Genetic diversity of hepatitis E virus (HEV) strains derived from humans, swine and wild boars in Croatia from 2010 to 2017.

BACKGROUND: To fulfill epidemiological data and investigate possible interspecies transmission, this study shall attempt to sequence representative HEV strains of human, swine and wild boar origin collected from 2010 to 2017 in Croatia. METHODS: In total, 174 anti-HEV antibody positive human sera samples; 1419 blood or faeces samples of swine, as well as 720 tissue and/or blood samples of wild boar originating from different counties (18 in total) in Croatia were tested for the presence of HEV RNA. RESULTS: HEV RNA was detected in 26 human sera samples (14.9%; 95% CI 10.4-21.0%). HEV RNA was detected in 216 tested swine (15.2%; 95% CI 13.5-17.1%), regardless of age, farm breeding system or geographical origin. Viral RNA was also detectable in faeces samples which prove that swine actively participate in shedding HEV into the environment. Of the total of 720 tested wild boar samples, 83 were HEV RNA positive (11.5, 95% CI 9.4-14.1%) originating from six counties. According to the sequence analysis all strains have shown to be members of Orthohepevirus A genotype HEV-3, regardless of host. The genotyping results confirm grouping of sequences into four subtypes of HEV strains of which subtypes 3a and 3c belong to the general cluster 3abchij, and were predominately detected during the study, while subtypes 3e and 3f fall within cluster 3efg. Strains within subtypes 3a and 3e were found in humans, swine and wild boars; subtype 3c strains were derived from humans and swine, whereas subtype 3f strains were found only in humans. Strains belonging to subtypes 3a and 3c were derived during the entire investigated period and may be considered endemic in Croatia, whereas strains within subtypes 3e and 3f were detected sporadically indicating the possibility of newly imported infections. CONCLUSIONS: All detected strains show to be genetically highly related to strains found in humans and/or animals from other European Countries, indicating that trade of live animals or wild boar movement increases the risk of HEV infection spread. Furthermore, homologous strains found in different investigated species within this study indicate interspecies transmission of HEV and/or an existence of an accessible mutual source of infection.

First recorded case of paramyxovirus infection introduced into a healthy snake collection in Croatia.

BACKGROUND: In the present study, we describe the first paramyxovirus infection in a snake collection in Croatia caused by an introduction of new snakes that were not previously tested and didn't show any signs of disease. CASE PRESENTATION: In less than a month after introduction into a healthy colony, new snakes began to show respiratory symptoms (i.e. mouth opening, wheezing, etc.) and died within a month and a half after antibiotic therapy was applied. The same symptoms and a high mortality rate were then observed in in-contact snakes from other collections belonging to different snake families. CONCLUSIONS: Two entries of new snakes in different time periods were recorded and recognized as possible sources of infection. We stress the need for veterinary health control and monitoring of snakes prior to transportation as well as implementing obligatory quarantine measures to minimize the risk of infection among newly established snake groups.

The silent spread of Porcine Bocavirus in Croatian pigs: should we be concerned?

A survey was conducted to evaluate the presence and prevalence of Porcine Bocavirus (PBoV) in Croatian domestic pigs by means of PCR targeting the NS1 gene fragment of PBoV. This study included testing of faecal samples collected from 10 small commercial farms and 11 small backyard holdings in Croatia. The presence of PBoV was confirmed by PCR in 24 out of 57 composite faecal samples from small commercial farms and in 12 out of 43 composite faecal samples from small backyard holdings. The PCR products of 18 positive samples were sequenced for genotyping. PBoV sequences grouped into the PBoV-a, PBoV-b and PBoV-c groups with 90.81% to 99.25% nucleotide identity. All Croatian PBoV sequences showed a high nucleotide and amino acid identity with PBoV sequences from China and Hong Kong, the United States, Sweden, and Slovenia. These results clearly show that PBoV is circulating among the domestic pig population in Croatia.

