Babesia ovis is a significant tick-borne parasite of sheep, capable of causing both acute disease and long-lasting, low-grade infections. Imidocarb dipropionate (IMDP) is commonly used against babesiosis, yet whether it can completely eliminate B. ovis remains uncertain. In this study, we examined whether the parasite persists after treatment and whether such residual infections can still be transmitted. Three sheep were experimentally infected, treated with IMDP once clinical signs appeared, and then monitored for 180 days by microscopy, nested PCR, and iELISA. Fever and microscopic parasitemia resolved soon after treatment, but nPCR intermittently detected parasite DNA for several weeks. By day 180, all treated sheep were negative by nPCR and microscopy, while two still showed detectable antibodies. Blood collected at this time was transfused into naïve sheep. Two of the three recipients showed nPCR positivity at scattered time points and later seroconverted while showing no clinical signs. In contrast, Rhipicephalus bursa ticks that fed on the treated donors neither acquired the parasite nor transmitted it to recipients, likely because post-treatment parasitemia remained below the acquisition threshold. Overall, these results indicate that IMDP controls clinical disease but may not fully clear B. ovis, allowing silent transmission through blood despite negative routine tests.

Diagnosis of rickettsial infections is challenging due to nonspecific clinical symptoms and limitations of current diagnostic methods. Molecular assays allow early detection but are limited by cost and technical demands, whereas conventional serological tests often exhibit cross-reactivity and low sensitivity during the early stages of infection. This study aimed to develop and evaluate a recombinant-antigen sandwich ELISA for improved antibody detection against Rickettsia spp. Three Rickettsia akari proteins, rGroEL, rDnaK, and rA8GP63 (uncharacterized protein), were produced and validated for immunogenicity. The assay was evaluated using 94 patient serum samples, including those with positive, negative, and unknown clinical course. The optimized ELISA demonstrated high reproducibility, with IgG sensitivity of 89.47–95.39% and specificity of 90%. IgM detection, also assessed, showed lower sensitivity (42.11–82.89%) but maintained strong specificity (83.33%). The diagnostic performance was comparable to that of a commercial indirect immunofluorescence assay, with no cross-reactivity detected in sera from patients with unrelated infections. rDnaK and rA8GP63 represent newly explored diagnostic candidates. These findings highlight the potential of this recombinant protein-based ELISA as an accessible, sensitive and specific diagnostic tool, with a meaningful clinical impact for improving the early and accurate detection of rickettsial infections.

People who use crack cocaine (PWUCC) constitute a key population due to vulnerability and marginalization, especially in a socio-ecologically diverse, relatively isolated region with limited public health infrastructure. This study aimed to perform a genetic characterization of circulating HCV among PWUCC in the municipality of Bragança, situated on the Brazilian Amazon coast, identifying viral genotypes, subtypes, resistance-associated substitutions (RAS)—naturally occurring mutations in the viral genome that can reduce the efficacy of direct-acting antiviral (DAA) agents—and predictions of phenotypic resistance. Methods: Between 2016 and 2018, biological samples and epidemiological data were obtained from 165 PWUCC. Viral detection was performed using RT-PCR, while genotyping, subtyping, and RAS profiling were conducted through nucleotide sequencing and fragment analysis. Results: In 165 PWUCC, 22 (13.3%) tested positive for HCV RNA. Most of them had not had access to public health services (91.5%), and more than half (57.0%) reported living in unstable housing conditions. HCV subtypes 1a (27.3%), 1b (40.9%), and 3a (31.8%) were detected. Evidence of resistance associated with DAAs, such as daclatasvir and dasabuvir, was detected in five PWUCC with HCV (22.7%). Conclusions: The high prevalence of HCV infection, predominantly subtype 1b, and significant levels of resistance are very concerning. This demonstrates the urgent need for targeted public health interventions to expand access to testing, treatment, and effective antiviral therapy in this vulnerable population of the Brazilian Amazon.

Measles, also known as rubeola, is a highly contagious and potentially life-threatening disease caused by the measles virus. It classically presents with fever, cough, coryza, conjunctivitis, and a maculopapular rash. Despite the availability of an effective vaccine for decades, measles outbreaks continue to occur globally, largely driven by declining vaccination coverage and increased international travel. With no specific antiviral therapy available, rapid and accurate diagnosis remains essential for timely clinical management and effective outbreak control. Diagnostic methods have evolved from traditional virus isolation in cell culture to serologic assays and, more recently, to molecular techniques such as real-time reverse transcriptase polymerase chain reaction (rRT-PCR). Each diagnostic method has unique advantages and limitations influenced by specimen type, timing of collection, and laboratory capacity. This minireview summarizes the progress of measles virus diagnostics, outlines current laboratory detection strategies, and discusses emerging technologies and ongoing challenges amid global measles resurgence and increasing public health demands.

