RESUMEN
SARS-CoV-2 infection outbreaks in minks have serious implications associated with animal health and welfare, and public health. In two naturally infected mink farms (A and B) located in Greece, we investigated the outbreaks and assessed parameters associated with virus transmission, immunity, pathology, and environmental contamination. Symptoms ranged from anorexia and mild depression to respiratory signs of varying intensity. Although the farms were at different breeding stages, mortality was similarly high (8.4% and 10.0%). The viral strains belonged to lineages B.1.1.218 and B.1.1.305, possessing the mink-specific S-Y453F substitution. Lung histopathology identified necrosis of smooth muscle and connective tissue elements of vascular walls, and vasculitis as the main early key events of the acute SARS-CoV-2-induced broncho-interstitial pneumonia. Molecular investigation in two dead minks indicated a consistently higher (0.3-1.3 log10 RNA copies/g) viral load in organs of the male mink compared to the female. In farm A, the infected farmers were responsible for the significant initial infection of 229 out of 1,000 handled minks, suggesting a very efficient human-to-mink transmission. Subsequent infections across the sheds wherein animals were being housed occurred due to airborne transmission. Based on a R0 of 2.90 and a growth rate equal to 0.293, the generation time was estimated to be 3.6 days, indicative of the massive SARS-CoV-2 dispersal among minks. After the end of the outbreaks, a similar percentage of animals were immune in the two farms (93.0% and 93.3%), preventing further virus transmission whereas, viral RNA was detected in samples collected from shed surfaces and air. Consequently, strict biosecurity is imperative during the occurrence of clinical signs. Environmental viral load monitoring, in conjunction with NGS should be adopted in mink farm surveillance. The minimum proportion of minks that need to be immunized to avoid outbreaks in farms was calculated at 65.5%, which is important for future vaccination campaigns.
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COVID-19/veterinaria , Visón/virología , Animales , COVID-19/epidemiología , COVID-19/genética , COVID-19/transmisión , Brotes de Enfermedades/veterinaria , Microbiología Ambiental , Granjas , Femenino , Grecia/epidemiología , Humanos , Masculino , Visón/genética , Exposición Profesional , Zoonosis Virales/transmisión , Zoonosis Virales/virologíaRESUMEN
The epidemiology of West Nile virus (WNV) is unpredictable and changing. Availability of whole genome sequences enables the detailed molecular epidemiology studies and the evaluation and design of diagnostic tools. In the present study we provide two PCR-based protocols which can be applied directly on biological samples from hosts infected by WNV strains belonging to lineage 1 or lineage 2. It was shown that the protocols worked successfully even on samples with relatively low viral load.
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Fiebre del Nilo Occidental , Virus del Nilo Occidental , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Filogenia , Reacción en Cadena de la Polimerasa , Virus del Nilo Occidental/genéticaRESUMEN
Lumpy skin disease (LSD) is a transboundary disease of economic importance affecting cattle and buffaloes. In South-Eastern Europe, immunization of cattle with homologous live attenuated vaccines for LSD control has prevented outbreaks since 2017, but has been associated with adverse reactions resembling disease symptoms. Thus, a diagnostic method suitable for disease surveillance in farms during vaccination campaigns with Neethling (Onderstepoort) and SIS type (Lumpyvax) live attenuated LSDV vaccines in Europe should be able to detect the wild type (WT) LSDV in animals with adverse reactions to the vaccines and samples with potentially high titers of the vaccine LSDV. To this end, a real-time PCR method targeting the EEV gene of LSDV was developed for the specific detection of WT strains, along with the use of beta-actin gene as an internal amplification control (IAC). Amplification efficiency of the WT virus target was 99.0% and 98.6%, in the presence and in the absence of high loads of vaccine LSDV, respectively. In the presence of 105.6 vaccine LSDV DNA copies, the limit of detection for WT LSDV was 12.6 DNA copies per reaction. The inter-assay CV was 0.04% for WT LSDV and 0.13% for beta-actin. The method can confirm diagnosis in suspect cases irrespective of the presence of the vaccine LSDV DNA by overcoming the masking effect of the WT LSDV. The simultaneous amplification of the beta-actin gene further assures the quality of diagnostic testing. The new method is a surveillance tool, complementing the DIVA real-time PCR during vaccination campaigns and can provide rapid insight on the targeted EEV gene in countries with novel and recombinant LSDV strains.
