Association between Exposure to Leptospira spp. and Abortion in Mares in Croatia.

Leptospirosis is one of the most common zoonotic infections and a major problem in terms of both veterinary medicine and public health. However, the disease is under-recognised and under-diagnosed worldwide, particularly in horses. Clinical leptospirosis in horses is mainly associated with recurrent uveitis (ERU), which has recently been studied more intensively, and reproductive disorders, the epidemiology of which is still relatively poorly understood. To enhance our comprehension of abortions caused by leptospirosis in horses and to identify the causative strains, a serological study was carried out with subsequent molecular characterisation of the isolate obtained. Using the microscopic agglutination test (MAT), serum samples from mares that aborted and foetal fluids (when available) were tested for antibodies against Leptospira spp. Furthermore, bacteria isolation from kidney cultures was conducted. Of 97 mare serum samples, 21 (21.64%) tested positive, with Grippotyphosa and Pomona being the most frequently detected serogroups. A significantly higher seroprevalence was found in aborting mares compared to the healthy horse population from the same geographical area, as well as a pronounced seasonal variation. Leptospiral antibodies were not detected in any of the foetal fluids, but isolation was successful in 1 case out of 39 (2.56%). Genotyping by multilocus sequence typing (MLST) and core genome multilocus sequence typing (cgMLST) identified the obtained isolate as Leptospira kirschneri, serogroup Pomona, serovar Mozdok. Further surveillance and molecular typing of Leptospira strains causing abortion in horses would be invaluable in understanding the prevalence and impact of leptospirosis on equine reproductive health in Europe.

Proinflammatory Chemokine Levels in Cerebrospinal Fluid of Patients with Neuroinvasive Flavivirus Infections.

Tick-borne encephalitis virus (TBEV) and West Nile virus (WNV) are the most important neuroinvasive arboviruses detected in Europe. In this study, we analyzed cerebrospinal fluid (CSF) concentrations of 12 proinflammatory chemokines (CCL2, CCL3, CCL4, CCL11, CCL17, CCL20, CXCL1, CXCL5, CXCL8, CXCL9, CXCL10, and CXCL11) in 77 patients with neuroinvasive diseases (NIDs). Flavivirus infection was confirmed in 62 patients (TBEV and WNV in 31 patients each), while in 15 patients the etiology of NID was not determined (NDE). Similar patterns of high-level expression of chemokines regulating monocyte/macrophage responses (CCL2), neutrophil recruitment (CXCL1 and CXCL8), and interferon-inducible chemoattractants for leukocytes (CXCL10 and CXCL11) have been observed in WNV and TBEV groups. None of the tested chemokines significantly differed between patients with TBEV or WNV. Concentrations of CCL17, CCL20, CXCL5, CXCL10, and CXCL11 were significantly lower in both WNV and TBEV groups compared to NID NDE patients. The logistic regression model showed that CSF concentrations of CXCL11, CXCL5, and CXCL10 could potentially be used for the classification of patients into the WNV or TBEV group versus groups with other NIDs. This study identified, for the first time, similar patterns of CSF chemokine expression in WNV and TBEV infections, suggesting common immunopathogenic mechanisms in neuroinvasive flavivirus infections that should be further evaluated.

An Overview of Tick-Borne Encephalitis Epidemiology in Endemic Regions of Continental Croatia, 2017-2023.

Tick-borne encephalitis (TBE) represents an important public health problem in Europe. We analyzed the epidemiology of TBE based on data from humans, animals, and Ixodes ricinus ticks in endemic regions of continental Croatia. In the period from 2017 to 2023, cerebrospinal fluid (CSF) and serum samples of 684 patients with neuroinvasive diseases, 2240 horse serum samples, and 300 sheep serum samples were tested for TBEV. In addition, 8751 I. ricinus ticks were collected. CSF samples were tested using RT-PCR. Serological tests (serum, CSF) were performed using commercial ELISA, with confirmation of cross-reactive samples by a virus neutralization test. Eighty-four autochthonous human TBEV cases were confirmed. The majority of patients were in the age group of 40-69 years (58.3%) with a male predominance (70.2%). TBE showed a bimodal seasonality with a large peak in April-August and a small one in October-November. In addition to humans, TBEV IgG antibodies were found in 12.2% of horses and 9.7% of sheep. Seasonal tick abundance corresponds to the reported number of human infections. Continental Croatia is still an active natural focus of TBE. Continuous monitoring of infections in humans, sentinel animals, and ticks is needed for the implementation of preventive measures.