Oz virus, an emerging tick-borne thogotovirus, has been reported to cause fatal human infection in Japan. However, its ecology and geographic distribution remain largely unknown. In this study, we developed a double-antigen sandwich enzyme-linked immunosorbent assay (DAgS ELISA) for detecting Oz virus antibodies in animals and used it to conduct a seroepidemiological survey of wild boars (Sus scrofa) in Miyazaki Prefecture, Japan. Recombinant Oz virus nucleoprotein was expressed in E. coli and used as both the capture and detection antigen. Relative to the neutralization test, the DAgS ELISA showed a sensitivity of 72.2%, a specificity of 88.2%, and an overall concordance rate of 79.0%. We used this assay to examine 1045 wild boar serum samples collected between November 2022 and May 2025, finding a seroprevalence of 33.5%. The seroprevalence did not significantly differ by sex, age, or region, but showed significant seasonal variation, peaking in summer (p < 0.0001). Oz virus RNA was detected by quantitative RT-PCR in one serum sample (0.09%). Phylogenetic analysis of the partial Oz virus glycoprotein gene showed that this strain shared 98.8% nucleotide identity with the EH8 strain, which was the first Oz virus isolate obtained from ticks in Ehime Prefecture. These findings suggest that wild boars in Miyazaki are frequently exposed to Oz virus and that ticks in the region harbor the virus. However, no human cases have been reported to date. The DAgS ELISA developed in this study provides a practical tool for serological surveillance in animals. Continuous monitoring of animal populations is warranted to clarify the epidemiology of Oz virus in the region and to identify potential reservoir species.

Salmonella is a Gram-negative enteric bacterium responsible for the foodborne and waterborne disease salmonellosis, which was the second most reported foodborne gastrointestinal infection in humans in the European Union in 2023. Animals represent the principal reservoir of this pathogen, with animal-derived food products serving as the main route of transmission to humans. In a household context, having numerous animals can be a crucial factor for contracting Salmonella spp. infection. In the present study, we report a case of a familiar outbreak of Salmonella Thompson that occurred in 2024 in central Italy, involving an infant and the companion animals (a dog, a cat and ten birds) of the family’s farm. To support the epidemiological investigations, antimicrobial susceptibility testing and whole-genome sequencing (WGS) were conducted on strains from the human case and from animals. Eleven strains were isolated in total, from fecal samples collected from the child and the animals at different times. WGS confirmed the genetic relatedness between human and animal isolates, supporting the hypothesis of a shared source of infection, but genes or plasmid involved in antibiotic resistance were not found. Moreover, AST revealed that isolates were fully susceptible to major antimicrobial classes tested. Despite being an uncommon serotype, the involved Salmonella Thompson serovar 6,7: k:1,5 O:7 (C1) demonstrated a high pathogenic potential, emphasizing the need for vigilance even toward serotypes not typically associated with major public health concerns. Moreover, these findings underscore the critical need for an integrated One Health approach to effectively monitor, prevent, and control zoonotic infections.

Tick bites acquired in the northern or southern hemisphere can transmit microbes that may cause illness. The most prevalent infection is Lyme borreliosis (LB), with all proven cases to date having been acquired in the northern hemisphere. The existence of endemic LB in Australia has not been proven explicitly, and there is uncertainty concerning the cause of “Lyme-like” disease (LLD) in Australia. As many tick-borne diseases (TBDs) are diagnosed by serology, validated assays for use in both the northern and southern hemispheres are required. Using a multiplex enzyme-linked immunosorbent assay (TICKPLEX®), two independent laboratories tested a total of 53 well-characterized reference sera that consisted of 33 samples from northern hemisphere patients with confirmed tick-borne disease (TBD) and 20 randomly selected sera from Australian patients with suspected TBDs, presenting with or without LLD. Antibody responses to multiple microbial antigens from causative agents of TBDs were found. High concordance between laboratories was demonstrated on this small set of samples. The results obtained provide the basis for further evaluation of TICKPLEX® on a larger number of samples from Australian patients with suspected TBDs. These findings should be considered preliminary, providing proof-of-concept evidence that warrants validation in larger, clinically diverse cohorts.

Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic flavivirus causing severe neurological disease across Asia, and genotype V (GV) is now predominant in Korea. Despite frequent detection of GV in mosquitoes, human-derived complete genome data remain scarce. To elucidate the molecular and antigenic characteristics of human GV infections, cerebrospinal fluid samples from unvaccinated patients positive for JEV RNA during 2018–2023 were subjected to virus isolation in LLC-MK2 cells (rhesus monkey kidney-derived epithelial cell line). Three human GV isolates (K18P80, K23P84, K23P88) were successfully obtained and their complete open reading frames (~10.3 kb) sequenced. Phylogenetic analysis with representative JEV strains (GI–GV) revealed that these isolates form a distinct lineage, clustering into two domestic clades (Clade I and II), suggesting endemic circulation and local evolution in Korea. Sequence identities with GIII-based vaccine strains were low (79% nucleotide, 91.1% amino acid), with notable divergence in nonstructural regions. Three consistent E protein substitutions (Q52E, S156T, D292E) near antigenic epitopes indicate possible immune escape. Additional clade-defining substitutions in NS3 (L31F) and NS5 (K269R, M330I) were shared with mosquito isolates, supporting human–vector molecular continuity. These findings provide fundamental genomic evidence of human JEV GV in Korea and highlight the need for genotype-specific surveillance and next-generation vaccine evaluation.

Equine bacterial abortion presents substantial economic and One Health challenges; however, comprehensive epidemiological data from China are limited. This study sought to ascertain the overall prevalence of key pathogens—namely, Chlamydia spp., Coxiella burnetii, Salmonella abortus equi, and Brucella spp.—in equine populations in northwestern China. In this study, we aimed to further elucidate the characteristics of co-infections, profile antimicrobial resistance genes, and identify associated risk factors. Conducted as a cross-sectional analysis across four provinces, we collected 508 blood samples and 24 abortion tissue samples from 15 farms. Pathogen detection was performed using ELISA and real-time PCR, complemented by a targeted PCR panel screening for 29 AMR genes. The highest prevalence was observed for S. abortus equi (serology: 35.03%; molecular: 23.03%), followed by C. burnetii (28.94%; 15.35%) and Chlamydia spp. (18.90%; 14.17%). No PCR-confirmed cases of Brucella spp. were detected, despite low-level seropositivity. Notably, donkeys and horses aged 5–10 years exhibited higher positivity rates, and co-infections were common, particularly S. abortus equi + C. burnetii (n = 44). Among the 196 PCR-positive samples, extended-spectrum beta-lactamase (ESBL) genes were predominant, with CTX-M (n = 158) and TEM-1 (n = 106) being the most prevalent. Additionally, we identified a high prevalence of genes conferring resistance to fluoroquinolones (qnrA/B), tetracyclines (tetM), macrolides (ermA/B/C), and sulfonamides (sul1), along with sporadic occurrences of carbapenemase genes. This study presents the inaugural comprehensive analysis of pathogen prevalence and associated antimicrobial resistance (AMR) gene carriage in equine abortion cases in northwest China. The findings highlight the imperative for integrated serological and molecular surveillance, revealing a significant discrepancy between empirical therapeutic approaches and the prevalent resistance genotypes. Consequently, this research lays the groundwork for evidence-based biosecurity measures and antimicrobial stewardship within a One Health framework.

The tick species Ixodes ricinus is the most widely distributed throughout Europe and serves as the primary vector for the spirochete Borrelia burgdorferi sensu lato (s.l.), which is responsible for Lyme borreliosis. The present study evaluated the prevalence of B. burgdorferi (s.l.) in I. ricinus ticks using nPCR amplification. Ticks were collected from three geographical regions in Bulgaria: the Black Sea Coast in the east, the Pleven region in the north, and the Stara Zagora region in the central south. This study focused on urban and peri-urban areas, including heavily traveled trails in parks. The results indicated statistically significant differences in infection rates, with the highest percentage of infected ticks found in Pleven (52.34%), followed by Stara Zagora (35.46%) and the lowest on the Black Sea Coast (23.08%). Furthermore, we conducted genospecies molecular identification of B. afzelii and B. garinii by PCR amplification of genospecies-specific nucleotide sequences in the 16S rRNA gene. The lowest prevalence of B. afzelii was recorded in Stara Zagora at 10.64%, followed by the Black Sea region at 12.5%, while the statistically highest frequency was observed in Pleven at 21.03%. The prevalence of B. garinii among the total number of I. ricinus ticks was greatest in Pleven at 15.89%, followed by the Black Sea region at 8.65%, and the lowest in the Stara Zagora region at 4.96%. Statistically significant differences were found only between Pleven and Stara Zagora (p = 0.002), but no significant differences in infection rates were observed between adults and nymphs in the regions examined. Overall, the prevalence of B. afzelii in each region exceeds that of B. garinii, and when considered collectively for Bulgaria, the frequency of B. afzelii (15.9%) is higher than that of B. garinii (10.89%) in infected ticks. In a controlled epidemiological context, managing the population of I. ricinus infected with B. burgdorferi s.l. can mitigate the health burden of Lyme disease, although this preventive strategy may not guarantee complete protection.