Evaluation of the performance of the IFN-γ release assay in bovine tuberculosis free herds from five European countries.

The diagnostic methods for granting and maintenance of the official tuberculosis-free (OTF) status and for intra-Community movement of cattle are the tuberculin skin tests (single or comparative) and the interferon-γ (IFN-γ) release assay (IGRA). However, until now, IGRAs have been primarily applied in infected farms in parallel to the skin test to maximize the number of infected animals detected. Therefore, an evaluation of the performance of IGRAs in OTF herds to assess whether if their specificity is equal to or higher than that of the skin tests is needed. For this, a panel of 4365 plasma samples coming from 84 OTF herds in six European regions (five countries) was assembled and analysed using two IGRA kits, the ID Screen® Ruminant IFN-g (IDvet) and the Bovigam™ TB Kit (Bovigam). Results were evaluated using different cut-offs, and the impact of herd and animal-level factors on the probability of positivity was assessed using hierarchical Bayesian multivariable logistic regression models. The percentage of reactors ranged from 1.7 to 21.0% (IDvet: S/P ≥ 35%), and 2.1-26.3% (Bovigam: ODbovis-ODPBS ≥ 0.1 and ODbovis-ODavium ≥ 0.1) depending on the region, with Bovigam disclosing more reactors in all regions. The results suggest that specificity of IGRAs can be influenced by the production type, age and region of origin of the animals. Changes in the cut-offs could lead to specificity values above 98-99% in certain OTF populations, but no single cut-off yielding a sufficiently high specificity (equal or higher than that of skin tests) in all populations was identified. Therefore, an exploratory analysis of the baseline IFN-γ reactivity in OTF populations could help to assess the usefulness of this technique when applied for the purpose of maintaining OTF status.

Whole genome sequencing and phylogenetic characterisation of rabies virus strains from Moldova and north-eastern Romania.

BACKGROUND: Rabies is the oldest fatal zoonotic disease recognised as a neglected tropical disease and is caused by an RNA virus belonging to the genus Lyssavirus, family Rhabdoviridae. METHODOLOGY/PRINCIPAL FINDINGS: A deep molecular analysis was conducted on full-length nucleoprotein (N) gene and whole genome sequences of rabies virus from 37 animal brain samples collected between 2012 and 2017 to study the circulation of rabies virus (RABV) variants. The overall aim was to better understand their distribution in Moldova and north-eastern Romania. Both Sanger and high throughput sequencing on Ion Torrent and Illumina platforms were performed. Phylogenetic analysis of the RABV sequences from both Moldova and Romania revealed that all the samples (irrespective of the year of isolation and the species) belonged to a single phylogenetic group: north-eastern Europe (NEE), clustering into three assigned lineages: RO#5, RO#6 and RO#7. CONCLUSIONS/SIGNIFICANCE: High throughput sequencing of RABV samples from domestic and wild animals was performed for the first time for both countries, providing new insights into virus evolution and epidemiology in this less studied region, expanding our understanding of the disease.

Insights into the impact of manure on the environmental antibiotic residues and resistance pool.

The intensive use of antibiotics in the veterinary sector, linked to the application of manure-derived amendments in agriculture, translates into increased environmental levels of chemical residues, AR bacteria (ARB) and antibiotic resistance genes (ARG). The aim of this review was to evaluate the current evidence regarding the impact of animal farming and manure application on the antibiotic resistance pool in the environment. Several studies reported correlations between the prevalence of clinically relevant ARB and the amount and classes of antibiotics used in animal farming (high resistance rates being reported for medically important antibiotics such as penicillins, tetracyclines, sulfonamides and fluoroquinolones). However, the results are difficult to compare, due to the diversity of the used antimicrobials quantification techniques and to the different amounts and types of antibiotics, exhibiting various degradation times, given in animal feed in different countries. The soils fertilized with manure-derived products harbor a higher and chronic abundance of ARB, multiple ARG and an enriched associated mobilome, which is also sometimes seen in the crops grown on the amended soils. Different manure processing techniques have various efficiencies in the removal of antibiotic residues, ARB and ARGs, but there is only a small amount of data from commercial farms. The efficiency of sludge anaerobic digestion appears to be dependent on the microbial communities composition, the ARB/ARG and operating temperature (mesophilic vs. thermophilic conditions). Composting seems to reduce or eliminate most of antibiotics residues, enteric bacteria, ARB and different representative ARG in manure more rapidly and effectively than lagoon storage. Our review highlights that despite the body of research accumulated in the last years, there are still important knowledge gaps regarding the contribution of manure to the AMR emergence, accumulation, spread and risk of human exposure in countries with high clinical resistance rates. Land microbiome before and after manure application, efficiency of different manure treatment techniques in decreasing the AMR levels in the natural environments and along the food chain must be investigated in depth, covering different geographical regions and countries and using harmonized methodologies. The support of stakeholders is required for the development of specific best practices for prudent - cautious use of antibiotics on farm animals. The use of human reserve antibiotics in veterinary medicine and of unprescribed animal antimicrobials should be stopped and the use of antibiotics on farms must be limited. This integrated approach is needed to determine the optimal conditions for the removal of antibiotic residues, ARB and ARG, to formulate specific recommendations for livestock manure treatment, storage and handling procedures and to translate them into practical on-farm management decisions, to ultimately prevent exposure of human population.

