Rabies outbreak in Greece during 2012-2014: use of Geographical Information System for analysis, risk assessment and control

The objectives of this work were (i) geographical analysis of the 2012–2014 outbreak of rabies in Greece using GIS and (ii) comparative analysis of animal cases with data of potential human exposure to rabies together with environmental data, in order to provide information for risk assessment, effective monitoring and control. Most animal cases (40/48) involved red foxes, while domestic animals were also diagnosed with rabies. Overall, 80% of the cases were diagnosed in central northern Greece; 75% of the cases were diagnosed in low altitudes (<343·5 m), within a distance of 1 km from human settlements. Median distance from livestock farms was 201·25 m. Most people potentially exposed to rabies (889/1060) presented with dog bite injuries. Maximum entropy analysis revealed that distance from farms contributed the highest percentage in defining environmental niche profiles for rabid foxes. Oral vaccination programmes were implemented in 24 administrative units of the country during 2013 and 2014, covering a total surface area of ~60 000 km2. Rabies re-occurrence in Greece emphasizes the need for ongoing surveillance in cross-border areas and in areas with intense human activity

SARS-CoV-2 IgG database

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Modelling Human Brucellosis Based on Infection Rate and Vaccination Coverage of Sheep and Goats

In this study, the vaccination coverage, serological sampling and infection rate of sheep and goats were evaluated as predictors for the modeling of human brucellosis in Greece. The human bru-cellosis disease frequency per local regional unit (RU) varied significantly (RR90) among consecutive years. The notification rate was higher (p &lt; 0.001) in the RUs with implementation of vaccination in sheep and goats (vaccination zone—VZ) with a median of 1.4 (IQR 0.0–3.1) compared with the RUs of the eradication zone (EZ) with a median of 0.0 (IQR 0.0–0.0). In VZ, the increased frequency of human cases was associated with delayed vaccine administration (estimate: 0.14 (0.04; 0.29), p = 0.03) and higher vaccination coverage of the animals (estimate: −0.349 (−0.72; −0.07), p &lt; 0.01). However, the flock sampling rate was highly heterogenous among RUs (IQR 10.56–52.93), and inconsistent within RUs throughout the period of the study 2013–2017 (p = 0.001), limiting the reliable estimation of the infection rate in livestock and the design of an integrated One Health model for human disease. © 2022 by the authors. Licensee MDPI, Basel, Switzerland

Modelling Human Brucellosis Based on Infection Rate and Vaccination Coverage of Sheep and Goats

In this study, the vaccination coverage, serological sampling and infection rate of sheep and goats were evaluated as predictors for the modeling of human brucellosis in Greece. The human bru-cellosis disease frequency per local regional unit (RU) varied significantly (RR90) among consecutive years. The notification rate was higher (p < 0.001) in the RUs with implementation of vaccination in sheep and goats (vaccination zone—VZ) with a median of 1.4 (IQR 0.0–3.1) compared with the RUs of the eradication zone (EZ) with a median of 0.0 (IQR 0.0–0.0). In VZ, the increased frequency of human cases was associated with delayed vaccine administration (estimate: 0.14 (0.04; 0.29), p = 0.03) and higher vaccination coverage of the animals (estimate: −0.349 (−0.72; −0.07), p < 0.01). However, the flock sampling rate was highly heterogenous among RUs (IQR 10.56–52.93), and inconsistent within RUs throughout the period of the study 2013–2017 (p = 0.001), limiting the reliable estimation of the infection rate in livestock and the design of an integrated One Health model for human disease. © 2022 by the authors. Licensee MDPI, Basel, Switzerland

Brucellosis underreporting in Greece: Assessment based on aggregated laboratory data of culture-confirmed cases from public hospitals

Background: Brucella spp. isolation is one of the mainstays of brucellosis diagnosis. Simultaneously, the true brucellosis disease rate may be underrepresented in notification systems. This study aims at assessing the nosocomial capacity for Brucella spp. isolation and the underreporting rate of brucellosis cases in Greece. Methods: Data for Brucella spp. culture capacity and the number of isolations were collected annually from public hospitals nationwide, during 2015-2018. The number of unreported cases was estimated after subtracting the National Mandatory Notification System cases from the survey-captured isolations, matched by hospital and year. Results: Feedback was provided by 112 public hospitals (response rate: 97.4 %). Brucella spp. isolation capacity was completely absent in 27.7 % of hospitals; during the four years of the study, 11.3 %, 13.9 %, 20.0 %, and 25.2 % of the hospitals had isolation competence for one, two, three, or four years, respectively. Underreporting assessment was possible in hospitals that declared at least one Brucella spp. isolation (n =35) and unreported cases were identified in 19 (54 %). Α mean underreporting of 28.9 % of total cases was estimated for the whole period of the study ranging annually from 24.1 % to 35.0 %. The number of unreported cases per hospital ranged from one to 12 per year (median: 2, IQR: 5). Conclusions: Interventions for improving diagnosis and reporting of the disease are recommended. Assessment of brucellosis underreporting by comparing raw numerical data of survey-captured isolations and officially notified cases lacks the case by case specificity, however, keeping required data to a minimum achieves high feedback rate from hospitals and provides a tentative estimation of the notification deficit. © 2019, Lithografia Antoniadis I - Psarras Th G.P.. All rights reserved