Salivary Flow Rate, Oral Yeast Colonization and Dental Status in Institutionalized and Non-Institutionalized Elderly

Elderly people, especially those institutionalized in long-term care facilities, are at risk of various oral diseases. The aim of the study was to determine the incidence of hyposalivation and colonization/oral fungal infection of oral cavity with yeasts, as well as dental status in institutionalized and non-institutionalized elderly. The study included 280 institutionalized and 61 non-institutionalized elderly people. Salivary flow rate, oral colonization with yeasts/oral infection and dental status were assessed and compared between the groups. The institutionalized elderly had a significantly lower salivary flow rate (p=0.035). Oral colonization with yeasts was more frequently found in institutionalized elderly (p<0.001) as compared with non-institutionalized elderly. A negative correlation was found between decreased salivary flow rate and oral yeast colonization and oral fungal infection in both the institutionalized (rs=-0.58; p<0.05) and non-institutionalized (rs=-0.52; p<0.05) groups. A significant difference in DMFT index was observed between the two groups (p<0.001). A negative correlation between decreased salivary flow rate and dental status was found in both the institutionalized (rs=-0.22; p<0.05) and non-institutionalized (rs=-0.56; p<0.05) groups. The results revealed a significantly higher level of hyposalivation and oral yeast colonization and poorer dental status in the institutionalized group as compared with the non-institutionalized group of elderly people.

Characterisation of pseudorabies virus in domestic pigs and wild boars in Croatia.

Serological data imply that pseudorabies (Aujeszky's disease) is present in domestic pigs and wild boars in Croatia. Therefore, this study included testing of brain tissue samples collected from 200 domestic pigs and 105 wild boars originating from seventeen districts of Croatia. The presence of pseudorabies virus (PrV) DNA was confirmed in samples originating from six domestic pigs (3%) and one wild boar (0.95%). Positive samples were sequenced and analysed on the basis of a gC genome fragment. PrV strains have shown to be genetically identical and they are strongly related to some representative strains in the relatively heterogeneous Clade A. The results clearly show that PrV is still circulating among the domestic pig population in Croatia. Furthermore, the presence of pseudorabies virus in wild boars underlines the importance of this species as a PrV reservoir. Continued surveillance is necessary to track the viral spread in order to achieve final eradication of the disease.

Phylogenetic comparison of porcine circovirus type 2 (PCV2) and porcine reproductive respiratory syndrome virus (PRRSV) strains detected in domestic pigs until 2008 and in 2012 in Croatia.

BACKGROUND: Porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) have been present for the last 2 decades in Croatia, causing large economical losses in the pig production. The clinical features of the infections are mostly manifested by the development of respiratory problems, weight loss and poor growth performance, as well as reproductive failure in pregnant sows. Even though the infections are continuously recognized in some regions in Croatia, the heterogeneity of the detected viral strains from 2012 has not yet been investigated. The objective of this study was to compare virus strains of PCV2 and PRRSV detected until 2008 in Croatia with strains isolated in 2012 to gain a better epidemiological understanding of these two infections. RESULTS: PCV2 and PRRSV strains detected in 2012 in fattening pigs from regions where these two diseases have been previously described were compared to strains that have been detected in the same regions within the past two decades. The phylogenetic analysis revealed that the circulating PCV2 and PRRSV strains are distantly related to the previously described Croatian viral strains. However, when compared to known isolates from the GenBank a high genetic identity of PRRSV isolates with isolates from Hungary, Denmark and the Netherlands was found. CONCLUSION: The results of this study reveal that even though PCV2 and PRRSV are constantly present in the investigated regions in Croatia, the viral strains found in 2012 genetically differ from those detected in earlier years. This indicates that new entries into the pig population appeared with regard to both infections, probably as a result of pig trade.

Periodontal Health Status in Adults Exposed to Tobacco Heating System Aerosol and Cigarette Smoke vs. Non-Smokers: A Cross-Sectional Study.