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Dermatosis Nodular Contagiosa , Virus de la Dermatosis Nodular Contagiosa , Actinas/genética , Animales , Bovinos , Dermatosis Nodular Contagiosa/diagnóstico , Dermatosis Nodular Contagiosa/prevención & control , Virus de la Dermatosis Nodular Contagiosa/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Vacunas AtenuadasRESUMEN
Small ruminant lentiviruses (SRLVs) are highly diverse retroviruses infecting sheep and goats. Although PCR-based testing is being utilized for diagnostics, its application is hampered by various factors. These include, among others, the exceptionally high genetic variability of SRLVs, as well as the low number of infected blood monocytes. For this reason, a highly sensitive and specific semi-nested real-time PCR for proviral DNA detection and quantification was developed. The method is innovative in that a) its design is based on selecting the preferred codon usage in the targeted conserved genomic regions and b) oligospermine-conjugated degenerate primers with increased Tm were utilized. Modifications permitted primer/template duplex formation in the cases of mismatches due to sporadic nucleotide polymorphisms in a number of variant SRLV strains and consequently, the detection of highly diverse SRLV strains. The potential loss of analytical sensitivity and specificity was counterbalanced by including a semi-nested step in combination with LNA probes. An in silico procedure for the evaluation of hybridization efficiency of the designed oligonucleotides to all known targeted variants was also implemented. The method presents a linear range of quantification over a 3-log10 range and a limit of detection of 3.9 proviral dsDNA copies per reaction. Its diagnostic performance was evaluated by testing field samples from seropositive and seronegative animals, followed by phylogenetic analysis of the strains detected. To further increase the diagnostic sensitivity, a DNA extraction protocol for blood leukocytes was developed and evaluated. A minimum of 500 ng input DNA is recommended for PCR-based detection of SRLV proviral DNA, given the low numbers of infected blood monocytes. The developed methodology may serve as a useful tool, which can be adjusted for the quantitative detection of viruses exhibiting high genetic variability.
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Enfermedades de las Cabras/diagnóstico , Lentivirus/genética , Provirus/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Enfermedades de las Ovejas/diagnóstico , Animales , Cartilla de ADN , ADN Viral/genética , ADN Viral/aislamiento & purificación , Enfermedades de las Cabras/sangre , Enfermedades de las Cabras/virología , Cabras , Lentivirus/aislamiento & purificación , Leucocitos/metabolismo , Leucocitos/virología , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/virología , Filogenia , Sensibilidad y Especificidad , Ovinos , Enfermedades de las Ovejas/sangre , Enfermedades de las Ovejas/virologíaRESUMEN
BACKGROUND: Canine inflammatory bowel disease (IBD) is a group of chronic gastrointestinal (GI) disorders of still largely unknown etiology. Canine IBD diagnosis is time-consuming and costly as other diseases with similar signs should be initially excluded. In human IBD microRNA (miR) expression changes have been reported in GI mucosa and blood. Thus, there is a possibility that miRs may provide insight into disease pathogenesis, diagnosis and even treatment of canine IBD. The aim of this study was to determine the colonic mucosal and serum relative expression of a miRs panel in dogs with large intestinal IBD and healthy control dogs. RESULTS: Compared to healthy control dogs, dogs with large intestinal IBD showed significantly increased relative expression of miR-16, miR-21, miR-122 and miR-147 in the colonic mucosa and serum, while the relative expression of miR-185, miR-192 and miR-223 was significantly decreased. Relative expression of miR-146a was significantly increased only in the serum of dogs with large intestinal IBD. Furthermore, serum miR-192 and miR-223 relative expression correlated to disease activity and endoscopic score, respectively. CONCLUSION: Our data suggest the existence of dysregulated miRs expression patterns in canine IBD and support the potential future use of serum miRs as useful noninvasive biomarkers.
