Mass mortality events associated with cyprinid herpesvirus 2 (CyHV-2) infection in wild Prussian carp Carassius gibelio in the Netherlands, and molecular biology of virus strains.

In 2011 and 2015, four mass mortalities of Prussian carp (Carassius gibelio) were observed in a recreational freshwater lake and open freshwater in the western part of the Netherlands. Cyprinid herpesvirus 2 (CyHV-2) infection was suspected in these cases, based on presumptive gross diagnosis. To elucidate the cause of the mass mortalities diagnostic PCR assays were performed for CyHV-2, based on the helicase gene. Furthermore, the viral isolates were genotyped by sequencing the enlarged marker A and marker B sequences. Diagnostic PCR revealed that three of four samples were positive for CyHV-2, indicating these three mass mortalities were associated with CyHV-2 infection. The marker A sequence from one of the isolates found in this study was identical to those from different locations such as Asia and Middle East, suggesting a link among the isolates. This is the first detailed report on mass mortalities of Prussian carp associated with CyHV-2 infection in natural aquatic environments in the Netherlands. Since 2015, additionally, in total three CyHV-2 associated outbreaks of Dutch Prussian carp were seen in 2016 and 2020. These outbreaks in Prussian carp from lakes and open water suggest that the virus has been spreading in natural freshwaters in the Netherlands.

Molecular Characterization of Serratia marcescens Strain Isolated from Yellow Mealworms, Tenebrio molitor, in The Netherlands.

Insect culture has developed rapidly worldwide; it faces important security and safety control issues, including animal infections and disease development. In the Netherlands, in 2021, a ~30% mortality of mealworms, Tenebrio molitor, occurred at one farm, where over-humid sites in the substrate were observed. Bacterial cultures from both the external and internal partsof fry and larger mealworms were identified by MALDI-TOF to predominantly Serratia marcescens, Staphylococcus xylosus and Staphylococus saprofyticus. Due to the important role of S. marcescens as a potential zoonotic bacterium, we performed a molecular characterization of the isolated strain. Genomic analysis showed a multidrug-resistant S. marcescens isolate carrying a tet (41), aac (6')-Ic, and blaSST-1 chromosomal class C beta-lactamase-resistantgenes, all located on the chromosome. Additionally, several virulence genes were identified. The phylogenetic tree revealed that the S. marcescens strain from this study was similar to other S. marcescens strains from different ecological niches. Although the entomopathogenic activity was not confirmed, this case demonstrates that T. molitor can act as a reservoir and as an alternative path for exposing clinically important antibiotic-resistant bacteria that can affect animals and humans. It underlines the need to keep management factors optimal, before insects and their products enter the feed and food chain.

Epidemiological cut-off values for Vibrio anguillarum MIC and disc diffusion data generated by standardised methods.

This work aims to generate the data needed to set epidemiological cut-off values for minimum inhibitory concentration (MIC) and disc-diffusion zone measurements of Vibrio anguillarum. A total of 261 unique isolates were tested, applying standard methods specifying incubation at 28°C for 24-28 h. Aggregated MIC distributions for a total of 247 isolates were determined in 9 laboratories for 11 agents. Data aggregations of the disc zone for the 10 agents analysed contained between 157 and 218 observations made by 4 to 7 laboratories. Acceptable ranges for quality control (QC) reference strains were available for 7 agents and the related multi-laboratory aggregated data were censored, excluding the data of a laboratory that failed to meet QC requirements. Statistical methods were applied to calculate epidemiological cut-off values. Cut-off values for MIC data were calculated for florfenicol (≤1 µg ml-1), gentamicin (≤4 µg ml-1), oxytetracycline (≤0.25 µg ml-1) and trimethoprim/sulfamethoxazole (≤0.125/2.38 µg ml-1). The cut-off values for disc zone data were calculated for enrofloxacin (≥29 mm), florfenicol (≥27 mm), gentamicin (≥19 mm), oxolinic acid (≥24 mm), oxytetracycline (≥24 mm) and trimethoprim/sulfamethoxazole (≥26 mm). MIC and disc-diffusion zone data for the other agents where not supported by QC, thus yielding only provisional cut-off values (meropenem, ceftazidime). Regardless of whether QC is available, some of the aggregated MIC distributions (enrofloxacin, oxolinic acid), disc zone (sulfamethoxazole), and MIC and disc-diffusion distributions (ampicillin, chloramphenicol) did not meet the statistical requirements. The data produced will be submitted to the Clinical Laboratory Standards Institute for their consideration in setting international consensus epidemiological cut-off values.