Maternal deprivation causes CaMKII downregulation and modulates glutamate, norepinephrine and serotonin in limbic brain areas in a rat model of single prolonged stress.

BACKGROUND: Early life stress is a major risk factor for later development of psychiatric disorders, including post-traumatic stress disorder (PTSD). An intricate relationship exists between various neurotransmitters (such as glutamate, norepinephrine or serotonin), calcium/calmodulin-dependent protein kinase II (CaMKII), as an important regulator of glutamatergic synaptic function, and PTSD. Here, we developed a double-hit model to investigate the interaction of maternal deprivation (MD) as an early life stress model and single prolonged stress (SPS) as a PTSD model at the behavioral and molecular levels. METHODS: Male Wistar rats exposed to these stress paradigms were subjected to a comprehensive behavioral analysis. In hippocampal synaptosomes we investigated neurotransmitter release and glutamate concentration. The expression of CaMKII and the content of monoamines were determined in selected brain regions. Brain-derived neurotrophic factor (BDNF) mRNA was quantified by radioactive in situ hybridization. RESULTS: We report a distinct behavioral phenotype in the double-hit group. Double-hit and SPS groups had decreased hippocampal presynaptic glutamatergic function. In hippocampus, double-hit stress caused a decrease in autophosphorylation of CaMKII. In prefrontal cortex, both SPS and double-hit stress had a similar effect on CaMKII autophosphorylation. Double-hit stress, rather than SPS, affected the norepinephrine and serotonin levels in prefrontal cortex, and suppressed BDNF gene expression in prefrontal cortex and hippocampus. LIMITATIONS: The study was conducted in male rats only. The affected brain regions cannot be restricted to hippocampus, prefrontal cortex and amygdala. CONCLUSION: Double-hit stress caused more pronounced and distinct behavioral, molecular and functional changes, compared to MD or SPS alone.

Neutralizing Activity of SARS-CoV-2 Antibodies in Patients with COVID-19 and Vaccinated Individuals.

BACKGROUND: Serological diagnosis of COVID-19 is complex due to the emergence of different SARS-CoV-2 variants. METHODS: 164 serum samples from (I) patients who recovered from COVID-19 (n = 62) as well as (II) vaccinated individuals (n = 52) and (III) vaccinated individuals who were infected with different SARS-CoV-2 variants after vaccination (n = 50) were included. All samples were tested using EIA (binding antibodies) and a virus neutralization test (VNT) using the Wuhan strain (NT antibodies). Group III was further tested with a VNT using the Alpha/Delta/Omicron strains. RESULTS: The highest antibody index (AI) was observed in vaccinated individuals infected with COVID-19 (median AI = 50, IQR = 27-71) and the lowest in vaccinated individuals (median AI = 19, IQR = 8-48). Similarly, NT antibody titer was highest in vaccinated individuals infected with COVID-19 (median 128; IQR = 32-256) compared to vaccinated individuals (median 32, IQR = 4-128) and patients with COVID-19 (median 32, IQR = 8-64). The correlation between AI and NT titer was strongly positive in vaccinated individuals and moderately positive in patients with COVID-19. No significant correlation was observed in vaccinated individuals infected with COVID-19. In patients infected with Alpha and Delta, the lowest VNT positivity rate was for the Omicron variant (85.0%/83.3%). Patients infected with the Alpha variant showed the lowest NT titer for the Omicron variant (median titer 32) compared to the Wuhan/Delta variants (64/128). Patients infected with the Delta variant had the lowest NT titer to the Omicron variant (median 32), compared to the Wuhan/Alpha variants (64/128). Patients infected with the Omicron variant showed similar titers to the Delta/Wuhan variants (128) and higher to the Alpha variant (256). CONCLUSIONS: The cross-immunity to SARS-CoV-2 is lowest for the Omicron variant compared to the Alpha/Delta variants.