Genotyping of African Swine Fever Virus (ASFV) Isolates in Romania with the First Report of Genotype II in Symptomatic Pigs.

The World Organisation for Animal Health has listed African swine fever as the most important deadly disease in domestic swine around the world. The virus was recently brought from South-East Africa to Georgia in 2007, and it has since expanded to Russia, Eastern Europe, China, and Southeast Asia, having a devastating impact on the global swine industry and economy. In this study, we report for the first time the molecular characterization of nine African swine fever virus (ASFV) isolates obtained from domestic pigs in Mures County, Romania. All nine Romanian samples clustered within p72 genotype II and showed 100% identity with all compared isolates from Georgia, Armenia, Russia, Azerbaijan, Ukraine, Belarus, Lithuania, and Poland. This is the first report of ASFV genotype II in the country.

Iron Oxide-Silica Core-Shell Nanoparticles Functionalized with Essential Oils for Antimicrobial Therapies.

Recent years have witnessed a tremendous interest in the use of essential oils in biomedical applications due to their intrinsic antimicrobial, antioxidant, and anticancer properties. However, their low aqueous solubility and high volatility compromise their maximum potential, thus requiring the development of efficient supports for their delivery. Hence, this manuscript focuses on developing nanostructured systems based on Fe3O4@SiO2 core-shell nanoparticles and three different types of essential oils, i.e., thyme, rosemary, and basil, to overcome these limitations. Specifically, this work represents a comparative study between co-precipitation and microwave-assisted hydrothermal methods for the synthesis of Fe3O4@SiO2 core-shell nanoparticles. All magnetic samples were characterized by X-ray diffraction (XRD), gas chromatography-mass spectrometry (GC-MS), Fourier-transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), zeta potential, scanning electron microscopy (SEM), transmission electron microscopy (TEM), thermogravimetry and differential scanning calorimetry (TG-DSC), and vibrating sample magnetometry (VSM) to study the impact of the synthesis method on the nanoparticle formation and properties, in terms of crystallinity, purity, size, morphology, stability, and magnetization. Moreover, the antimicrobial properties of the synthesized nanocomposites were assessed through in vitro tests on Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans. In this manner, this study demonstrated the efficiency of the core-shell nanostructured systems as potential applications in antimicrobial therapies.

Preparation and Characterization of Dextran Coated Iron Oxide Nanoparticles Thin Layers.

In the present study, we report the synthesis of a dextran coated iron oxide nanoparticles (DIO-NPs) thin layer on glass substrate by an adapted method. The surface morphology of the obtained samples was analyzed by Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM), optical, and metallographic microscopies. In addition, the distribution of the chemical elements into the DIO-NPs thin layer was analyzed by Glow Discharge Optical Emission Spectrometry (GDOES). Furthermore, the chemical bonds formed between the dextran and iron oxide nanoparticles was investigated by Fourier Transform Infrared Spectroscopy (FTIR). Additionally, the HepG2 viability incubated with the DIO-NPs layers was evaluated at different time intervals using MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The goal of this study was to obtain a DIO-NPs thin layer which could be used as a coating for medical devices such as microfluidic channel, microchips, and catheter. The results of the surface morphology investigations conducted on DIO-NPs thin layer suggests the presence of a continuous and homogeneous layer. In addition, the GDOES results indicate the presence of C, H, Fe, and O signal intensities characteristic to the DIO-NPs layers. The presence in the IR spectra of the Fe-CO metal carbonyl vibration bonds prove that the linkage between iron oxide nanoparticles and dextran take place through carbon-oxygen bonds. The cytotoxicity assays highlighted that HepG2 cells morphology did not show any noticeable modifications after being incubated with DIO-NPs layers. In addition, the MTT assay suggested that the DIO-NPs layers did not present any toxic effects towards HEpG2 cells.