Fast and accurate identification by MALDI-TOF of the zoonotic serovar E of Vibrio vulnificus linked to eel culture.

Vibrio vulnificus is a zoonotic pathogen that can cause death by septicaemia in farmed fish (mainly eels) and humans. The zoonotic strains that have been isolated from diseased eels and humans after eel handling belong to clade E (or serovar E (SerE)), a clonal complex within the pathovar (pv.) piscis. The aim of this study was to evaluate the accuracy of MALDI-TOF mass spectrometry (MS) in the identification of SerE, using the other two main pv. piscis-serovars (SerA and SerI) from eels as controls. MALDI-TOF data were compared with known serologic and genetic data of five pv. piscis isolates or strains, and with the non pv. piscis reference strain. Based on multiple spectra analysis, we found serovar-specific peaks that were of ~3098 Da and ~ 4045 Da for SerE, of ~3085 Da and ~ 4037 Da for SerA, and of ~3085 Da and ~ 4044 Da for SerI. Therefore, our results demonstrate that MALDI-TOF can be used to identify SerE and could also help in the identification of the other serovars of the species. This means that zoonosis due to V. vulnificus could be prevented by using MALDI-TOF, as action can be taken immediately after the isolation of a possible zoonotic V. vulnificus strain.

Biogeography of the fish pathogen Aeromonas salmonicida inferred by vapA genotyping.

A recently described typing system based on sequence variation in the virulence array protein (vapA) gene, encoding the A-layer surface protein array, allows unambiguous subtyping of Aeromonas salmonicida. In the present study, we compile A-layer typing results from a total of 675 A. salmonicida isolates, recovered over a 59-year period from 50 different fish species in 26 countries. Nine novel A-layer types (15-23) are identified, several of which display a strong predilection towards certain fish hosts, including e.g. Cyprinidae and Pleuronectidae species. Moreover, we find indications that anthropogenic transport of live fish may have aided the near global dissemination of two cyprinid-associated A-layer types. Comparison of whole genome phylogeny and A-layer typing for a subset of strains further resulted in compatible tree topologies, indicating the utility of vapA as a phylogenetic as well as an epizootiological marker in A. salmonicida. A Microreact project (microreact.org/project/r1pcOAx9m) has been created, allowing public access to the vapA analyses and relevant metadata. In sum, the results generated provide valuable insights into the global population structure of A. salmonicida, particularly in relation to its piscine host spectrum and the geographic distribution of these hosts.

Ranavirus genotypes in the Netherlands and their potential association with virulence in water frogs (Pelophylax spp.).

Ranaviruses are pathogenic viruses for poikilothermic vertebrates worldwide. The identification of a common midwife toad virus (CMTV) associated with massive die-offs in water frogs (Pelophylax spp.) in the Netherlands has increased awareness for emerging viruses in amphibians in the country. Complete genome sequencing of 13 ranavirus isolates collected from ten different sites in the period 2011-2016 revealed three CMTV groups present in distinct geographical areas in the Netherlands. Phylogenetic analysis showed that emerging viruses from the northern part of the Netherlands belonged to CMTV-NL group I. Group II and III viruses were derived from the animals located in the center-east and south of the country, and shared a more recent common ancestor to CMTV-amphibian associated ranaviruses reported in China, Italy, Denmark, and Switzerland. Field monitoring revealed differences in water frog host abundance at sites where distinct ranavirus groups occur; with ranavirus-associated deaths, host counts decreasing progressively, and few juveniles found in the north where CMTV-NL group I occurs but not in the south with CMTV-NL group III. Investigation of tandem repeats of coding genes gave no conclusive information about phylo-geographical clustering, while genetic analysis of the genomes revealed truncations in 17 genes across CMTV-NL groups II and III compared to group I. Further studies are needed to elucidate the contribution of these genes as well as environmental variables to explain the observed differences in host abundance.

Importation of CyHV-2-infected goldfish into the Netherlands.