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Enfermedades de los Perros/sangre , Enfermedades Inflamatorias del Intestino/veterinaria , MicroARNs/genética , Animales , Biomarcadores/sangre , Colon/metabolismo , Enfermedades de los Perros/genética , Enfermedades de los Perros/metabolismo , Enfermedades de los Perros/patología , Perros , Femenino , Perfilación de la Expresión Génica/veterinaria , Enfermedades Inflamatorias del Intestino/sangre , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/metabolismo , Mucosa Intestinal/metabolismo , Masculino , MicroARNs/metabolismoRESUMEN
A viability quantitative PCR (qPCR) utilizing propidium monoazide (PMA) is presented for rapid quantification of viable cells using the foodborne pathogen Campylobacter coli as a bacterial model. It includes optimized spheroplast formation via lysozyme and EDTA, induction of a mild osmotic shock for enhancing the selective penetration of PMA into dead cells, and exploitation of an internal sample process control (ISPC) involving cell inactivation to assess residual false-positive signals within each sample. Spheroplasting of bacteria in exponential phase did not permit PMA entrance into viable cells since a strong linear relationship was detected between simple qPCR and PMA-qPCR quantification, and no differences were observed regardless of whether spheroplasting was utilized. The PMA-qPCR signal suppression of dead cells was elevated using spheroplast formation. With regard to the ISPC, cell inactivation by hydrogen peroxide resulted in higher signal suppression during qPCR than heat inactivation did. Viability quantification of C. coli cells by optimized spheroplasting-PMA-qPCR with ISPC was successfully applied in an aging pure culture under aerobic conditions and artificially inoculated meat. The same method exhibited a high linear range of quantification (1.5 to 8.5 log10 viable cells ml-1), and results were highly correlated with culture-based enumeration. PMA-qPCR quantification of viable cells can be affected by their rigidity, age, culture media, and niches, but spheroplast formation along with osmotic shock and the use of a proper ISPC can address such variations. The developed methodology could detect cells in a viable-but-nonculturable state and might be utilized for the quantification of other Gram-negative bacteria.IMPORTANCE There is need for rapid and accurate methods to detect viable bacterial cells of foodborne pathogens. Conventional culture-based methods are time-consuming and unable to detect bacteria in a viable-but-nonculturable state. The high sensitivity and specificity of the quantitative PCR (qPCR) are negated by its inability to differentiate the DNAs from viable and dead cells. The combination of propidium monoazide (PMA), a DNA-intercalating dye, with qPCR assays is promising for detection of viable cells. Despite encouraging results, these assays still encounter various challenges, such as false-positive signals by dead cells and the lack of an internal control identifying these signals per sample. The significance of our research lies in enhancing the selective entrance of PMA into dead Campylobacter coli cells via spheroplasting and in developing an internal sample process control, thus delivering reliable results in pure cultures and meat samples, approaches that can be applicable to other Gram-negative pathogens.
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Azidas/química , Campylobacter coli/aislamiento & purificación , Microbiología de Alimentos/métodos , Carne/microbiología , Viabilidad Microbiana , Propidio/análogos & derivados , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Esferoplastos/aislamiento & purificación , Microbiología de Alimentos/instrumentación , Propidio/química , Reacción en Cadena en Tiempo Real de la Polimerasa/instrumentaciónRESUMEN
West Nile virus (WNV) is an arbovirus causing neuroinvasive disease to humans and equines. Since 2004, lineage 2 WNV strains have been identified in Europe and have been implicated in severe outbreaks, with that of 2018 exceeding the total number from the previous seven years. The aim of this study was to explore the evolutionary process that shapes the genetic diversity of lineage 2 WNV strains (belonging to the Central European/Hungarian subclade) and reconstruct the origin and transmission routes in Europe, and especially in the Balkans. For this purpose, a high number of whole genome sequences (WGSs) were analyzed, along with newly characterized sequences, including strains from the 2018 WNV transmission season in Greece. Maximum likelihood and Bayesian inference methods were used to perform the phylogenetic and phylodynamic analyses and phylogeographic reconstruction. The majority of the Central European/Hungarian lineage 2 strains are grouped in 2 phylogenetic subgroups (Central/South-West European and Balkan) with bush-like topology. Purifying selection shapes their evolution, however, strong evidence of positive selection was revealed in 7 non-structural protein codons of NS1, NS4B and NS5. Thirty-two amino-acid substitutions were fixed in different phylogenetic subgroups, indicating that random genetic drift is responsible for the majority of evolutionary changes. Virus migration, followed by subsequent local evolution is responsible for continuously evolving strains throughout Europe. In total, 10 virus transitions between discrete geographical locations, involving virus spread from Central Europe to other regions, were highly supported. Three novel, independent introductions from Hungary and Bulgaria were responsible for the 2018 re-emergence of WNV in Northern Greece, indicating that Hungary remains an important ecological niche for the virus and has a central role for the dissemination of novel strains in the Balkans. In Northern Greece, tMRCA estimations indicated that a 1-to 2-year period of silent enzootic transmission precedes spread to dead-end hosts. Reconstruction of WNV population dynamics, from WGS data, revealed epidemic patterns characterized by 3- to 5-year oscillations in Europe. Future studies are necessary to determine the possible driving factors for these fluctuations i.e. avian herd immunity and climatic conditions affecting mosquito and bird populations. Maintaining adequate epidemiological surveillance with emphasis on obtaining WGS data, in areas at risk, is crucial for understanding the epidemiology and transmission patterns of WNV. It can further support integrated programs for risk assessment of virus circulation dynamics, aiming to targeted prevention and response measures for veterinary and public health in Europe.
