated with the Presence of Antigen and/or Antibodies Against Anaplasma phagocytophilum in Sheep.

Anaplasma phagocytophilum is the causative agent of a disease known as tick borne fever in sheep, although fever is not always present. Due to inconclusive clinical signs, diagnosis is based on the cytological or molecular detection of the microorganism in blood and/or the determination of antibodies against A. phagocytophilum. The aim of the study was to determine the alterations caused by the presence of antibodies and/or the antigen of A. phagocytophilum in the blood cell count and morphology in sheep. Cytology and indirect immunofluorescence assay were performed for detection of antibodies and the antigen of A. phagocytophilum, respectively. The samples were divided into four groups depending on the result of the antigen and antibody detection. The samples that were only positive for antigen detection had mild anemia, leukopenia (lymphopenia), and thrombocytopenia. The samples that were positive in both assays had anemia, leukopenia (neutropenia and lymphopenia), and thrombocytopenia. Samples that were positive only for antibody detection had mild leukopenia. Morphological findings in infected sheep included band neutrophils, toxic neutrophils, reactive lymphocytes, and activated monocytes. The hematological findings along with cytological and serological tests can contribute to the assessment of the stage of the disease. A combination of leukopenia and thrombocytopenia raises a strong suspicion of the disease. When the microorganism and antibodies are simultaneously present, sheep are more susceptible to secondary complications. The first reported morphological findings and the quantitative hematological alterations are indicative of an inflammatory reaction, antigenic stimulation, and stress.

Trichinella britovi as a risk factor for alternative pig production systems in Greece and Europe.

Trichinellosis is an important zoonosis and the most common source of human infection is meat from pigs and wild boars. The European Union (EU) supports alternative pig production systems (organic and free-ranging) as sustainable farming systems. However, these systems that allow outdoor access for farm animals, may create new or reintroduce old risks to public health. During the last years, alternative pig production systems (free-ranging or organic pig) are growing in popularity in Greece due to the increasing interest of consumers for organic products. The majority of the trichinellosis outbreaks in the EU were associated with pork and meat products including wild boars. In Greece, from 2009 to 2012, 37 Trichinella spp. positive free-ranging pigs were reported in free-ranging pig farms of Northern-Eastern Greece (31 were identified as T. britovi). The recent re-emerging present of the Trichinella spp. infections in free-ranging pigs and wild boars are a high risk for the consumers and should alarm the Public Health Authorities in Greece and the EU. During the last years, the organic or free-ranging pig production systems are growing in popularity in the EU. However, these systems increase the risk of Trichinella spp. infections, since pigs are possible to be infected by feeding on carcasses or the offal of hunted or dead wild animals. For this reason, it is important for Public Health Authorities to be focused on the training of hunters and farmers in order to avoid the transmission among free-ranging pigs and prevent the cases of human infection.

Correction to: The tree that hides the forest: cryptic diversity and phylogenetic relationships in the Palaearctic vector Obsoletus/Scoticus complex (Diptera: Ceratopogonidae) at the European level.

An amendment to this paper has been published and can be accessed via the original article.

The tree that hides the forest: cryptic diversity and phylogenetic relationships in the Palaearctic vector Obsoletus/Scoticus Complex (Diptera: Ceratopogonidae) at the European level.