Cyprinid herpesvirus 2 (CyHV-2) is known as the causative agent of herpesviral haematopoietic necrosis in goldfish Carassius auratus auratus. However, the virus has also been detected in Prussian carp C. gibelio and crucian carp C. carassius from European and Asian countries. To prevent spread of the causative virus to other areas, investigation of the risk factors of spread of this virus is important. In this study, 8 batches of goldfish imported into the Netherlands by airfreight from Asia and the Middle East were investigated for the presence of the virus. CyHV-2 DNA was detected by PCR in the pooled kidneys of 4 of the 8 imported goldfish batches, of which 1 was from a CyHV-2 disease case at a Dutch importer's quarantine facility. Sequence analysis of the CyHV-2 strains from this study and from previous reports showed that there were at least 6 different lengths in the mA region, resulting in tentatively at least 4 genotypes. Virus isolation was positive for only 1 (Amsterdam Schiphol-1 [AMS-1]) of the 8 samples. It was shown that the AMS-1 isolate was highly virulent to Ryukin goldfish after 100.3 TCID50 fish-1 intraperitoneal injection. The viral titre of the AMS-1 isolate for goldfish fin cells at several temperatures was similar to that of a Japanese CyHV-2 isolate. Our results prove that one of the routes of spread of various CyHV-2 strains is through the global trade of apparently healthy infected goldfish.

Complete Genome Sequence of Frog virus 3, Isolated from a Strawberry Poison Frog (Oophaga pumilio) Imported from Nicaragua into the Netherlands.

Frog virus 3 was isolated from a strawberry poison frog (Oophaga pumilio) imported from Nicaragua via Germany to the Netherlands, and its complete genome sequence was determined. Frog virus 3 isolate Op/2015/Netherlands/UU3150324001 is 107,183 bp long and has a nucleotide similarity of 98.26% to the reference Frog virus 3 isolate.

Phylogeny of the infectious hematopoietic necrosis virus in European aquaculture.

Infectious hematopoietic necrosis (IHN)-a highly lethal infectious salmonid disease-has caused substantial economic losses in the European production of rainbow trout (Oncorhynchus mykiss) since the late 1980s. The causal agent of IHN is the IHN virus (IHNV) introduced from overseas. However, until today, its phylogeographic spread in Europe remains poorly understood. We therefore sought to elucidate this unresolved topic by using the largest ever compiled dataset of European IHNV isolates (E isolates) (193 GenBank E isolates and 100 isolates from this study) for the complete glycoprotein (G) gene sequence. Our results clearly revealed that the active trout trade has left its traces in the E phylogeny. For example, the spread by trade of IHNV-infected trout was apparently the cause for the exposure of the E lineage to different local scenarios of selection and genetic drift, and therefore has led to the split of this lineage into various subordinated lineages. Accordingly, we also found evidence for E isolates being mixed Europe-wide by cross-border introduction events. Moreover, there were indications that this propagation of the E lineage within Europe corresponded with an extensive and rapid spread event, already during or shortly after its formation. Finally, in accordance with the high substitution rate of IHNV determined by previous studies, our dataset indicates that the mean period of occurrence of a single E haplotype is typically not longer than one calendar year.

Chromosome-Based blaOXA-48-Like Variants in Shewanella Species Isolates from Food-Producing Animals, Fish, and the Aquatic Environment.

Carbapenems are considered last-resort antibiotics in health care. Increasing reports of carbapenemase-producing bacteria in food-producing animals and in the environment indicate the importance of this phenomenon in public health. Surveillance for carbapenemase genes and carbapenemase-producing bacteria in Dutch food-producing animals, environmental freshwater, and imported ornamental fish revealed several chromosome-based blaOXA-48-like variants in Shewanella spp., including two new alleles, blaOXA-514 and blaOXA-515 Carbapenemase genes were not associated with mobile genetic elements or Enterobacteriaceae.

Phylogeny of the Viral Hemorrhagic Septicemia Virus in European Aquaculture.

One of the most valuable aquaculture fish in Europe is the rainbow trout, Oncorhynchus mykiss, but the profitability of trout production is threatened by a highly lethal infectious disease, viral hemorrhagic septicemia (VHS), caused by the VHS virus (VHSV). For the past few decades, the subgenogroup Ia of VHSV has been the main cause of VHS outbreaks in European freshwater-farmed rainbow trout. Little is currently known, however, about the phylogenetic radiation of this Ia lineage into subordinate Ia clades and their subsequent geographical spread routes. We investigated this topic using the largest Ia-isolate dataset ever compiled, comprising 651 complete G gene sequences: 209 GenBank Ia isolates and 442 Ia isolates from this study. The sequences come from 11 European countries and cover the period 1971-2015. Based on this dataset, we documented the extensive spread of the Ia population and the strong mixing of Ia isolates, assumed to be the result of the Europe-wide trout trade. For example, the Ia lineage underwent a radiation into nine Ia clades, most of which are difficult to allocate to a specific geographic distribution. Furthermore, we found indications for two rapid, large-scale population growth events, and identified three polytomies among the Ia clades, both of which possibly indicate a rapid radiation. However, only about 4% of Ia haplotypes (out of 398) occur in more than one European country. This apparently conflicting finding regarding the Europe-wide spread and mixing of Ia isolates can be explained by the high mutation rate of VHSV. Accordingly, the mean period of occurrence of a single Ia haplotype was less than a full year, and we found a substitution rate of up to 7.813 × 10-4 nucleotides per site per year. Finally, we documented significant differences between Germany and Denmark regarding their VHS epidemiology, apparently due to those countries' individual handling of VHS.