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Filogenia , Filogeografía , Selección Genética , Virus del Nilo Occidental/clasificación , Sustitución de Aminoácidos , Animales , Teorema de Bayes , Codón/genética , Europa (Continente) , Humanos , Funciones de Verosimilitud , Factores de Tiempo , Fiebre del Nilo OccidentalRESUMEN
Lineage 2 West Nile virus (WNV) strains have been implicated in severe encephalitis outbreaks in humans and equines residing in Europe. WNV molecular characterization is important for the development of diagnostic assays, as well as for obtaining molecular information, which is necessary for epidemiological investigations of virus transmission in areas at risk. For whole genome sequencing of lineage 2 WNV strains, directly from biological specimens, a PCR-based NGS protocol was developed. The method was applied in WNV-positive specimens obtained from animal, human and mosquito hosts in Greece. The results of its application indicate that, even in cases of low virus titers, the developed PCR-based NGS approach is able to provide whole genome sequences of lineage 2 WNV strains.
Asunto(s)
Genoma Viral/genética , Filogenia , Fiebre del Nilo Occidental/genética , Virus del Nilo Occidental/genética , Animales , Culicidae/virología , Brotes de Enfermedades , Europa (Continente) , Grecia , Secuenciación de Nucleótidos de Alto Rendimiento , Caballos/genética , Caballos/virología , Humanos , ARN Viral/genética , Fiebre del Nilo Occidental/diagnóstico , Fiebre del Nilo Occidental/virología , Virus del Nilo Occidental/patogenicidad , Secuenciación Completa del GenomaRESUMEN
Enzootic nasal adenocarcinoma (ENA) is a contagious neoplasm of sheep and goats, associated with the oncogenic retroviruses enzootic nasal tumor virus (ENTV) 1 and 2, respectively. It appears to be common in countries with substantial small ruminant-production. ENA diagnosis in goats is based on autopsy and histopathology, and there is no real-time PCR method available for ENTV-2 detection. Here, a novel one-tube real-time RT-PCR (RT-qPCR) method for the detection and quantification of ENTV-2 in nasal swabs is presented. The method targets the env gene/U3 region. For the design of ENTV-2-specific oligonucleotides, molecular characterization of seven Greek ENTV-2 strains was performed. Phylogenetic analysis revealed three distinct phylogenetic clades of ENTV-2 that correlate with the country of sample collection. Evaluation of the analytical performance of the RT-qPCR revealed an amplification efficiency of 92.8% and a linear range of quantification between 2 × 108 and 2 × 102 RNA transcripts. Analysis of nasal swabs from 23 histopathologically confirmed, naturally occurring ENA cases via RT-qPCR yielded positive results. Moreover, modification of the method for use in a real-time PCR (qPCR) assay enables detection of proviral DNA in tumor specimens. Both methods are highly specific and can be used for the confirmation of ENA-suspected cases. Future applications could include ante-mortem diagnosis, verification of the ENTV-2-free status in animal trade, disease surveillance, and control programs.
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Betaretrovirus/aislamiento & purificación , Enfermedades de las Cabras/virología , Neoplasias Nasales/veterinaria , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Infecciones por Retroviridae/veterinaria , Infecciones Tumorales por Virus/veterinaria , Animales , Betaretrovirus/clasificación , Betaretrovirus/genética , Enfermedades de las Cabras/diagnóstico , Cabras , Neoplasias Nasales/diagnóstico , Neoplasias Nasales/virología , Filogenia , Infecciones por Retroviridae/diagnóstico , Infecciones por Retroviridae/virología , Infecciones Tumorales por Virus/diagnóstico , Infecciones Tumorales por Virus/virologíaRESUMEN
Betanodaviruses are small ssRNA viruses that cause viral encephalopathy and retinopathy, a severe neuropathological infectious disease in marine fish species worldwide. In the present study, the occurrence of betanodaviruses was investigated in wild and cultured populations of fishes and invertebrates of the Greek territorial waters. Betanodaviruses were detected in 35 species belonging to 21 families and 12 orders. To our knowledge, 23 of those are reported for the first time in Greek waters, while 11 of them are reported for the first time globally. The positive samples were subjected to sequencing and phylogenetic analysis of partial segments of RNA1 and RNA2 genes. Almost all the viruses circulating in Greece fell within RGNNV genotype, while reassortant viruses were detected in three samples, namely two inter-RGNNV and one RGNNV/SJNNV. A novel unclassified Betanodavirus sequence was also identified. Most of the Greek sequence types have a restricted geographic distribution except for two RNA1 and one RNA2 sequence types that are widespread throughout the Mediterranean basin. The results of this study indicate the range of reservoirs/hosts of betanodaviruses and also their wide spread in the Greek territorial waters and reinforce the hypothesis that wild fish species transmit the virus to cultured ones and vice versa.