Current Status of Vector-Borne Diseases in Croatia: Challenges and Future Prospects.

Different vector-borne pathogens are present or have (re-)emerged in Croatia. Flaviviruses tick-borne encephalitis (TBEV), West Nile (WNV), and Usutu (USUV) are widely distributed in continental regions, while Toscana virus (TOSV) and sandfly fever viruses are detected at the Croatian littoral. Recently, sporadic clinical cases of Tahyna orthobunyavirus (TAHV) and Bhanja bandavirus infection and seropositive individuals have been reported in continental Croatia. Acute infections and serologic evidence of WNV, TBEV, USUV, and TAHV were also confirmed in sentinel animals and vectors. Autochthonous dengue was reported in 2010 at the Croatian littoral. Lyme borreliosis is the most widely distributed vector-borne bacterial infection. The incidence is very high in northwestern and eastern regions, which correlates with numerous records of Ixodes ricinus ticks. Acute human Anaplasma phagocytophilum infections are reported sporadically, but there are many records of serologic evidence of anaplasmosis in animals. Mediterranean spotted fever (Rickettsia conorii) and murine typhus (Rickettsia typhi) are the main rickettsial infections in Croatia. Human leishmaniasis is notified sporadically, while serologic evidence of leishmaniasis was found in 11.4% of the Croatian population. After the official eradication of malaria in 1964, only imported cases were reported in Croatia. Since vector-borne diseases show a growing trend, continuous monitoring of vectors is required to protect the population from these infections.

Detection of Bhanja Bandavirus in Patients with Neuroinvasive Disease of Unknown Etiology in Croatia.

BACKGROUND: Although the Bhanja bandavirus (BHAV) is widely distributed in some European countries, human infections are rarely reported. This study analyzed the prevalence of BHAV antibodies in patients with neuroinvasive diseases of unsolved etiology. METHODS: A total of 254 Croatian patients who developed neurological symptoms during the four consecutive arbovirus transmission seasons (April 2017-October 2021) were tested. Cerebrospinal fluid (CSF) and urine samples were tested using RT-qPCR. In addition, CSF and serum samples were tested using a virus neutralization test. RESULTS: BHAV RNA was not detected in any samples, while neutralizing (NT) antibodies were detected in serum samples of 53/20.8% of patients (95% CI = 16.0-26.3). In two patients, BHAV NT antibodies were detected in the CSF, indicating a recent infection. Both patients were inhabitants of rural areas in continental Croatia, and one reported a tick bite two weeks before symptoms onset. The seropositivity was high in all age groups (15.2-29.1%). The majority of seropositive patients (94.3%) resided at altitudes less than 200 m above sea level. The prevalence rates correlated positively with population density and negatively with certain climate parameters (temperature, number of hot/warm days). CONCLUSIONS: The presented results indicate that BHAV is distributed in Croatia. Further studies are needed to determine the clinical significance of this neglected arbovirus.

Detection of SARS-CoV-2 Antibodies: Comparison of Enzyme Immunoassay, Surrogate Neutralization and Virus Neutralization Test.

