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.

Brucella spp. distribution, hosting ruminants from Greece, applying various molecular identification techniques.

BACKGROUND: Brucellosis still remains an endemic disease for both livestock and human in Greece, influencing the primary sector and national economy in general. Although farm animals and particularly ruminants constitute the natural hosts of the disease, transmission to humans is not uncommon, thus representing a serious occupational disease as well. Under this prism, knowledge concerning Brucella species distribution in ruminants is considered a high priority. There are various molecular methodologies for Brucella detection with however differential discriminant capacity. Hence, the aim of this survey was to achieve nationally Brucella epidemiology baseline genotyping data at species and subtype level, as well as to evaluate the pros and cons of different molecular techniques utilized for detection of Brucella species. Thirty-nine tissue samples from 30 domestic ruminants, which were found positive applying a screening PCR, were tested by four different molecular techniques i.e. sequencing of the 16S rRNA, the BP26 and the OMP31 regions, and the MLVA typing panel 1 assay of minisatellite markers. RESULTS: Only one haplotype was revealed from the 16S rRNA sequencing analysis, indicating that molecular identification of Brucella bacteria based on this marker might be feasible solely up to genus level. BP26 sequencing analysis and MLVA were in complete agreement detecting both B. melitensis and B. abortus. An interesting exception was observed in 11 samples, of lower quality extracted DNA, in which not all expected MLVA amplicons were produced and identification was based on the remaining ones as well as on BP26. On the contrary OMP31 failed to provide a clear band in any of the examined samples. CONCLUSIONS: The present study reveals the constant circulation of Brucella bacteria in ruminants throughout Greece. Further, according to our results, BP26 gene represents a very good alternative to MLVA minisatellite assay, particularly in lower quality DNA samples.

Molecular detection of Brucella spp. in ruminant herds in Greece.

Brucellosis is a worldwide distributed infectious disease. Ruminants and other animal species (swine, dogs, equids, etc.), as well as wild mammals, can be affected. The disease can be transmitted to humans through the food chain or by direct contact with infected animals. Because of the relatively high economic burden due to abortions within a herd, significant efforts have been employed and hence the disease in most European countries has been eradicated. Accordingly, Greece applies both control and eradication programs concerning small ruminants (sheep and goats) and bovines depending on the geographical area. Current challenges in the standard antibody-based laboratory methods used for Brucella detection are the failure to differentiate antibodies against the wild strain from the ones against the vaccine strain Rev1 and antibodies against B. melitensis from those against B. abortus. The aim of the study was to reexamine and combine previously published protocols based on PCR analysis and to generate a rapid, not expensive, and easy to perform diagnostic tool able to confirm the doubtful results delivered from serology. For this reason, 264 samples derived from 191 ruminants of the farm and divided in 2 groups (male/female) were examined with a modified DNA extraction and PCR protocol. Molecular examination revealed the presence of Brucella spp. in 39 out of 264 samples (derived from 30 animals). In addition, Brucella spp. was detected in infected tissues such as testicles, inguinal lymph nodes, fetal liver, and fetal stomach content.

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.