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Peces/virología , Invertebrados/virología , Nodaviridae/clasificación , Animales , Enfermedades de los Peces/virología , Genotipo , Grecia , Filogenia , Infecciones por Virus ARN/veterinaria , Infecciones por Virus ARN/virología , ARN Viral/genética , Virus ReordenadosRESUMEN
We analyzed the highly pathogenic avian influenza (HPAI) H5 epizootic of 2016-17 in Europe by epidemiologic and genetic characteristics and compared it with 2 previous epizootics caused by the same H5 Guangdong lineage. The 2016-17 epizootic was the largest in Europe by number of countries and farms affected and greatest diversity of wild birds infected. We observed significant differences among the 3 epizootics regarding region affected, epidemic curve, seasonality, and outbreak duration, making it difficult to predict future HPAI epizootics. However, we know that in 2005-06 and 2016-17 the initial peak of wild bird detections preceded the peak of poultry outbreaks within Europe. Phylogenetic analysis of 2016-17 viruses indicates 2 main pathways into Europe. Our findings highlight the need for global surveillance of viral changes to inform disease preparedness, detection, and control.
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Virus de la Influenza A/clasificación , Gripe Aviar/epidemiología , Gripe Aviar/virología , Animales , Animales Salvajes , Aves , Brotes de Enfermedades , Europa (Continente)/epidemiología , Genoma Viral , Geografía Médica , Historia del Siglo XXI , Virus de la Influenza A/patogenicidad , Gripe Aviar/historia , Gripe Aviar/transmisión , Morbilidad , Mortalidad , Filogenia , Enfermedades de las Aves de Corral/epidemiología , Enfermedades de las Aves de Corral/virología , Análisis Espacio-Temporal , ZoonosisRESUMEN
The main purpose of this work was to establish an experimental model for immunosuppression in sheep, and evaluate its possible effects on bluetongue viremia. Animals were allocated in 4 groups: Cy (cyclophosphamide), BT (bluetongue), CyBT (combined Cy and BT) and Co (control), and underwent clinical evaluations, virological testing, peripheral blood immunophenotyping and determination of antiviral humoral immune responses. Intravenous administration of cyclophosphamide (37.5 mg/kg body weight) resulted in immunosuppresion induction, as significant drops were observed in blood leukocytes and lymphocyte subset counts (CD2+, CD4+, CD8+, CD19+), lasting 3-10 days after its administration. Reduction in B-cell (CD19+) counts was more pronounced than in T-/NK-cell (CD2+) counts (92% and 59%, respectively). BTV-9 inoculation resulted in pronounced lymphocytopenia observed from day 1 post-inoculation. Their combined administration resulted in a more intense immunosuppressive effect, as indicated by the greater reduction in lymphocyte, granulocyte, CD4+ and CD8+ cell counts. In group CyBT, earlier initiation of fever by one day (day 6 p.i.) compared to group BT (day 7 p.i.), and delay in antibody responses by one day was observed, compared to group BT. Neutralizing antibodies in both groups (BT, CyBT) were detectable from day 10 p.i., but no significant titer differences were observed. Infectious virus titers were detected from day 4 p.i. in group BT and from day 3 in group CyBT. Statistical significances in virus titers were also observed (greatest mean titer difference: 1.4 log10 CEID50/ml RBCs at day 5 p.i., P < 0.001), indicating possible impact of immunosuppression on virus transmission and epidemiology of bluetongue.