BACKGROUND: Since sensitivity and specificity vary widely between tests, SARS-CoV-2 serology results should be interpreted with caution. METHODS: The study included serum samples from patients who had recovered from COVID-19 (n = 71), individuals vaccinated against SARS-CoV-2 (n = 84), and asymptomatic individuals (n = 33). All samples were tested for the presence of binding antibodies (enzyme immunoassay; EIA), neutralizing (NT) antibodies (virus neutralization test; VNT), and surrogate NT (sNT) antibodies (surrogate virus neutralization test; sVNT) of SARS-CoV-2. RESULTS: SARS-CoV-2-binding antibodies were detected in 71 (100%) COVID-19 patients, 77 (91.6%) vaccinated individuals, and 4 (12.1%) control subjects. Among EIA-positive samples, VNT was positive (titer ≥ 8) in 100% of COVID-19 patients and 63 (75.0%) of the vaccinated individuals, while sVNT was positive (>30% inhibition) in 62 (87.3%) patients and 59 (70.2%) vaccinated individuals. The analysis of antibody levels showed a significant moderate positive correlation between EIA and VNT, a moderate positive correlation between EIA and sVNT, and a strong positive correlation between VNT and sVNT. The proportion of positive sVNT detection rate was associated with VNT titer. The lowest positivity (72.4%/70.8%) was detected in samples with low NT titers (8/16) and increased progressively from 88.2% in samples with titer 32 to 100% in samples with titer 256. CONCLUSIONS: sVNT appeared to be a reliable method for the assessment COVID-19 serology in patients with high antibody levels, while false-negative results were frequently observed in patients with low NT titers.

A retrospective cohort study of canid herpesvirus-1 seroprevalence and epidemiology in kennel dogs in Croatia.

Canid alphaherpesvirus-1 (CaHV-1) is an endemic pathogen of dogs worldwide. CaHV-1 is often related to abortions, neonatal mortality, and the death of puppies. Since the first description of the virus in 1965, there has been no generally accepted method for diagnosing CaHV-1. Many authors used the virus neutralisation test (VNT) as a 'reference standard' due to its high specificity. Nasal, vaginal, preputial swabs and serum samples for this study were collected in the population of kennel dogs in Croatia. To determine the optimal VNT protocol, three modifications of the VNT were compared. These modifications were: VNT using native serum samples, VNT using thermally inactivated serum samples, and VNT using thermally inactivated serum samples with the addition of the complement. The correlation between the results of the VNT methods was significant (P < 0.001). Of all three modifications of VNT, the one using native serum samples was the one that increased VNT sensitivity. The overall seroprevalence of CaHV-1 was 32.02%. The PCR did not confirm the CaHV-1 presence in collected swabs. According to analysed anamnestic data, significant risk factors for CaHV-1 infection were: kennel size, attendance at the dog shows, hunt trials, kennel disinfection protocols, and mating. The oestrus cycle had no significant effect on seropositivity. The study results indicate that CaHV-1 spreads horizontally between dams living in kennels and in males during mating. Although there was no correlation between seropositivity and a history of reproductive disorders, significantly more stillborn puppies were recorded in seronegative dames (P < 0.01).

Seroprevalence Trends and Molecular Epidemiology of Viral Hepatitis in Croatia.

Viral hepatitis is a significant cause of morbidity and mortality worldwide. In Croatia, hepatitis B virus (HBV) and hepatitis C virus (HCV) are widely distributed, especially in some high-risk groups such as people who inject drugs (PWID), prisoners, and highly promiscuous groups. The seroprevalence of HBV ranges from 7.0% in the general population to 38.8% in PWID, depending on the region. The seroprevalence of HCV is highest among PWID (29-75.5%) as compared to 0.9% in the general population. Analyzing the distribution of HCV genotypes, no substantial changes in the molecular epidemiology of the two most frequent HCV genotypes (1 and 3) in the past 20 years were observed. However, the predominance of subtype 1b compared to subtype 1a as detected in 1996-2005 was not confirmed in 2008-2015. Hepatitis A virus (HAV) incidence was high in the past with a decreasing trend since the 2000s, except for an outbreak in 2017-2018 as part of the large European outbreak, which was mainly among men who have sex with men. Hepatitis E virus (HEV) is an emerging virus detected for the first time in Croatia in 2012. The seroprevalence of HEV is high among hemodialysis patients (27.9%) and liver transplant recipients (19.3-24.4%). In addition, higher seroprevalence rates were observed in animal-related professions (e.g., veterinarians, 15.2%; hunters, 14.9%). All detected HEV strains belonged to genotype 3.

Cross-Reactive Antibodies in Tick-Borne Encephalitis: Case Report and Literature Review.