Serosurvey of IgG antibodies against bartonella henselae and rickettsia typhi in the population of Attica, Greece

Rickettsia typhi and Bartonella henselae are the causative agents of murine typhus and cat-scratch disease, respectively. A small-scale survey (N = 202) was conducted in the Attica region, Greece, for determining the prevalence rates of IgG antibodies against B. henselae and R. typhi by indirect fluorescence antibody test. IgG against B. henselae and R. typhi were present in 17.8% (36/202) and 4.5% (9/202) of the participants, respectively; co-occurring IgG against both B. henselae and R. typhi were detected in 3.5% (7/202), whereas only anti-B. henselae IgG in 14.3% (29/202), and only anti-R. typhi IgG in 1.0% (2/202). Titres 1/64, 1/128, 1/256, and 1/512, of anti-B. henselae IgG were identified in 6.4%, 4.5%, 4.5%, and 2.4%, whereas titres 1/40 and 1/80 of anti-R. typhi IgG were detected in 4.0%, and 0.5%, respectively. A positive association of anti-B. henselae IgG prevalence with a coastal area featuring a major seaport (p = 0.009) and with younger age (p = 0.046) was identified. The findings of this survey raise concern for exposure of the population of Attica to B. henselae and R. typhi, which should be considered in the differential diagnosis when compatible symptoms are present. Our results also suggest that seaports may represent high-risk areas for exposure to Bartonella spp. © 2020 by the authors

Molecular detection of Rickettsia felis in common fleas in Greece and comparative evaluation of genotypic methods

Introduction: Rickettsia felis is the causative agent of flea-borne spotted fever (FBSF), an emerging zoonosis. Although there is evidence of FBSF in Greece, fleas, the classic vectors of R. felis, have not been adequately studied. Thus, the aim of this study was to detect and characterize bacteria of genus Rickettsia and especially R. felis from common fleas parasitizing domestic cats and dogs in Greece and evaluate the efficiency of established molecular techniques. Materials and methods: DNA of flea-pools (samples) by animal-host was investigated by quantitative real-time PCRs (qPCR), and 16S metagenomics (16S). Determination of Rickettsia spp., Rickettsia felis-like organisms (RFLOs), and R. felis was based on a combination of qPCRs targeting gltA and ompB genes, 16S automated metagenomics and manual comparison of 16S sequences for &gt;99% similarity with the publicly available 16S R. felis GenBank sequences using the Basic Local Alignment Search Tool (BLAST&gt;99). Information for the animal-hosts was available and statistically analyzed. Results: Among 100 flea-pools, R. felis was detected in 14 samples with a combination of six, five and three assays in 10, two and two samples, respectively. The sensitivity of the assays for Rickettsia genus (16S, and genus specific qPCRs) ranged from 62.5% to 93.8% and the specificity from 65.0% to 100%. R. felis-targeting qPCRs for gltA and ompB demonstrated sensitivity and specificity of 92.9% and 100%, and 100.0% and 87.5%, respectively. 16S metagenomics using the assay software was not able to identify R. felis positive specimens, although manual BLAST&gt;99 did identify the species, but demonstrated sensitivity of 92.9% and specificity of 65.0%. No association of the detection rate of Rickettsia genus or R. felis, with the epidemiological data collected, was identified. Conclusions: These observations suggest the occurrence of R. felis in fleas from pets in Attica, Greece, but PCR and sequencing assays varied considerably in sensitivity and specificity and a consensus methodology for assigning the positivity status is required to be established. © 2020 Elsevier B.V

Evidence of Brucella melitensis DNA in the Microbiome of Ctenocephalides felis from Pet Cats in Greece