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Virus de la Lengua Azul/crecimiento & desarrollo , Lengua Azul/virología , Ciclofosfamida/administración & dosificación , Tolerancia Inmunológica , Terapia de Inmunosupresión , Inmunosupresores/administración & dosificación , Viremia , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Lengua Azul/inmunología , Virus de la Lengua Azul/inmunología , Virus de la Lengua Azul/aislamiento & purificación , Recuento de Leucocitos , Subgrupos Linfocitarios/inmunología , Ovinos , Carga ViralRESUMEN
OBJECTIVE: To investigate the prevalence of Leishmania infection in rodents from various areas of northern Greece. METHODS: Ninety-seven rodents (66 Mus musculus, 19 Rattus norvegicus and 12 R. rattus) were collected during pest control programmes and examined by cytology (spleen and liver smears), serology (ELISA) and PCR (real-time and gel-based) for Leishmania. Date, environment, sex, existence of dogs in the close environment were recorded for each rodent. RESULTS: All cytological preparations were negative, whereas specific IgG was detected in 54.5% in total; 70% of R. norvegicus; 50% of R. rattus; and 50% of M. musculus. In at least one molecular method, 19.6% of the samples in total were positive: 25% of R. rattus, 24% of M. musculus, but no R. norvegicus was found positive. Environment (semi-urban areas, P = 0.037) and species (M. musculus, P = 0.032) were associated with positive PCR. All infected animals showed evidence of low parasite burden, demonstrated by the negative cytological examinations and the high Ct values observed in real-time PCR. CONCLUSION: Due to the proximity of rodents to humans and dogs, these animals may be important in the epidemiology of leishmaniosis, especially if proven that they can infect sand flies.
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Reservorios de Enfermedades , Leishmania/crecimiento & desarrollo , Leishmaniasis/parasitología , Enfermedades de los Roedores/parasitología , Roedores/parasitología , Animales , Vectores de Enfermedades , Perros , Ambiente , Grecia , Humanos , Inmunoglobulina G/sangre , Leishmaniasis/sangre , Leishmaniasis/epidemiología , Leishmaniasis/transmisión , Ratones , Prevalencia , Psychodidae/parasitología , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Enfermedades de los Roedores/sangre , Roedores/sangre , Especificidad de la EspecieRESUMEN
During 2014, an outbreak of Bluetongue virus (BTV) infections attributed to serotype 4 occurred in Greece and spread to south-eastern Europe. In the present article, the clinical and epidemiological data of 15 sheep flocks and 5 dairy cattle herds affected in Greece are described. In sheep, the most frequent clinical signs observed were fever, hyporexia, and edema of the face. A number of clinically affected sheep had chronic laminitis resulting in chronic lameness. Confirmation of suspect clinical cases was performed using BTV-specific real-time RT-PCR, and serotype 4-specific RT-PCR. The average morbidity of bluetongue in the sheep flocks was estimated to be 15.3 % (95 % C.I. 6.8-23.8 %) and the average mortality and case fatality were 4.5 % (95 % C.I. 1.5-7.6 %) and 32.0 % (95 % C.I. 18.1-42.9 %), respectively. The BTV seroprevalence and the ratio of clinical manifestations-to-infections determined in seven of these flocks, were on average 36.5 % (95 % C.I. 15.7-57.3 %) and 24.6 % (95 % C.I. 12.8-36.3 %). BTV ratio of clinical manifestations-to-infections was higher in the imported western European sheep breeds examined compared to the local ones. In dairy cattle, the average herd prevalence of viremia was 48.8 % (95 % C.I. 15.3-82.4 %) and none had signs associated with bluetongue. The results of this study indicate that the 2014 Greek BTV-4 has significant impact on the health status and the viability of sheep in affected flocks but does not cause clinical signs in cattle, despite the high prevalence of viremia.
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Virus de la Lengua Azul/aislamiento & purificación , Lengua Azul/epidemiología , Brotes de Enfermedades/veterinaria , Animales , Lengua Azul/mortalidad , Lengua Azul/virología , Virus de la Lengua Azul/clasificación , Bovinos , Femenino , Grecia/epidemiología , Reacción en Cadena de la Polimerasa/veterinaria , Estudios Seroepidemiológicos , OvinosRESUMEN
During March 2013, we investigated the presence and the levels of Schmallenberg virus (SBV) circulation in three dairy cow herds and three sheep flocks in Central Macedonia, Greece. In two cow herds, a high number of abortions had been observed during the winter. Six bulk-tank milk samples and 147 individual sera were screened for SBV-specific antibodies by ELISA. Positive reactions were obtained from 5 out of 6 bulk-tank milk samples, 58 out of 90 sera from the 3 cow herds, and 2 sera from 2 of the 3 sheep flocks. Twenty-two ELISA-positive sera were tested by serum neutralization test (SNT). SNT confirmed the presence of neutralizing antibodies against SBV in all samples tested, with titers ranging between 1:32 and ≥1:256. No neutralizing antibodies against Akabane virus (AKAV) or Shamonda virus (SHAV) were detected, indicating that neutralizing antibodies against SBV do not cross react with AKAV or SHAV in SNT. ELISA testing of bulk-tank milk samples proved to be convenient and reliable. None of the tested sera was found positive for SBV by real-time RT-PCR, indicating that the sampling was conducted past the viremia stage. This is the first report of SBV circulation in Greece.