Species composition and relative abundance of the genus Culicoides (Diptera: Ceratopogonidae) in Romania.

BACKGROUND: Culicoides biting midges are vectors involved in the biological transmission cycle of important animal diseases such as bluetongue and African horse sickness. In Romania, the first outbreaks of bluetongue were reported in 2014, leading to increased activities within the existing entomological surveillance network. The main goals of the surveillance activities were the establishment of the vector free period in relation to animal trade and the identification of Culicoides species involved in the transmission of the pathogen. This study was conducted on the composition and relative abundance of the species belonging to the genus Culicoides (Diptera: Ceratopogonidae) in certain regions of Romania and provided the opportunity to update the existing checklist of Culicoides species of this country. METHODS: The study was conducted in 33 of the 42 administrative units (counties), including a total of 659 catches, in 102 locations. The collections were carried out with UV blacklight suction traps (OVI type). The collected insects were preserved in 70% ethanol. Morphological insect identification was carried out using a stereomicroscope, according to established identification keys. In ten localities the relative abundance of the cryptic species of the Obsoletus complex was determined by multiplex PCR assay based on the ITS2 segment. The identification of the Culicoides chiopterus (Meigen) species by morphological examination was confirmed by PCR assay based on the ITS1 segment. RESULTS: Eleven species were identified using morphological and PCR tools. The rest of the individuals were separated into five taxa. The species of the Obsoletus complex (grouping Culicoides obsoletus (Meigen) and Culicoides scoticus Downes & Kettle) were the most abundant, accounting for 59% of the total number of captured Culicoides spp. Three of the identified species are mentioned, according to our knowledge, for the first time in Romania: Culicoides newsteadi Austen, Culicoides flavipulicaris Dzhafarov and Culicoides bysta Sarvasová, Kocisová, Candolfi & Mathieu. CONCLUSIONS: Our study demonstrates that the Culicoides species most commonly cited as being involved in the transmission of arboviruses in Europe (i.e. bluetongue and Schmallenberg viruses) make up a high proportion of adult Culicoides trapped in Romania.

New 2-(4-(4-bromophenylsulfonyl)phenyl)-4-arylidene-oxazol-5(4H)-ones: analgesic activity and histopathological assessment.

This paper reports the synthesis, analgesic activity, acute toxicity and histopathological (HP) assessment of four new compounds from oxazol-5(4H)-ones class that contain in their molecule a diarylsulfone moiety. The new 2-(4-(4-bromophenylsulfonyl)phenyl)-4-arylidene-oxazol-5(4H)-ones were obtained by reaction of 2-(4-(4-bromophenyl-sulfonyl)benzamido)acetic acid intermediate with aromatic aldehydes (benzaldehyde, 4-methoxy, 4-nitro or 4-bromobenzaldehyde), in acetic anhydride and in the presence of anhydrous sodium acetate. The new compounds have been characterized by spectral techniques, such as: Fourier-transform infrared spectroscopy (FT-IR), mass spectrometry (MS), proton nuclear magnetic resonance (1H-NMR) and by elemental analysis. The acute toxicity of the new oxazol-5(4H)-ones in mice was assessed through "acute toxic class" method, according to Organization for Economic Co-operation and Development (OECD) Guidelines. The HP assessment of some preserved organs collected from mice has been performed. The analgesic activity of all new synthesized compounds was carried out with two pharmacological tests: the writhing test and the hot plate test. In order to predict the binding affinities of the synthesized oxazol-5(4H)-ones derivatives against molecular targets involved in pain and inflammation, molecular docking simulations were performed. The results of the writhing test indicated that the most active compound was the oxazolone that contains in the molecule a methoxy group. The acute oral toxicity study revealed no lethal effect of new compounds. The HP assessment of the preserved organs collected from mice did not indicate any cytohistopathological aspects that can be linked to any inflammatory, neoplastic or cytotoxic process, demonstrating the low toxicity of new compounds.