Flaviviruses are a heterogeneous group of viruses that may induce broad antigenic cross-reactivity. We present a patient who was admitted to the infectious disease department with symptoms suggestive of aseptic meningitis. During the clinical workup, the patient reported a tick bite two weeks before the disease onset. High titers of IgM and IgG antibodies to tick-borne encephalitis virus (TBEV) were found in both serum and cerebrospinal fluid (CSF) samples, indicating acute TBEV infection. West Nile virus (WNV) and Usutu virus (USUV) IgM and/or IgG antibodies were also detected, and a virus neutralization test (VNT) was performed. A high titer of TBEV neutralizing (NT) antibodies (640) was detected, which confirmed acute TBE. However, NT antibodies to WNV and USUV were also detected (titer 80 for both viruses). After TBEV and WNV IgG avidity evaluation, previous flavivirus infection was highly suspected (avidity index 82% and 89%, respectively). Blood, CSF, and urine samples were negative for respective viruses' RNA. The presented case highlights the challenges in flavivirus serodiagnosis. In the published literature, different degrees of cross-reactivity or cross-neutralization between TBEV and dengue, louping ill, Omsk hemorrhagic fever, Langat, and Powassan virus were also observed. Therefore, the serology results should be interpreted with caution, including the possibility of cross-reactivity. In areas where several flaviviruses co-circulate VNT is recommended for disease confirmation.

Quantification of Antiviral Cytokines in Serum, Cerebrospinal Fluid and Urine of Patients with Tick-Borne Encephalitis in Croatia.

BACKGROUND: Tick-borne encephalitis virus (TBEV) is one of the most significant arboviruses affecting the human central nervous system (CNS) in Europe. Data on cytokine response in TBEV infection are limited. METHODS: We analyzed the cytokine response in serum, cerebrospinal fluid (CSF) and urine samples of patients with TBE. The control group consisted of patients with 'febrile headache' who had normal CSF cytology. The panel included 12 cytokines: TNF-α, IL-6, Th1 (IL-2, IFN-γ), Th2 (IL-4, IL-5, IL-13), Th9 (IL-9), Th17 (IL-17A, IL-17F), Th22 (IL-22) cytokines and IL-10. RESULTS: TBE patients were more likely to have increased levels of IL-6 and IFN-γ in CSF compared to controls (85.7% vs. 58.8% and 85.7% vs. 47.1%, respectively). However, concentrations of IL-6 (the most abundant cytokine in the CSF of both groups), IL-10 and IL-9 were lower in TBEV patients compared with controls, but the difference was statistically significant for IL-9 only (p = 0.001). By analyzing the cytokine levels in different clinical samples, all measured cytokines were detected in the serum, with the highest concentrations found for IFN-γ, TNF-α, IL-10, IL-17F and IL-22. Higher concentrations of cytokines in the CSF compared with serum were observed for IL-5, IL-6 and IL-22. All cytokines except IL-13 were detectable in urine but in a small proportion of patients, except for IL-22, which was detectable in 95.8% of patients. CONCLUSIONS: Cytokine composition in different clinical samples of TBE patients reveals a different network of early innate immune response cytokines, Th1, Th2, Th9, Th22, Th17 and anti-inflammatory cytokines.

Insights into hepatitis E virus epidemiology in Croatia.

