A Multidisciplinary "One Health" Tool for Tackling Brucellosis.

Introduction: The Epizootiological Investigation Form (EIF) is a document issued for every notified human brucellosis case, with the aim to convey information from public health to veterinary authorities for farm animals epidemiologically linked with the patient. We assessed the integration of EIF to the routine collaboration among stakeholders and the efficiency in directing the veterinary efforts to identify Brucella-infected animals. Methods: EIFs were evaluated for the implementation, timeliness, and completeness of the shared information provided by the public health and the veterinary authorities. The efficiency of EIFs in identifying infected farms was compared with the Brucella infection rate of routinely screened farms in the frame of the national brucellosis program. Results: During 2017-2022, 344 EIFs were issued for equal number of human brucellosis cases and 118 (34.3%) were circulated successfully among all stakeholders, whereas 226 (65.7%) went missing. The highest rate of intersectoral circulation occurred in May (47.8%, p = 0.007). Veterinary investigation was performed, and result was provided in 62 (57.4%) of the 108 circulated EIFs that disclosed the contact details of the epidemiologically linked animal farms. Brucella was detected at a significantly higher rate (51.7%) in the investigated sheep and goats' farms than the infection rate (2.7%) of the national brucellosis program (p < 0.00001). Among the screened bovine herds, two were found infected of the eight tested (25%). The circulation among all competent authorities of EIFs with a farm screening outcome required a median (interquartile range) of 50 days (22, 88). The likelihood of a "complete" EIF per human case differed among geographic Regions (p = 0.010), and was higher for patients diagnosed in April (p = 0.001) and occupied as stockbreeders (p = 0.025). Conclusions: EIF is a useful tool for pinpointing suspected animals for brucellosis screening. Training of the collaborating personnel is essential for improving the implementation of EIF in the everyday practice.

Epidemiology of reported cases of leptospirosis in the EU/EEA, 2010 to 2021.

BackgroundLeptospirosis is a zoonotic disease caused by bacteria of the genus Leptospira. Humans are infected by exposure to animal urine or urine-contaminated environments. Although disease incidence is lower in Europe compared with tropical regions, there have been reports of an increase in leptospirosis cases since the 2000s in some European countries.AimWe aimed to describe the epidemiology of reported cases of leptospirosis in the European Union/European Economic Area (EU/EEA) during 2010-2021 and to identify potential changes in epidemiological patterns.MethodsWe ran a descriptive analysis of leptospirosis cases reported by EU/EEA countries to the European Centre for Disease Prevention and Control with disease during 2010-2021. We also analysed trends at EU/EEA and national level.ResultsDuring 2010-2021, 23 countries reported 12,180 confirmed leptospirosis cases corresponding to a mean annual notification rate of 0.24 cases per 100,000 population. Five countries (France, Germany, the Netherlands, Portugal and Romania) accounted for 79% of all reported cases. The highest notification rate was observed in Slovenia with 0.82 cases per 100,000 population. Overall, the notification rate increased by 5.0% per year from 2010 to 2021 (95% CI: 1.2-8.8%), although trends differed across countries.ConclusionThe notification rate of leptospirosis at EU/EEA level increased during 2010-2021 despite including the first 2 years of the COVID-19 pandemic and associated changes in population behaviours. Studies at (sub)national level would help broaden the understanding of differences at country-level and specificities in terms of exposure to Leptospira, as well as biases in diagnosis and reporting.

Human Brucellosis in Greece, 2005-2020: A Persistent Public Health Problem.

Brucellosis remains an important zoonosis in various parts of the world. In Greece, brucellosis is endemic in sheep and goats. Since 1998, animal control measures include vaccination of sheep and goats older than 3 months of age in the mainland (vaccination zone) and slaughtering of infected livestock on the islands (eradication zone). We retrieved data from the mandatory notification system for 2005-2020 to describe the epidemiology in humans and assess current public health measures. Overall, 1786 notifications and an additional 111 cases related to an outbreak in 2008 (eradication zone) were recorded. The mean annual notification rate was 0.9/100,000 population (standard deviation [SD]: 0.35). A substantial mean annual decline in notification rate was reported in 2019 and 2020. A statistically significant decreasing trend of brucellosis notification rate was observed over the whole 16-year period (p < 0.001). The mean annual notification rate was significantly higher in the vaccination zone (1.2/100,000) (SD: 036) compared with the eradication zone (0.2/100,000) (SD: 0.14) (p < 0.001). Seventy percent of cases was male (n = 1175) and the highest mean notification rate was recorded in the 45-64 age group (1.3/100,000). The majority of cases (71%) reported animal contact and almost half of the cases (49%) were stockbreeders by occupation. Consumption of unpasteurized dairy products, mainly of their own production, was reported by 57% of cases; for 115 (6.5%) cases, animal exposure or consumption of unpasteurized dairy products were not recorded. Despite animal control measures, brucellosis remains endemic in Greece with cases in the vaccination zone, as well as the eradication zone. We recommend re-evaluation and strengthening of animal control programs and training of people working in stock breeding regarding disease transmission in co-operation with local farmers' associations.