BACKGROUND: Culicoides obsoletus is an abundant and widely distributed Holarctic biting midge species, involved in the transmission of bluetongue virus (BTV) and Schmallenberg virus (SBV) to wild and domestic ruminants. Females of this vector species are often reported jointly with two morphologically very close species, C. scoticus and C. montanus, forming the Obsoletus/Scoticus Complex. Recently, cryptic diversity within C. obsoletus was reported in geographically distant sites. Clear delineation of species and characterization of genetic variability is mandatory to revise their taxonomic status and assess the vector role of each taxonomic entity. Our objectives were to characterize and map the cryptic diversity within the Obsoletus/Scoticus Complex. METHODS: Portion of the cox1 mitochondrial gene of 3763 individuals belonging to the Obsoletus/Scoticus Complex was sequenced. Populations from 20 countries along a Palaearctic Mediterranean transect covering Scandinavia to Canary islands (North to South) and Canary islands to Turkey (West to East) were included. Genetic diversity based on cox1 barcoding was supported by 16S rDNA mitochondrial gene sequences and a gene coding for ribosomal 28S rDNA. Species delimitation using a multi-marker methodology was used to revise the current taxonomic scheme of the Obsoletus/Scoticus Complex. RESULTS: Our analysis showed the existence of three phylogenetic clades (C. obsoletus clade O2, C. obsoletus clade dark and one not yet named and identified) within C. obsoletus. These analyses also revealed two intra-specific clades within C. scoticus and raised questions about the taxonomic status of C. montanus. CONCLUSIONS: To our knowledge, our study provides the first genetic characterization of the Obsoletus/Scoticus Complex on a large geographical scale and allows a revision of the current taxonomic classification for an important group of vector species of livestock viruses in the Palaearctic region.

Phlebotomine sandflies and factors associated with their abundance in the leishmaniasis endemic area of Attiki, Greece.

Leishmaniasis is a parasitic disease of animals and humans caused by several Leishmania species and transmitted by phlebotomine sandflies. The aim of the present study was to identify the species of field collected phlebotomine sandflies in the endemic area of the Attiki during 4 consecutive years, to isolate the Leishmania parasites from the infected sandflies, and identify possible factors associated with sandfly abundance in the area. A total of 542 trappings were made in 46 collection sites, in purely urban areas, periurban areas, and purely rural areas in Attiki. Out of the 3254 sandflies trapped, 1448 (44.43%) were female and 241 (16.64%) of the females were blood fed while Leishmania infantum DNA was detected in the 0.41% of them. Regarding sandfly species, the most prevalent was Phlebotomus tobbi (41.52%) followed by Sergentomyia minuta (27.44%), P. neglectus (14.83%), P. simici (11.08%), P. papatasi (3.68%), P. similis (0.89%), and P. alexandri (0.56%). Periurban areas were found to have the highest density of sandfly populations.

Cross-Sectional Serosurvey and Risk Factors Associated with the Presence of Toxoplasma gondii Antibodies in Pigs in Greece.

INTRODUCTION: Toxoplasmosis is a worldwide reported zoonotic infection caused by the protozoon Toxoplasma gondii. Pigs may become infected by ingesting feed or water contaminated with cat faeces, by cannibalism, and/or by eating infected rodents. T. gondii infected edible tissues of pigs are a source of infection for humans. This study was undertaken to detect seropositivity of pigs against T. gondii and identify possible risk factors as well as to compare two serological methods. METHODS: A total of 609 blood samples were collected from 10% of the sows from 65 pig farms located in mainland Greece. Information about the geographical location, size, and biosecurity level of the herd was recorded. Samples were tested for the presence of antibodies against T. gondii employing the indirect fluorescent antibody test and the enzyme-linked immunosorbent assay. RESULTS: Twenty-six positive samples were detected (4.3%) in 17 out of the 65 farms tested (26.2%). The average seroprevalence in affected herds was 26.8% (95% confidence interval 21.0-32.6%). Among the categorical data evaluated, the biosecurity level (odds ratio 0.17, p < 0.01) and the geographical characteristics (odds ratio 13.55, p < 0.05) significantly affected the presence of toxoplasmosis in the herd. Toxoplasmosis was recorded in significantly higher percentages in mountainous farms as compared to lowlands (p < 0.05) and in those with poor biosecurity levels than in those with very good (p < 0.05). A very good agreement (κ = 0.958) was observed between the two serological methods. CONCLUSION: The presence of antibodies in pigs is indirect information on the risk of the infection and an indication of the necessity of biosecurity measures to be taken in order to control infection at least in the areas at highest risk.