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Infecciones por Bunyaviridae/veterinaria , Enfermedades de los Bovinos/virología , Orthobunyavirus/aislamiento & purificación , Aborto Veterinario/epidemiología , Aborto Veterinario/virología , Animales , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/química , Infecciones por Bunyaviridae/epidemiología , Infecciones por Bunyaviridae/virología , Bovinos , Enfermedades de los Bovinos/epidemiología , Industria Lechera , Ensayo de Inmunoadsorción Enzimática/veterinaria , Femenino , Grecia/epidemiología , Leche/química , Leche/inmunología , Orthobunyavirus/inmunología , Embarazo , Pruebas Serológicas , OvinosRESUMEN
A novel strategy employing the rhabdovirus untranslated conserved intergenic regions was developed and applied successfully for the determination of the complete nucleotide sequence of Eggplant mottled dwarf virus (EMDV). The EMDV genome contains seven open reading frames with the same organization as Potato yellow dwarf virus (PYDV), the type species of the genus Nucleorhabdovirus. These two species encode five core genes [nucleocapsid (N), phosphoprotein (P), matrix (M), glycoprotein (G), and the polymerase (L)] like other viruses of the genus and an additional one (X), located between N and P, giving rise to a protein with currently unknown function. Furthermore, both EMDV and PYDV contain a gene (Y), inserted between P and M, which probably encodes the virus movement protein, in concordance with the rest of the plant-infecting rhabdoviruses. Phylogenetic analysis of the polymerase gene confirmed the classification of EMDV within the genus Nucleorhabdovirus and showed a close evolutionary relationship to PYDV. The novel sequencing strategy developed is a useful tool for the genome determination of yet uncharacterized rhabdoviruses.
Asunto(s)
Genoma Viral , Enfermedades de las Plantas/virología , Rhabdoviridae/genética , Análisis de Secuencia de ADN/métodos , Solanum melongena/virología , Sistemas de Lectura Abierta , Filogenia , Rhabdoviridae/clasificación , Rhabdoviridae/aislamiento & purificación , Proteínas Virales/genéticaRESUMEN
BACKGROUND: The occurrence of paratuberculosis in Ugandan cattle has recently been reported but there is no information on the strains of Mycobacterium avium subspecies paratuberculosis (MAP) responsible for the disease. The aim of this study was to isolate and characterise MAP from seropositive cattle and paratuberculosis lesions in tissues obtained from slaughtered cattle in Uganda. RESULTS: Twenty one isolates of MAP were differentiated into 11 genotype profiles using seven genotyping loci consisting of Insertion Sequence 1311(IS1311), Mycobacterial interspersed repeat units (MIRU) (loci 2, 3), Variable number tandem repeats (VNTR) locus 32 and Short sequence repeats (SSR) (loci 1, 2 and 8). Three different IS1311 types and three MIRU 2 profiles (7, 9, 15 repeats) were observed. Two allelic variants were found based on MIRU 3 (1, 5 repeats), while VNTR 32 showed no polymorphism in any of the isolates from which it was successfully amplified. SSR Locus 1 revealed 6 and 7 G1 repeats among the isolates whereas SSR locus 2 revealed 10, 11 and 12 G2 repeats. SSR locus 8 was the most polymorphic locus. Phylogenetic analysis of SSR locus 8 sequences based on their single nucleotide polymorphisms separated the isolates into 8 genotypes. We found that the use of Ethylene glycol as a PCR additive improved the efficiency of the PCR reactions for MIRUs (2, 3), VNTR 32 and SSR (loci 1 and 2). CONCLUSIONS: There is a high strain diversity of MAP in Uganda since 21 isolates could be classified into 11 genotypes. The combination of the seven loci used in this study results into a very precise discrimination of isolates. However analysis of SNPs on locus alone 8 is very close to this combination. Most of the genotypes in this study are novel since they differed in one or more loci from other isolates of cattle origin in different studies. The large number of MAP strains within a relatively small area of the country implies that the epidemiology of paratuberculosis in Uganda may be complicated and needs further investigation. Finally, the use of Ethylene glycol as a PCR additive increases the efficiency of PCR amplification of difficult templates.