Complete genome sequence of a common midwife toad virus-like ranavirus associated with mass mortalities in wild amphibians in the Netherlands.

A ranavirus associated with mass mortalities in wild water frogs (Pelophylax spp.) and other amphibians in the Netherlands since 2010 was isolated, and its complete genome sequence was determined. The virus has a genome of 107,772 bp and shows 96.5% sequence identity with the common midwife toad virus from Spain.

Evolutionary dynamics and genetic diversity from three genes of Anguillid rhabdovirus.

Wild freshwater eel populations have dramatically declined in recent past decades in Europe and America, partially through the impact of several factors including the wide spread of infectious diseases. The anguillid rhabdoviruses eel virus European X (EVEX) and eel virus American (EVA) potentially play a role in this decline, even if their real contribution is still unclear. In this study, we investigate the evolutionary dynamics and genetic diversity of anguiillid rhabdoviruses by analysing sequences from the glycoprotein, nucleoprotein and phosphoprotein (P) genes of 57 viral strains collected from seven countries over 40 years using maximum-likelihood and Bayesian approaches. Phylogenetic trees from the three genes are congruent and allow two monophyletic groups, European and American, to be clearly distinguished. Results of nucleotide substitution rates per site per year indicate that the P gene is expected to evolve most rapidly. The nucleotide diversity observed is low (2-3 %) for the three genes, with a significantly higher variability within the P gene, which encodes multiple proteins from a single genomic RNA sequence, particularly a small C protein. This putative C protein is a potential molecular marker suitable for characterization of distinct genotypes within anguillid rhabdoviruses. This study provides, to our knowledge, the first molecular characterization of EVA, brings new insights to the evolutionary dynamics of two genotypes of Anguillid rhabdovirus, and is a baseline for further investigations on the tracking of its spread.

Thermal preference of juvenile Dover sole (Solea solea) in relation to thermal acclimation and optimal growth temperature.

Dover sole (Solea solea) is an obligate ectotherm with a natural thermal habitat ranging from approximately 5 to 27°C. Thermal optima for growth lie in the range of 20 to 25°C. More precise information on thermal optima for growth is needed for cost-effective Dover sole aquaculture. The main objective of this study was to determine the optimal growth temperature of juvenile Dover sole (Solea solea) and in addition to test the hypothesis that the final preferendum equals the optimal growth temperature. Temperature preference was measured in a circular preference chamber for Dover sole acclimated to 18, 22 and 28°C. Optimal growth temperature was measured by rearing Dover sole at 19, 22, 25 and 28°C. The optimal growth temperature resulting from this growth experiment was 22.7°C for Dover sole with a size between 30 to 50 g. The temperature preferred by juvenile Dover sole increases with acclimation temperature and exceeds the optimal temperature for growth. A final preferendum could not be detected. Although a confounding effect of behavioural fever on temperature preference could not be entirely excluded, thermal preference and thermal optima for physiological processes seem to be unrelated in Dover sole.

Viral diseases of wild and farmed European eel Anguilla anguilla with particular reference to the Netherlands.

Diseases are an important cause of losses and decreased production rates in freshwater eel farming, and have been suggested to play a contributory role in the worldwide decline in wild freshwater eel stocks. Three commonly detected pathogenic viruses of European eel Anguilla anguilla are the aquabirnavirus eel virus European (EVE), the rhabdovirus eel virus European X (EVEX), and the alloherpesvirus anguillid herpesvirus 1 (AngHV1). In general, all 3 viruses cause a nonspecific haemorrhagic disease with increased mortality rates. This review provides an overview of the current knowledge on the aetiology, prevalence, clinical signs and gross pathology of these 3 viruses. Reported experimental infections showed the temperature dependency and potential pathogenicity of these viruses for eels and other fish species. In addition to the published literature, an overview of the isolation of pathogenic viruses from wild and farmed A. anguilla in the Netherlands during the past 2 decades is given. A total of 249 wild A. anguilla, 39 batches of glass eels intended for farming purposes, and 239 batches of farmed European eels were necropsied and examined virologically. AngHV1 was isolated from wild yellow and silver A. anguilla from the Netherlands from 1998 until the present, while EVEX was only found sporadically, and EVE was never isolated. In farmed A. anguilla AngHV1 was also the most commonly isolated virus, followed by EVE and EVEX.