BACKGROUND: Hepatitis E virus (HEV) is an emerging virus of global health concern. The seroprevalence rates differ greatly according to geographic region and population group. AIM: To analyze the seroprevalence of HEV in exposed (animal-related professions) and nonexposed populations, as well as solid organ and hematopoietic stem cell transplant patients. METHODS: Forestry workers (n = 93), hunters (n = 74), and veterinarians (n = 151) represented the exposed population. The general population (n = 126) and pregnant women (n = 118) constituted the control group. Transplant patients included liver transplant recipients (LTRs) (n = 83), kidney transplant recipients (KTRs) (n = 43), and hematopoietic stem cell transplant recipients (HSCRs) (n = 39). HEV immunoglobulin G antibodies were detected using the enzyme-linked immunosorbent assay and confirmed by the immunoblot test. RESULTS: The HEV seroprevalence significantly differed between groups: Veterinarians 15.2%, hunters 14.9%, forestry workers 6.5%, general population 7.1%, and pregnant women 1.7%. In transplant patients, the seropositivity was highest in LTRs (19.3%), while in KTRs and HSCRs, the seroprevalence was similar to the general population (6.9% and 5.1%, respectively). A significant increase in seropositivity with age was observed from 2.9% in individuals less than 30 years to 23.5% in those older than 60 years. Sociodemographic characteristics (sex, educational level, area of residence, and number of household members), eating habits (game meat, offal, and pork products consumption), and environmental and housing conditions (drinking water supply, type of water drainage/sewer, waste disposal, domestic animals) were not associated with HEV seropositivity. However, individuals who reported a pet ownership were more often seropositive compared to those who did not have pet animals (12.5% vs 7.0%). CONCLUSION: The results of this study showed that individuals in professional contact with animals and LTRs are at higher risk for HEV infection. In addition, age is a significant risk factor for HEV seropositivity.

The Impact of Latent Toxoplasma gondii Infection on Spontaneous Abortion History and Pregnancy Outcomes: A Large-Scale Study.

BACKGROUND: Toxoplasma gondii (TG), a zoonotic protozoan parasite, belongs to a group of TORCH infectious agents, which can cause severe damage to the fetus if a primary infection occurs during pregnancy. After primary infection, TG rests lifelong in human organisms causing a latent infection. Most studies have analyzed the consequences of acute, but not latent, TG infection. This study analyzed the impact of latent toxoplasmosis on spontaneous abortion history, pregnancy complication rate and neonatal outcome. METHODS: IgG and IgM anti-TG antibodies were tested in 806 pregnant women who were consulted at the Timisoara Clinical Emergency Hospital between 2008 and 2010. Demographic data, obstetrical history, and data about the pregnancy complications, birth and neonate were collected for each woman and comparisons between the groups, with and without latent TG infection, were made. RESULTS: This study did not show differences between groups regarding the history of spontaneous abortion (OR = 1.288, p = 0.333), cesarean section (OR = 1.021, p = 0.884), placental abruption (OR 0.995, p = 0.266), pregnancy-induced hypertension rate (OR 1.083, p = 0.846), secondary sex ratio (1.043, p = 0.776), 1' APGAR score at birth (p = 0.544), gestational age at birth (p = 0.491) or birth weight (p = 0.257). CONCLUSIONS: The observed differences between the rate of pregnancy complications in the two groups of pregnant women with and without latent infection with TG, did not reach a statistical significance.

Surveillance of Tahyna Orthobunyavirus in Urban Areas in Croatia-The "One Health" Approach.

BACKGROUND: Tahyna orthobunyavirus (TAHV) is a neglected mosquito-borne bunyavirus. Although the virus is widespread in continental Europe, TAHV infections are rarely reported. We analyzed the prevalence of TAHV in humans and different animal species as well as mosquitoes collected in urban areas of Zagreb and its surroundings in the period from 2020 to 2022. METHODS: The study included 32 patients with neuroinvasive disease (NID), 218 asymptomatic individuals, 98 horses, 94 pet animals (dogs and cats), and 4456 Aedes vexans mosquitoes. Cerebrospinal fluid (CSF) and urine samples of patients with NID were tested for the TAHV RNA using a real-time reverse transcription-polymerase chain reaction (RT-qPCR). Human and animal serum samples were tested for TAHV-neutralizing (NT) antibodies using a virus-neutralization test (VNT). Mosquito pools were tested for TAHV RNA using an RT-qPCR. RESULTS: TAHV NT antibodies were detected in 3/9.4% of patients with NID, 8/3.7% of asymptomatic individuals, 29/29.6% of horses, and 11/11.7% of pet animals. There was no difference in the seroprevalence according to age, sex, and area of residence in asymptomatic individuals. In addition, TAHV seropositivity did not differ according to age and sex in pet animals. None of the tested mosquito pools was TAHV RNA-positive. CONCLUSIONS: The presented results highlight the importance of interdisciplinary surveillance ("One Health") of this neglected viral zoonosis.