Tobacco heating systems (THS) are new products on the market, advertised as a less harmful alternative for smokers, in which tobacco is heated and not burned like in conventional cigarettes. This research explored the effect on periodontal tissues in contact with heating and burning tobacco residual products (smoke and tobacco). METHODS: The sample included 66 subjects, patients of the Clinic of Dentistry in Rijeka, Croatia, aged 26-56 (median 38), 64% females. Three age- and gender-matched groups were formed (each N = 22): non-smokers, classic cigarettes smokers and THS smokers. Probing depth (PD) and clinical attachment loss (CAL) were primary research parameters. RESULTS: Three groups differed in average PD and CAL (p ≤ 0.002), with cigarette smokers having the highest and non-smokers the lowest values (p ≤ 0.002). THS consumers generally had lower values of periodontal indices than smokers, but only CAL differed significantly (p = 0.011). Periodontal indices CAL and PD were worse in THS consumers than non-smokers, but they did not reach a level of statistical significance. Cigarette smoking was the only predictor of periodontitis (average CAL ≥ 4 mm) in logistic regression models, with an odds ratio of 4.7 (95% confidence interval 1.2-18.3; p = 0.027). CONCLUSIONS: Exposure to nicotine-containing aerosol of THS in adults has a less harmful effect on periodontal tissues, measurable through periodontal indices (PD and CAL), compared to burning tobacco of conventional cigarettes. THS, presented as an alternative product to classic cigarettes, also has a detrimental effect on the periodontium.

Association of obesity with periodontitis, tooth loss and oral hygiene in non-smoking adults.

BACKGROUND: Periodontitis was found to be significantly related to obesity as well as the number of missing teeth and oral hygiene. However, the studies addressing these relationships often included smokers and diabetics, and none was performed in Eastern European patients. The aim of this cross-sectional study was to investigate associations between obesity and periodontitis, oral hygiene, and tooth loss in a sample of non-smoking Croatian subjects aged 31-75 years. METHODS: A total of 320 patients were recruited by convenient sampling at the Dental Clinic, Clinical Hospital Centre in Rijeka, Croatia. Periodontal examination and data on tooth loss were completed in 292 subjects and each participant completed a structured written questionnaire with questions regarding oral hygiene, education, height, and weight. Periodontitis was categorized as early, moderate and advanced. In multiple regression analysis, periodontitis was used as predictor variable, and BMI, oral hygiene, tooth loss, and education level were used as dependent variables. RESULTS: Use of interdental brushes/flossing and number of missing teeth correlated significantly with BMI, but the same could not be proven for periodontitis and frequency of tooth brushing. However, logistic regression proved that the subset of obese, poorly educated women aged 36-55 years were 5-6 times more likely to develop severe forms of periodontal disease. CONCLUSIONS: Obesity was associated with tooth loss, oral hygiene, and education level in the investigated group. BMI could not be correlated with severity of periodontal disease, except in poorly educated women aged 36-55 years.

Hepatitis E Virus (HEV) Infection among Humans and Animals: Epidemiology, Clinical Characteristics, Treatment, and Prevention.

The public health significance of hepatitis E is very important [...].

Absence of Hepatitis E Virus (HEV) Circulation in the Most Widespread Wild Croatian Canine Species, the Red Fox (Vulpes vulpes) and Jackal (Canis aureus moreoticus).

Hepatitis E virus (HEV) can infect a wide range of domestic and wild animals, and the identification of new host species is reported successively worldwide. Nevertheless, its zoonotic potential and natural transmission, especially in wildlife remains unclear, primarily due to the discrete nature of HEV infections. Since the red fox (Vulpus vulpus) is the most widespread carnivore worldwide, and has been recognized as a potential HEV reservoir, its role as a potent host species is of increasing interest. Another wild canine species, the jackal （Canis aureus moreoticus）, is becoming more important within the same habitat as that of the red fox since its number and geographical distribution have been rapidly growing. Therefore, we have chosen these wild species to determine their potential role in the epidemiology and persistence of HEV in the wilderness. The main reason for this is the finding of HEV and a rather high HEV seroprevalence in wild boars sharing the same ecological niche as the wild canine species, as well as the risk of the spread of HEV through red foxes into the outskirts of cities, where possible indirect and even direct contact with people are not excluded. Therefore, our study aimed to investigate the possibility of natural HEV infection of free-living wild canines, by testing samples for the presence of HEV RNA and anti-HEV antibodies to gain better epidemiological knowledge of the disease. For this purpose, 692 red fox and 171 jackal muscle extracts and feces samples were tested. Neither HEV RNA nor anti-HEV antibodies were detected. Although HEV circulation was not detected in the tested samples, to our knowledge, these are the first results that include jackals as a growing and important omnivore wildlife species for the presence of HEV infection in Europe.