Copper Bead Therapy in Severe Bone Infection: A Rabbit Tibial Model.

OBJECTIVE: We investigated the benefits of a local preventive therapy based on copper beads against severe bone infection using a rabbit open tibial fracture model. MATERIALS AND METHODS: Cotton mesh balls soaked in a very high concentration of Staphylococcus aureus ATCC 6538 culture were inoculated in drilled holes of the tibiae of treated and control groups. The treated group was also implanted with small copper beads simultaneously, as prevention therapy. RESULTS: Survival rate in the treated group was 67% compared with 25% in the control group (difference 40%, for a 95% confidence interval: 40%, 93.4%). The few remaining animals in the control group had bone lesions which developed into osteomyelitis, while the tibiae of treated group had clear signs of reparatory processes. Sixty days after inoculation, signs of local-only toxicity were observed in healthy tibia of a separate non-infected control group. Drawbacks of copper toxicity were weighed against the threat of septicaemia and also against prolonged use of powerful systemic antibiotic medications in severe bone contamination. CINICAL SIGNIFICANCE: It was found that the proposed therapy prevented septicaemia and the spread of infection, and it also induced reparatory processes. The findings of this study may be relevant in antisepsis of open fractures in less appropriate medical settings (such as military camps or remote locations), as well as in severe bone infections.

Properties of Basil and Lavender Essential Oils Adsorbed on the Surface of Hydroxyapatite.

The research conducted in this study presented for the first time results of physico-chemical properties and in vitro antimicrobial activity of hydroxyapatite plant essential oil against Gram-positive bacteria (methicillin-resistant Staphylococcus aureus (MRSA) and S. aureus 0364) and Gram-negative bacteria (Escherichia coli ATCC 25922). The samples were studied by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy to determine the morphology and structure of the nanocomposites of hydroxyapatite coated with basil (HAp-B) and lavender (HAp-L) essential oils (EOs). The values of the BET specific surface area (SBET), total pore volume (VP) and pore size (DP) were determined. The results for the physico-chemical properties of HAp-L and HAp-B revealed that lavender EOs were well adsorbed on the surface of hydroxyapatite, whereas basil EOs showed a poor adsorption on the surface of hydroxyapatite. We found that the lavender EOs hydroxyapatite (HAp-L) exhibited a very good inhibitory growth activity. The value of the minimum inhibitory concentration (MIC) related to growth bacteria was 0.039 mg/mL for MRSA, 0.02 mg/mL for S. aureus and 0.039 mg/mL E. coli ATCC 25922. The basil EO hydroxyapatite (HAp-B) showed poor inhibition of bacterial cell growth. The MIC value was 0.625 mg/mL for the HAp-B sample in the presence of the MRSA bacteria, 0.313 mg/mL in the presence of S. aureus and 0.078 mg/mL for E. coli ATCC 25922.

Oral toxicity study of certain plant extracts containing pyrrolizidine alkaloids.

Pyrrolizidine alkaloids (PAs) are a class of toxic compounds which are found in plants. Poisoning caused by these toxins is associated with acute and chronic liver damage. Tussilago farfara (coltsfoot), Petasites hybridus (common butterbur), Senecio vernalis (eastern groundsel) and Symphytum officinale (comfrey) are traditional phytotherapic species, which beside the therapeutic bioactive compounds contain PAs. The aim of the paper was to assess the safety of some dry extracts obtained from these species. For the determination of acute toxicity, Organization for Economic Cooperation and Development (OECD) Guideline No. 423 was used. For the determination of repeated dose oral toxicity, Senecionis vernalis herba and Symphyti radix extracts (250 mg÷kg) were administrated, by gavage, for 28 days, and their effects on animal weight, liver and biliary functions, hepatic tissue and oxidative stress were investigated. After the acute toxicity testing, the dry extracts were placed in the GHS Category V (LD50>5000 mg÷kg, p.o.). For the subacute toxicity testing, no death or any signs of toxicity were observed. Also, no significant differences in biochemical parameters were observed between control and treated groups. The observed histopathological lesions were non-specific and were not consistent with the data reported in the literature for PAs exposure. In conclusion, the administration for 28 days, of the tested extracts, in a dose which correspond to a PAs concentration over the limits imposed in some countries, produced no hepatic and biliary toxic effects. Further studies, extended over a longer period of time, are needed in order to determine the safety of plant extracts containing PAs.