Behavioural interaction of pyrazoloquinolinone positive allosteric modulators at α6GABAA receptors and diazepam in rats: Anti-diazepam-induced ataxia action as a structure-dependent feature.

Several pyrazoloquinolinone (PQ) ligands were recently discovered as functionally selective positive modulators at the PQ site of α6-containing GABAA receptors. PQs are also neutral modulators at the benzodiazepine site. We assessed the influence of PQ compounds from three structural groups (PZ-II-029 and related deuterated analogues DK-I-56-1, RV-I-029, DK-I-60-3 and DK-I-86-1; LAU 463 and related analogues DK-I-58-1 and DK-II-58-1; and DK-I-87-1), alone and in combination with diazepam, on the behaviour of male Sprague-Dawley rats. An excellent behavioural safety profile of all tested PQs was demonstrated in the spontaneous locomotor activity, rotarod, loss of righting reflex and pentylenetetrazol tests. In interaction studies, only PZ-II-029 and its analogues prevented the ataxic effects of the benzodiazepine, as assessed in the rotarod test and during monitoring of rat locomotor activity after awakening from the loss of righting reflex. Published electrophysiological profiles of PQ ligands imply that positive modulation elicited at α6-GABAA receptors that contain the γ2 and δ subunit, rather than their neutral modulatory action at the benzodiazepine site, may prevent the ataxic action of diazepam. Thus, PZ-II-029 and its deuterated analogues are not prone to untoward interactions with benzodiazepines and may indeed completely abolish their ataxic action, seen at therapeutic, and especially toxic concentrations.

Detection of Tahyna Orthobunyavirus-Neutralizing Antibodies in Patients with Neuroinvasive Disease in Croatia.

BACKGROUND: Tahyna orthobunyavirus (TAHV) is widely distributed in continental Europe. Very few studies have analyzed TAHV seroprevalence in Croatia. We analyzed the prevalence of TAHV RNA and antibodies in Croatian patients with neuroinvasive disease (NID). METHODS: A total of 218 patients with unsolved NID detected during five consecutive arbovirus transmission seasons (April 2017-October 2021) were tested. Cerebrospinal fluid (CSF) and urine samples were tested for TAHV RNA using RT-PCR. In addition, CSF and serum samples were tested for TAHV antibodies using a virus neutralization test (VNT). RESULTS: Clinical presentations in patients with NID were meningitis (141/64.7%), meningoencephalitis (56/25.7%), myelitis (8/3.7%), and 'febrile headache' (13/5.9%). TAHV RNA was not detected in any of the tested CSF or urine samples; however, TAHV-neutralizing (NT) antibodies were detected in 22/10.1% of patients. Detection of NT antibodies in the CSF of two patients presenting with meningitis suggested recent TAHV infection. TAHV seropositivity increased significantly with age, from 1.8% to 24.4%. There was no difference in seroprevalence between genders or areas of residence (urban, suburban/rural). The majority of seropositive patients (90.9%) resided in floodplains along the rivers in continental Croatia. CONCLUSIONS: The presented results confirm that TAHV is present in Croatia. The prevalence and clinical significance of TAHV infection in the Croatian population have yet to be determined.

Effects of α5 GABAA receptor modulation on social interaction, memory, and neuroinflammation in a mouse model of Alzheimer's disease.