Canine Distemper Virus Infection in the Free-Living Wild Canines, the Red Fox (Vulpes vulpes) and Jackal (Canis aureus moreoticus), in Croatia.

The canine distemper virus (CDV), a paramyxovirus that is closely related to the human measles virus and rinderpest virus of cattle, is a highly contagious viral disease in dogs and wild carnivores worldwide. CDV represents a serious threat to domestic and wild animals, especially to the conservation of endangered wild carnivores. Our study aims to investigate the occurrence of CDV in free-living wild canines in Croatia. For this purpose, 176 red foxes and 24 jackal brain samples collected in the frame of the active surveillance of rabies during winter 2021/2022 were tested. This study provided the first comprehensive overview of the prevalence and spatial distribution of CDV in the wildlife of Croatia, including the molecular phylogenetic analysis of the H gene sequence of field CDV strains circulating in red fox and jackal populations of Croatia. The molecular characterization of hemagglutinin gene genomic regions confirmed the phylogenetic clustering of obtained sequences into the Europa 1 genotype. The obtained CDV red fox sequences were mutually very similar (97.60%). This study indicates the high genetic similarity of Croatian CDV red fox sequences and CDV red fox sequences from Italy and Germany, badger sequences from Germany, polecat sequences from Hungary, and dog sequences from Hungary and Germany.

Obesity and oral health--is there an association?

Obesity has been associated with several chronic diseases, such as coronary heart disease, stroke, adverse pregnancy outcomes, diabetes, and mortality; however it has not been until recently that an increased body mass index (BMI) was also related to dental health, especially periodontitis. We conducted a research to determine whether oral health was related to BMI using a cross-sectional design. Of 320 non-smoking subjects aged 31-60 years recruited from the patients referred to Dental Clinic at the Clinical Hospital Center in Rijeka, Croatia, a detailed dental health status was completed for 292 subjects. Measurements of weight and height, education level and frequency of toothbrushing were also recorded. Dental index comprising information on caries, periodontitis, periapical lesions, and missing teeth was used as a measure of dental health. Dental index and education level both correlated significantly with BMI, however for the dental index this correlation was rather weak. The same could not be proven for the frequency of tooth brushing. Multivariate linear analysis showed that BMI was most dependent upon the number of missing teeth (88.6%), followed by the number of carious lesions (8.3%). Persons with an increased BMI had slightly worse dental health, as represented by higher dental index, regardless of their toothbrushing routines, and lower levels of education. Prevention programs should aim at rising both the general health awareness and improving oral health.

The Influence of Hyaluronic Acid Adjunctive Therapy of Periodontitis on Salivary Markers of Oxidative Stress: Randomized, Controlled Clinical Trial.

Periodontitis is a common oral disease affecting the tooth-supporting tissues. Bacteria have been long viewed as the main causative factor in its development; however, many investigations have proved that aberrant immune and inflammatory response and the resulting misbalance between the damage caused by reactive oxygen species and the antioxidant capacity of tissues may be an underlying factor in disease progression that reduces healing potential. The objective of the current trial is to assess the outcomes of the addition of hyaluronic acid (HA) to standard non-surgical periodontal therapy (NST) on some major oxidative stress markers in saliva. HA-based gel designed for dental application was used and the measurements were taken after 3 months. HA adjunctive therapy had a significantly greater increase in markers with antioxidant properties as well as total antioxidant capacity compared to standard NST alone. Furthermore, clinically measured levels of gingival inflammation (bleeding on probing-BOP) and periodontal destruction (clinical attachment loss-CAL) were significantly correlated with these markers, and the correlation was negative. This investigation demonstrates that HA may indeed express antioxidant properties and improve the antioxidant capacity of periodontal tissues, thus improving the prognosis for the teeth and the results of periodontal therapy. Further investigations will be necessary to determine the duration of these effects over time.

Immunohistochemical and Histopathological Features of Persistent Gingival Enlargement in Relation to Metal Allergic Sensitisation during Orthodontic Treatment.