Asunto(s)
Técnicas Bacteriológicas/veterinaria , Dermatoglifia del ADN/métodos , Mycobacterium avium subsp. paratuberculosis/genética , Paratuberculosis/microbiología , Animales , Bovinos , ADN Bacteriano/clasificación , ADN Bacteriano/genética , Variación Genética , Genotipo , Mycobacterium avium subsp. paratuberculosis/clasificación , Paratuberculosis/epidemiología , Filogenia , Polimorfismo de Longitud del Fragmento de Restricción , Uganda/epidemiologíaRESUMEN
Cutaneous defects in cats are commonly encountered in clinical practice, and healing can be accomplished by first or second intention. Platelet-rich plasma (PRP) is characterized by a plasma concentration containing a large number of platelets in a small volume of plasma. The objective of the present study was to record the efficacy of PRP infiltration in open wounds in laboratory cats. Six wounds were created in the dorsal midline of eight laboratory cats, with the wounds of one side designated as the PRP group and the wounds of the other side as the control group. Wound healing was evaluated by daily clinical examination, planimetry, laser Doppler flowmetry, and histologic examination on days 0, 7, 14, and 25, and by measurement of metalloproteinases (MMPs)-2 and -9 and tissue inhibitor metalloproteinase (TIMP)-1 on days 0, 14, and 25. Based on the results of the present study, the mean time for full coverage with granulation tissue was shorter in the PRP group, the mean contraction and total wound healing percentage were increased compared to the control group, and finally, the perfusion measured with laser Doppler flowmetry was higher in the PRP group during all examination days. In conclusion, this is the first study focusing on the topical application of PRP in the treatment of open wounds in laboratory cats, and our results are encouraging-showing a more rapid healing in the PRP group.
RESUMEN
In the present study, the course of SARS-CoV-2 natural infection in two asymptomatic cats, which were negative for immunosuppressive retroviral infections, is investigated. The source of the virus for the cats was their COVID-19-affected owner, with whom they were in continuous proximity in a small household setting. The owner's signs included fatigue, sneezing, anosmia and loss of taste, and diagnosis was confirmed 4 days after symptom onset. Oropharyngeal and faecal swabs were collected from the cats, to investigate the course of SARS-CoV-2 RNA concentrations, as well as the directionality of the chain of virus transmission. Both infected cats were real-time RT-PCR-positive on various time-points. Pharyngeal shedding of at least 6 days was observed in them, with high SARS-CoV-2 titres (> 7 Log10 copies/swab) on the first sampling time-point, that is, 7 days after the onset of owner's clinical signs. In one cat, after the initial decline, slightly increasing virus titres were measured 3 to 6 days after the first real-time RT-PCR-positive swab. Serological testing of this cat revealed absence of seroconversion. The course of viral RNA concentrations in the faecal swabs of the other cat was similar to that in its pharynx. The detected SARS-CoV-2 strains, from both infected cats and their owner, underwent whole-genome sequencing, revealing the absence of emergence of cross-species adaptive mutations in cats. The results support the notion that human SARS-CoV-2 strains are relatively well-adapted to cats. It is still unclear whether asymptomatic animals could play a role in COVID-19 epidemiology, in case of interaction with naïve animals and/or people. Our findings highlight difficulties in SARS-CoV-2 transmission to cats, as neither the two infected cats nor their owner was able to transmit the virus to a third cat living in the same small flat, despite their very close contact during the days corresponding to high virus shedding.
Asunto(s)
COVID-19 , Enfermedades de los Gatos , Animales , COVID-19/veterinaria , Enfermedades de los Gatos/diagnóstico , Gatos , Humanos , Mutación , ARN Viral/genética , SARS-CoV-2/genética , Esparcimiento de VirusRESUMEN
Epidemic spread models are useful tools to study the spread and the effectiveness of the interventions at a population level, to an epidemic. The workhorse of spatially homogeneous class models is the SIR-type ones comprising ordinary differential equations for the unknown state variables. The transition between different states is expressed through rate functions. Inspired by -but not restricted to- features of the COVID-19 pandemic, a new framework for modeling a disease spread is proposed. The main concept refers to the assignment of properties to each individual person as regards his response to the disease. A multidimensional distribution of these properties represents the whole population. The temporal evolution of this distribution is the only dependent variable of the problem. All other variables can be extracted by post-processing of this distribution. It is noteworthy that the new concept allows an improved consideration of vaccination modeling because it recognizes vaccination as a modifier of individuals response to the disease and not as a means for individuals to totally defeat the disease. At the heart of the new approach is an infection age model engaging a sharp cut-off. This model is analyzed in detail, and it is shown to admit self-similar solutions. A hierarchy of models based on the new approach, from a generalized one to a specific one with three dominant properties, is derived. The latter is implemented as an example and indicative results are presented and discussed. It appears that the new framework is general and versatile enough to simulate disease spread processes and to predict the evolution of several variables of the population during this spread.