Cat fleas (Ctenocephalides felis) are the most prevalent ectoparasites of pet animals with cosmopolitan distribution, obligatory hematophagous, and may prey on humans to receive bloodmeals. We studied the microbiota of 100 flea-pools, containing C. felis, and collected from equal number of cats and dogs in the region of Attica, Greece, including Athens. The 16S metagenomics technique detected Brucella spp. nucleotide sequence that was identified as Brucella melitensis DNA by a real-time PCR, in five flea-pools, corresponding to five cats, one owned and the remaining four stray, residing in semiurban and urban areas, respectively. No definite conclusions can be drawn as to the pathway that led to the presence of B. melitensis in common fleas parasitizing cats. We suspect flea or cat contact with wild rodents, ubiquitous in various environments, which participate in the B. melitensis biology. The proximity of the cats and their fleas with humans and previous observations of flea potential to transmit B. melitensis in laboratory animals warrant a more elaborate research as to the vectorial dynamics, the ecological pathways resulting in pathogen carriage, and the risk for public health. © Copyright 2020, Mary Ann Liebert, Inc., publishers 2020

Molecular detection of Rickettsia felis in common fleas in Greece and comparative evaluation of genotypic methods

Introduction: Rickettsia felis is the causative agent of flea-borne spotted fever (FBSF), an emerging zoonosis. Although there is evidence of FBSF in Greece, fleas, the classic vectors of R. felis, have not been adequately studied. Thus, the aim of this study was to detect and characterize bacteria of genus Rickettsia and especially R. felis from common fleas parasitizing domestic cats and dogs in Greece and evaluate the efficiency of established molecular techniques. Materials and methods: DNA of flea-pools (samples) by animal-host was investigated by quantitative real-time PCRs (qPCR), and 16S metagenomics (16S). Determination of Rickettsia spp., Rickettsia felis-like organisms (RFLOs), and R. felis was based on a combination of qPCRs targeting gltA and ompB genes, 16S automated metagenomics and manual comparison of 16S sequences for &amp;gt;99% similarity with the publicly available 16S R. felis GenBank sequences using the Basic Local Alignment Search Tool (BLAST&amp;gt;99). Information for the animal-hosts was available and statistically analyzed. Results: Among 100 flea-pools, R. felis was detected in 14 samples with a combination of six, five and three assays in 10, two and two samples, respectively. The sensitivity of the assays for Rickettsia genus (16S, and genus specific qPCRs) ranged from 62.5% to 93.8% and the specificity from 65.0% to 100%. R. felis-targeting qPCRs for gltA and ompB demonstrated sensitivity and specificity of 92.9% and 100%, and 100.0% and 87.5%, respectively. 16S metagenomics using the assay software was not able to identify R. felis positive specimens, although manual BLAST&amp;gt;99 did identify the species, but demonstrated sensitivity of 92.9% and specificity of 65.0%. No association of the detection rate of Rickettsia genus or R. felis, with the epidemiological data collected, was identified. Conclusions: These observations suggest the occurrence of R. felis in fleas from pets in Attica, Greece, but PCR and sequencing assays varied considerably in sensitivity and specificity and a consensus methodology for assigning the positivity status is required to be established. © 2020 Elsevier B.V

Molecular evidence of a broad range of pathogenic bacteria in ctenocephalides spp.: Should we re-examine the role of fleas in the transmission of pathogens?

The internal microbiome of common cat and dog fleas was studied for DNA evidence of pathogenic bacteria. Fleas were grouped in pools by parasitized animal. DNA was extracted and investigated with 16S metagenomics for medically relevant (MR) bacteria, based on the definitions of the International Statistical Classification of Diseases and Related Health Problems (WHO). The MR bacterial species totaled 40, were found in 60% of flea-pools (N = 100), and included Acinetobacter baumannii, Bacteroides fragilis, Clostridium perfringens, Enterococcus faecalis, E. mundtii, Fusobacterium nucleatum, Haemophilus aegyptius, Kingella kingae, Klebsiella pneumoniae, Leptotrichia buccalis, L. hofstadii, Moraxella lacunata, Pasteurella multocida, Propionibacterium acnes, P. propionicum, Proteus mirabilis, Pseudomonas aeruginosa, Rickettsia australis, R. hoogstraalii, Salmonella enterica, and various Bartonella, Staphylococcus, and Streptococcus species. B. henselae (p = 0.004) and B. clarridgeiae (p = 0.006) occurred more frequently in fleas from cats, whereas Rickettsia hoogstraalii (p = 0.031) and Propionibacterium acnes (p = 0.029) had a preference in fleas from stray animals. Most of the discovered MR species can form biofilm, and human exposure may theoretically occur through the flea-host interface. The fitness of these pathogenic bacteria to cause infection and the potential role of fleas in the transmission of a broad range of diseases should be further investigated. © 2021 by the authors. Licensee MDPI, Basel, Switzerland