Identification and localization of the structural proteins of anguillid herpesvirus 1.

Many of the known fish herpesviruses have important aquaculture species as their natural host, and may cause serious disease and mortality. Anguillid herpesvirus 1 (AngHV-1) causes a hemorrhagic disease in European eel, Anguilla anguilla. Despite their importance, fundamental molecular knowledge on fish herpesviruses is still limited. In this study we describe the identification and localization of the structural proteins of AngHV-1. Purified virions were fractionated into a capsid-tegument and an envelope fraction, and premature capsids were isolated from infected cells. Proteins were extracted by different methods and identified by mass spectrometry. A total of 40 structural proteins were identified, of which 7 could be assigned to the capsid, 11 to the envelope, and 22 to the tegument. The identification and localization of these proteins allowed functional predictions. Our findings include the identification of the putative capsid triplex protein 1, the predominant tegument protein, and the major antigenic envelope proteins. Eighteen of the 40 AngHV-1 structural proteins had sequence homologues in related Cyprinid herpesvirus 3 (CyHV-3). Conservation of fish herpesvirus structural genes seemed to be high for the capsid proteins, limited for the tegument proteins, and low for the envelope proteins. The identification and localization of the structural proteins of AngHV-1 in this study adds to the fundamental knowledge of members of the Alloherpesviridae family, especially of the Cyprinivirus genus.

The alloherpesviral counterparts of interleukin 10 in European eel and common carp.

Viral interleukin 10 (IL-10) like open reading frames have been identified in several pox- and herpesviruses, including the fish herpesviruses Anguillid herpesvirus 1 (AngHV-1) and Cyprinid herpesvirus 3 (CyHV-3). European eel (Anguilla anguilla) IL-10 was sequenced, in order to compare European eel and common carp (Cyprinus carpio) IL-10 with their alloherpesviral counterparts. Homology between the virus and host IL-10 amino acid sequences is low, which is confirmed by phylogenetic analysis. However, the three dimensional structures of the fish and alloherpesviral IL-10 proteins as predicted by modeling are highly similar to human IL-10. Closely related AngHV-1 and CyHV-3 are expected to have obtained their viral IL-10 genes independently in the course of coexistence with their respective hosts. The presence and structural conservation of these alloherpesviral IL-10 genes suggest that they might play an important role in the evolution of pathogenesis.

[Zoonotic bacterium: duty to notify advisable]. / Zoönose Vibrio vulnificus: meldingsplicht raadzaam.

Vibrio vulnificus is a zoonotic bacterium that can be found in raw fish (mainly eel and oysters) and seawater. Human infections may extend from wound infections to fasciitis necroticans or primary sepsis with a mortality rate of more than 50%. Although V. vulnificus is mainly found in the USA, its worldwide spread is also likely to involve the Netherlands, as demonstrated by an increasing number of infected fish farms. Since 2007, V. vulnificus infections have become a notifiable infectious disease in the USA. Due to the high mortality rate and an increase in the number of elderly people with known risk factors for infection, we argue that human V. vulnificus infections should become a notifiable infectious disease in the Netherlands as well. This would provide reliable information on the epidemiology and facilitate correct risk assessment for public health.

Development and validation of a two-step real-time RT-PCR for the detection of eel virus European X in European eel, Anguilla anguilla.

Eel virus European X (EVEX) is one of the most common pathogenic viruses in farmed and wild European eel (Anguilla anguilla) in the Netherlands. The virus causes a hemorrhagic disease resulting in increased mortality rates. Cell culture and antibody-based detection of EVEX are laborious and time consuming. Therefore, a two-step real-time reverse transcriptase (RT-)PCR assay was developed for rapid detection of EVEX. Primers and probe for the assay were designed based on a sequence of the RNA polymerase or L gene of EVEX. The real-time RT-PCR assay was validated both for use with SYBR Green chemistry and for use with a TaqMan probe. The assay is sensitive, specific, repeatable, efficient and has a high r²-value. The real-time RT-PCR assay was further evaluated by testing field samples of European eels from the Netherlands, which were positive or negative for EVEX by virus isolation followed by an indirect fluorescent antibody test. The real-time RT-PCR assay allows rapid, sensitive and specific laboratory detection of EVEX in RNA extracts from 10% eel organ suspensions and cell cultures with cytopathic effects, and is a valuable contribution to the diagnosis of viral diseases of eel.