Vaccine-induced rabies case in a cow (Bos taurus): Molecular characterisation of vaccine strain in brain tissue.

Rabies is a fatal neuropathogenic zoonosis caused by the rabies virus of the Lyssavirus genus, Rhabdoviridae family. The oral vaccination of foxes - the main reservoir of rabies in Europe - using a live attenuated rabies virus vaccine was successfully conducted in many Western European countries. In July 2015, a rabies vaccine strain was isolated from the brain tissues of a clinically suspect cow (Bos taurus) in Romania. The nucleotide analysis of both N and G gene sequences showed 100% identity between the rabid animal, the GenBank reference SAD B19 strain and five rabies vaccine batches used for the national oral vaccination campaign targeting foxes.

The influence of foreign body surface area on the outcome of chronic osteomyelitis.

Reproducible animal models of osteomyelitis close to the clinical scenario are difficult to obtain as the animals either die shortly after inoculation of bacteria or the bone cures itself of infection. Additional materials used as foreign bodies offer increased chances for localized infection due to bacterial attachment and are closer to clinical pathology. Through in vivo experimentation we investigated here the influence of surface area of a series of foreign bodies on the final outcome of the animal model, in terms of reproducibility, survival rate and time necessary for onset of chronic disease. Stainless steel Kirschner wire segments, stainless steel balls and cotton meshes were employed for this purpose. The clinical, microbiological, radiological and histological results obtained were compared with the simple case where no foreign body was used. The follow-up period was 57days. The cotton meshes, which had the highest surface area, were observed to provide the best outcome, with the lowest disease onset time interval (of 1week earlier than the others), the highest survival (of 90%) and disease reproduction rate (90%). The only clinical pattern of the mesh group rabbits was short lived inflammation while the other rabbits presented also some other clinical signs such as rhinorrheas, abscesses, rush and/or dyspnea. Moreover, this model is the most suitable for further treatment studies, as the cotton meshes could be easily removed after disease onset, without any intervention on the bone. This is important, as the treatment would address the bacteria present within the bone parts (marrow, cortex, periosteum etc.) not those forming the biofilm.

Zoonotic ocular onchocercosis caused by Onchocerca lupi in dogs in Romania.

Onchocerca lupi is a filarial nematode, which infects the scleral conjunctival tissue of dogs, wolves and cats. Whilst adult nematodes localize in the conjunctive tissue of sclera or in the retrobulbar, microfilariae are found in the skin, and they are rarely diagnosed in asymptomatic animals. Since the first report of human ocular infection 5 years ago, up to 10 zoonotic cases have been identified in patients worldwide. We report, for the first time in Romania, three cases of canine ocular onchocercosis in dogs. Fragments of the harvested worms were characterized morphologically and molecularly. This article expands knowledge on the distribution of this parasite in Eastern Europe and sounds an alarm bell for ophthalmologists about the possible occurrence of human cases of O. lupi infection.

Design, Synthesis, Characterization and Antimicrobial Evaluation of Some Heterocyclic Condensed Systems with Bridgehead Nitrogen from Thiazolotriazole Class.