This study aimed to analyse the immunohistochemical profile of inflammatory infiltrates in the gingival tissue of patients undergoing orthodontic treatment in relation to patients' titanium and/or nickel allergy status. Patients with gingival enlargement received initial periodontal therapy, followed by external gingivectomy in the case of persistent gingival enlargement. The sample included 44 patients (22 had metal allergic sensitisation). Histopathological changes were assessed, and an immunohistochemical analysis was performed on formalin-fixed and paraffin-embedded gingival samples using antibodies against CD1a, CD3, CD4, CD8, CD20, CD68, and CD138. Computer-assisted image analysis was performed to evaluate the positive cell count in the gingival tissue. The gingiva of the sensitised patients was characterised by the absence of multifocal inflammatory infiltrates (p < 0.05), while pronounced exocytosis and band-like inflammatory infiltrates were more frequently observed in sensitised patients. In addition, there was an increase in Langerhans cells and T-helper lymphocytes and a decrease in naïve T-lymphocytes, cytotoxic T-lymphocytes, macrophages, and plasma cells in the sensitised subjects compared to non-sensitised. However, the differences were only statistically significant for macrophages, with a moderate effect size (82.8 vs. 133.9; p = 0.041; r = 0.308). The absence of multifocal inflammation appears to be the most characteristic histopathological feature of the gingiva of sensitised patients. Although their gingiva presented certain characteristics of late hypersensitivity immune reactions the observed changes imply dominant irritative effect e.

Oral Health Knowledge, Attitude, and Behavior of Nursing and Technical Students in Croatia.

OBJECTIVE: The aim of this study was to test knowledge, attitudes, and behavior of the students from the Bjelovar University of Applied Sciences in relation to oral health, and to determine the respectable differences between students of nursing and technical study programs. MATERIALS AND METHODS: Students were randomly selected to represent a convenient sample. A total of 140 students from nursing and technical studies were interviewed by using the World health organization Oral Health Questionnaire, adapted to fit the study purposes. STATISTICAL ANALYSIS: Descriptive statistics were used to determine frequency distribution and percentages for all variables. Pearson's Chi-square test was used to compare the proportions. A p-value less than 0.05 was considered to be statistically significant. RESULTS: The distribution of participants regarding gender was significantly different between the study programs (p = 0.000). Significant difference was also observed in their perceived socioeconomic status (p = 0.001). A significant difference was found between the study programs regarding the knowledge whether bad teeth could impair general health (p = 0.001), could tooth decay and periodontal disease may be prevented (p = 0.002), as well as the importance of regular dental check-ups for prevention of tooth decay (p = 0.028). There were significant differences regarding dietary habits and alcohol consumption in the past 30 days between the observed study programs (p = 0.000) while no significant differences were found regarding tobacco and tobacco product use (p = 0.170). CONCLUSION: Results obtained and presented in this paper show better knowledge and more favorable habits and behaviors among the students from nursing study program compared with the technical ones. Still all students' habits should be changed to improve oral health. To change attitudes and influence habits, effective oral health promotion programs are needed; not only in colleges, but also in primary and secondary schools.

Oral Mucosal Lesions in Childhood.

Childhood diseases are a continuous source of interest in all areas of general and dental medicine. Congenital, developmental, and hereditary diseases may either be present upon birth or appear in early childhood. Developmental anomalies, although often asymptomatic, may become grounds for different infections. Furthermore, they can indicate certain systemic disorders. Childhood age frequently brings about benign tumors and different types of traumatic lesions to the oral mucosa. Traumatic lesions can be caused by chemical, mechanical, or thermal injury. Mucocele and ranula are, by definition, traumatic injuries of the salivary glands or their ducts. Recurrent aphthous lesions are the most common type of ulcerations in childhood, and their etiology is considered multifactorial. Oral mucosal lesions in children require different treatment approaches depending on etiological factors and clinical presentation. Clinicians should have adequate knowledge of oral anatomy in order to diagnose and treat pathological conditions.

Biomolecules in Dental Applications: Randomized, Controlled Clinical Trial Evaluating the Influence of Hyaluronic Acid Adjunctive Therapy on Clinical Parameters of Moderate Periodontitis.