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 brucellosis 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.

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 Acinetobacterbaumannii, Bacteroidesfragilis, Clostridiumperfringens, Enterococcusfaecalis, E. mundtii, Fusobacteriumnucleatum, Haemophilusaegyptius, Kingellakingae, Klebsiellapneumoniae, Leptotrichiabuccalis, L. hofstadii, Moraxellalacunata, Pasteurellamultocida, Propionibacteriumacnes, P. propionicum, Proteusmirabilis, Pseudomonasaeruginosa, Rickettsiaaustralis, R. hoogstraalii, Salmonellaenterica, 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 Rickettsiahoogstraalii (p = 0.031) and Propionibacteriumacnes (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.

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 >99% similarity with the publicly available 16S R. felis GenBank sequences using the Basic Local Alignment Search Tool (BLAST>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>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.

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.

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.

Antibody Response Following Pre-Exposure Immunization Against Rabies in High-Risk Professionals.

Vaccination against rabies and routine antibody testing of subjects participating in programs for the surveillance and control of rabies in animals is strongly recommended. The scope of this study is to describe the antibody level as measured by a commercial enzyme-linked immunosorbent assay (ELISA) after primary and booster intramuscular vaccination with a purified vero-cell rabies vaccine (PVRV) in high-risk professionals and to determine the influence of an array of factors on antibody level, that is, time elapsed since primary immunization series and booster dose, sex, age, pathologic conditions, high-risk occupation, and peak antibody level after initial scheme and booster dose. A primary series of three doses of PVRV was administered and a commercial ELISA was recommended 14 days postimmunization with continuous repetition at 6 months and yearly intervals for the laboratory personnel and the rest of the professionals, respectively. The protective antibody titer was defined as a minimum of 0.5 equivalent units/mL (EU/mL) (seroconvertion) and a booster dose was applied if the titer was determined nonprotective. The seroconversion rate (SCR) after primary vaccination was 100%, with a geometric mean titer (GMT) of 2.90 EU/mL (interquartile range [IQR]: 1.85-3.45). After booster vaccination due to nonprotective titer, the SCR was 100% and the GMT increased by 678% (95% confidence interval [CI]: 514-887) reaching 4.25 EU/mL (IQR: 4.00-4.60), 2.5 times higher than the GMT elicited by the primary vaccine scheme in the respective recipients. The titer dropped by 1.20% per month (95% CI: 0.52-1.89) regardless of booster administration or any other factor. Women had 51% higher titer compared with men (95% CI: 6-116). High-risk professionals should be verified for adequate antibody titers, but routine administration of a single booster dose of PVRV 1 year after the primary series could be considered; more evidence is needed to support the benefit in terms of immunity and logistics.

Surveillance of Rabies Postexposure Prophylaxis in Greece: 4 Years Experience.

Rabies reemerged in Greek fauna during October 2012, 25 years after the last report in animals and 42 after the last human case. This study examined the administration of rabies postexposure prophylaxis (PEP) in humans over the period 2012-2016. A total of 1,454 individuals (62.6% males) received PEP. The vast majority (92.3%) of regimens was initiated in high risk for animal rabies areas (11.1 PEP per 100,000 residents per year). Among the exposed, 77.0% presented at a healthcare setting during the first 24 h after the incident; more severe injuries were associated with faster presentation (p < 0.05). A vaccine series was administered in 54.5% of exposed persons whereas 43.7% received both vaccine and immunoglobulin and 1.7% immunoglobulin only. Exposure to stray dogs represented 68.4% of all incidents. In exposures occurring in nonhigh risk for rabies areas, bat was the second most frequently involved animal (13.4% of incidents). All dogs and cats evaluated by a veterinarian and laboratory-confirmed rabid were initially deemed suspect for rabies during clinical examination or according to history. No human rabies cases were recorded during the period of the study. Surveillance of PEP represents a valuable tool for aiding assessment of present and future demands in prophylaxis biologicals, outlining the epidemiological profile of exposures and planning effective policies for the management of exposure incidents.

Outbreak of West Nile virus infection in Greece, 2010.

During 2010, an outbreak of West Nile virus infection occurred in Greece. A total of 197 patients with neuroinvasive disease were reported, of whom 33 (17%) died. Advanced age and a history of heart disease were independently associated with death, emphasizing the need for prevention of this infection in persons with these risk factors.