In the present study, a series of new heterocyclic condensed systems with bridgehead nitrogen from the thiazolo[3,2-b][1,2,4]triazoles class was synthesized starting from some 4-(4-X-phenylsulfonyl)phenyl)-4H-1,2,4-triazole-3-thioles 1a-c (X=H, Cl, Br). The intermediates of S-alkylated 1,2,4-triazoles, 2-(5-(4-(4-X-phenylsulfonyl)phenyl)-2H-1,2,4-triazol-3-ylthio)-1-(4-fluorophenyl)ethanones 2a-c, were obtained by treatment of triazoles 1a-c with 2-bromo-4'-fluoroacetophenone. The 2-(4-(4-X-phenylsulfonyl)phenyl)-6-(4-fluorophenyl)thiazolo[3,2-b][1,2,4]triazoles 3a-c were obtained by cyclization of S-alkylated 1,2,4-triazoles 2a-c in sulfuric acid media, at 0 °C. For the synthesis of 2-(4-(4-X-phenylsulfonyl)phenyl)-5-(4-fluorobenzylidene)-thiazolo[3,2-b][1,2,4]triazol-6(5H)-ones 4a-c, the triazoles 1a-c were treated with 4-fluorobenzaldehyde, chloroacetic acid and anhydrous sodium acetate, in the presence of acetic acid and acetic anhydride. The structures of the newly synthesized compounds have been confirmed by elemental analysis and spectral methods (IR, 1H-NMR, 13C-NMR, MS). The antimicrobial activity of all new compounds has been screened against some bacteria and yeasts.

First evidence of Anaplasma platys and Hepatozoon canis co-infection in a dog from Romania--a case report.

Anaplasma platys was first identified and described in North America as a Rickettsia-like, platelet-specific organism in dogs with infectious canine cyclic thrombocytopenia. In Europe, A. platys has so far mainly been described for some Mediterranean countries. Here, we describe a case of A. platys infection in a dog from Romania, confirmed by PCR. Additionally, the dog had a co-infection with Hepatozoon canis. To the best of our knowledge, this is the first case of A. platys infection in Romania and the first case of a co-infection with A. platys and H. canis altogether. Both pathogens should be considered as possible disease agents in dogs suffering from disease associated with tick bite in south-eastern Europe.

New heterocyclic compounds from 1,2,4-triazole and 1,3,4-thiadiazole class bearing diphenylsulfone moieties. Synthesis, characterization and antimicrobial activity evaluation.

Some new 5-(4-(4-X-phenylsulfonyl)phenyl)-4-(R)-2H-1,2,4-triazol-3(4H)-thiones 4a,b; 5a,b and 5-(4-(4-X-phenylsulfonyl)phenyl)-N-(R)-1,3,4-thiadiazol-2-amines 6a,b; 7a,b were obtained by cyclization of new N(1)-[4-(4-X-phenylsulfonyl)benzoyl]-N(4)-(R)-thiosemicarbazides 2a,b; 3a,b (X=H, Br). The 1,2,4-triazoles were synthesized by intramolecular cyclization of acylthiosemicarbazides, in basic media. On the other hand, 1,3,4-thiadiazoles were obtained from same acylthiosemicarbazides, in acidic media. These new intermediates from thiosemicarbazide class were afforded by the reaction of 4-(4-X-phenylsulfonyl)benzoic acids hydrazides (X=H, Br) 1a,b with 4-trifluoromethoxyphenyl or 3,4,5-trimethoxyphenyl isothiocyanate. The newly synthesized compounds were characterized by IR, (1)H NMR, (13)C NMR, MS and elemental analysis. All the new compounds were screened for their antimicrobial activity against some bacteria (Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC 13061, Escherichia coli ATCC 25922, Enterobacter cloacae ATCC 49141, Acinetobacter baumannii ATCC 19606 and Pseudomonas aeruginosa ATCC 27853) and yeasts (Candida albicans ATCC 90028 and Candida parapsilosis ATCC 22019).

Genetic diversity of porcine circovirus type 2 (PCV2) in the Romanian wild boar population.

In this study, we have analyzed 23 PCV2 ORF2 sequences recovered from wild boar population in Romania. The PCV2 sequences were originated from different geographical regions in Romania, and collected between 2008 and 2009 during the classical swine fever virus (CSFV) surveillance campaign. Complete open reading frame 2 (ORF2) nucleotide sequences were obtained and compared with sequences mainly from European and Asian isolates. The Romanian sequences were identified as belonging to previously described clusters 2a and 2b, with high degree of heterogeneity (PCV2 ORF2 nucleotide homology ranged between 90.1% and 100%). Interestingly, for cluster 2a, the majority of the sequences (8 from a total number of 9) clustered mainly with the Asian isolates (especially China, but also India and South Korea), with three exceptions from Europe previously reported in Germany, Belgium and The Netherlands.