The biological activity of hyaluronic acid (HA) has been well-researched during the past decades; however, there are few randomized, controlled trials of its clinical effects in periodontal therapy. The purpose of this study was to evaluate the effect of hyaluronic acid on the principal parameters of periodontal healing. A specific, commercially available formulation designed and registered for professional dental application, composed of 16 mg/mL of cross-linked and 2 mg/mL of non-cross-linked HA, was used as an adjunctive to non-surgical periodontal therapy, and clinical parameters were evaluated after 3 months. The addition of HA to periodontal therapy demonstrated more favorable clinical results regarding reduction in inflammation, measured by bleeding on probing (-6% compared to the control group) and gain in periodontal attachment (1 mm more than control group), while it had no effect on probing depth reduction. No side effects were reported. Our study demonstrated that HA is a safe and easy-to-use biological agent; due to its wide array of properties, it may significantly improve the results of periodontal therapy. However, more long-term studies are needed to investigate whether these favorable effects remain over time.

Oral Candidal Colonization in Patients with Different Prosthetic Appliances.

Oral infections caused by Candida species are becoming more common, which may be related to an increase in the number of immunologically compromised patients as well as favorable conditions in the oral cavity that often include removable prosthetic appliances. The purpose of this study was to determine the presence of a particular Candida species in patients with PMMA and Cr-Co prosthetic appliances, as well as the salivary flow rate, and oral signs and symptoms. This investigation included a total of 120 subjects with different kinds of removable dentures. A sample of concentrated oral rinse was collected from all subjects in order to detect Candida colonization and identify the Candida species, a quantum of salivation was measured, and subjects were examined clinically. Candida spp. was predominant among the subjects who were denture wearers (p < 0.0001). In all subjects, the most frequently detected species was C.albicans. A statistically significant difference was found between the prevalence of C.albicans (p < 0.001) and C.krusei (p < 0.001) in denture wearers. Subjects with PMMA-based removable prosthetic appliances mostly demonstrated a significant decrease in salivation (p < 0.001), an increase in burning sensations (p < 0.001), and dry mouth (p < 0.001) compared to the subjects who wore partial dentures with Co-Cr metallic frameworks. Red oral lesions were more frequently found among the subjects with partial dentures with Co-Cr metallic frameworks (p < 0.001). Regardless of the material used for the denture, patients must be regularly checked by their dentists in order to prevent the development of oral lesions.

Improving Current Knowledge on Seroprevalence and Genetic Characterization of Swine Influenza Virus in Croatian Pig Farms: A Retrospective Study.

In a total of 1536 blood serum samples analysed by ELISA, antibodies for IAV nucleoprotein (NP) were detected in 30.3%. Results from HI show that the most common subtype of swIAV in the Croatian pig population was H1N1 (44.6%), followed by H3N2 (42.7%) and H1N2 (26.3%). Antibodies to at least one subtype were detected in 62.19% of blood serum samples. Detection of swIAV antigen was performed by IHC and detected in 8 of 28 lung samples collected post-mortem. The matrix (M) gene was detected in nine of one hundred and forty-two lung tissue samples and in seven of twenty-nine nasopharyngeal swabs. Phylogenetic analysis of amplified HA and NA gene fragments in Croatian isolates suggests the presence of swIAV H1avN1av.

Treatment of Residual Periodontal Pockets Using a Hyaluronic Acid-Based Gel: A 12 Month Multicenter Randomized Triple-Blinded Clinical Trial.

The aim of the present study was to evaluate the adjunctive effect of hyaluronic acid (HA) gel in the treatment of residual periodontal pockets over a 12-month period. Periodontal patients presenting at least one residual periodontal pocket 5-9 mm of depth in the anterior area were recruited from six university-based centers. Each patient was randomly assigned to subgingival instrumentation (SI) with the local adjunctive use of HA for test treatment or adjunctive use of local placebo for control treatment at baseline and after 3 months. Clinical parameters ( )probing depth (PD), bleeding on probing (BoP), plaque index (PI), recession (REC), clinical attachment level (CAL)) and microbiological samples for the investigation of the total bacterial count (TBC) and presence of specific bacterial species (Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Fusobacterium nucleatum) were taken at baseline and every 3 months, until study termination. PD was determined as the primary outcome variable. From a total of 144 enrolled, 126 participants (53 males, 73 females) completed the entire protocol. Both treatments resulted in statistically significant clinical and microbiological improvements compared to baseline. Although the local application of HA showed a tendency for better results, there was a lack of statistically significant differences between the groups.