AIMS: GABAergic modulation involved in cognitive processing appears to be substantially changed in Alzheimer's disease (AD). In a widely used 5xFAD model of AD, we aimed to assess if negative and positive allosteric modulators of α5 GABAA receptors (NAM and PAM, respectively) would affect social interaction, social, object and spatial memory, and neuroinflammation. METHODS: After 10-day treatment with PAM, NAM, or solvent, 6-month-old transgenic and non-transgenic 5xFAD mice underwent testing in a behavioral battery. Gene expressions of IL-1ß, IL-6, TNF-α, GFAP, and IBA-1 were determined in hippocampus and prefrontal cortex by qPCR. RESULTS: PAM treatment impaired spatial learning in transgenic females compared to solvent-treated transgenic females, and social recognition in transgenic and non-transgenic males. NAM treatment declined social interaction in transgenic and non-transgenic males, while had beneficial effect on cognitive flexibility in non-transgenic males compared to solvent-treated non-transgenic males. Transgenic animals have not fully displayed cognitive symptoms, but neuroinflammation was confirmed. NAM reduced proinflammatory gene expressions in transgenic females and astrogliosis in transgenic males compared to pathological controls. CONCLUSION: PAM and NAM failed to exert favorable behavioral effects in transgenic animals. Suppression of neuroinflammation obtained with NAM calls for more studies with GABAergic ligands in amyloid beta- and/or tau-dependent models with prominent neuroinflammation.

SARS-CoV-2 Omicron Variant in Croatia-Rapid Detection of the First Case and Cross-Border Spread.

BACKGROUND: Due to rapid spread, the Omicron variant has become the dominant SARS-CoV-2 variant responsible for infections worldwide. We present the first detection of the Omicron variant in Croatia which resulted in rapid cross-border spreading. METHODS: Whole-genome sequencing was performed using the Illumina MiniSeq sequencing system. SARS-CoV-2 lineages were identified using the PANGOLIN and GISAID databases. RESULTS: The first case of the Omicron variant (BA.1.17) emerged in Croatia after a workshop held in Zagreb in November 2021. The patient reported a history of previous COVID-19 and received two doses of an mRNA vaccine. Three additional cases were detected among Croatian participants of the workshop. At the beginning of December, SARS-CoV-2 infection was confirmed in one participant from Montenegro and her husband. Phylogenetic analysis showed that the detected Omicron variants were closely related to the first Croatian case, confirming the connection with the workshop outbreak and rapid cross-border spreading. Subsequent analyses of SARS-CoV-2 positive samples in Croatia showed the rapid introduction of the Omicron variant and depletion of the Delta variant resulting in the fifth pandemic wave. CONCLUSIONS: Genomic monitoring and early detection of novel SARS-CoV-2 variants are essential to implement timely epidemiological interventions and reduce further transmission in the population.

Genetic Variability in Balkan Paleoendemic Resurrection Plants Ramonda serbica and R. nathaliae Across Their Range and in the Zone of Sympatry.

The genus Ramonda includes three Paleoendemic and Tertiary relict species that survived in refugial habitats of the Balkan Peninsula (R. nathaliae and R. serbica) and the Iberian Peninsula (R. myconi). They are all "resurrection plants," a rare phenomenon among flowering plants in Europe. Ramonda myconi and R. nathaliae are diploids (2n = 2x = 48), while R. serbica is a hexaploid (2n = 6x = 144). The two Balkan species occur in sympatry in only two localities in eastern Serbia, where tetraploid potential hybrids (2n = 4x = 96) were found. This observation raised questions about the existence of gene flow between the two species and, more generally, about the evolutionary processes shaping their genetic diversity. To address this question, genetic markers (AFLP) and an estimate of genome size variation were used in a much larger sample and at a larger geographic scale than previously. The combination of AFLP markers and genome size results suggested ongoing processes of interspecific and interploidy hybridization in the two sites of sympatry. The data also showed that interspecific gene flow was strictly confined to sympatry. Elsewhere, both Ramonda species were characterized by low genetic diversity within populations and high population differentiation. This is consistent with the fact that the two species are highly fragmented into small and isolated populations, likely a consequence of their postglacial history. Within sympatry, enormous variability in cytotypes was observed, exceeding most reported cases of mixed ploidy in complex plant species (from 2x to >8x). The AFLP profiles of non-canonical ploidy levels indicated a diversity of origin pathways and that backcrosses probably occur between tetraploid interspecific hybrids and parental species. The question arises whether this diversity of cytotypes corresponds to a transient situation. If not, the question arises as to the genetic and ecological mechanisms that allow this diversity to be maintained over time.