Emergence of Bluetongue Virus Serotype 3, the Netherlands, September 2023.

Since 1998, notifiable bluetongue virus (BTV) serotypes 1-4, 6, 8, 9, 11, and 16 have been reported in Europe. In August 2006, a bluetongue (BT) outbreak caused by BTV serotype 8 began in northwestern Europe. The Netherlands was declared BT-free in February 2012, and annual monitoring continued. On September 3, 2023, typical BT clinical manifestations in sheep were notified to the Netherlands Food and Product Safety Consumer Authority. On September 6, we confirmed BTV infection through laboratory diagnosis; notifications of clinical signs in cattle were also reported. We determined the virus was serotype 3 by whole-genome sequencing. Retrospective analysis did not reveal BTV circulation earlier than September. The virus source and introduction route into the Netherlands remains unknown. Continuous monitoring and molecular diagnostic testing of livestock will be needed to determine virus spread, and new prevention strategies will be required to prevent BTV circulation within the Netherlands and Europe.

H5N1 clade 2.3.4.4b avian influenza viruses replicate in differentiated bovine airway epithelial cells cultured at air-liquid interface.

Highly pathogenic avian influenza (HPAI) H5N1 viruses are responsible for disease outbreaks in wild birds and poultry, resulting in devastating losses to the poultry sector. Since 2020, an increasing number of outbreaks of HPAI H5N1 was seen in wild birds. Infections in mammals have become more common, in most cases in carnivores after direct contact with infected birds. Although ruminants were previously not considered a host species for HPAI viruses, in March 2024 multiple outbreaks of HPAI H5N1 were detected in goats and cattle in the United States. Here, we have used primary bronchus-derived well-differentiated bovine airway epithelial cells (WD-AECs) cultured at air-liquid interface to assess the susceptibility and permissiveness of bovine epithelial cells to infection with European H5N1 virus isolates. We inoculated bovine WD-AECs with three low-passage HPAI clade 2.3.4.4b H5N1 virus isolates and detected rapid increases in viral genome loads and infectious virus during the first 24 h post-inoculation, without substantial cytopathogenic effects. Three days post-inoculation infected cells were still detectable by immunofluorescent staining. These data indicate that multiple lineages of HPAI H5N1 may have the propensity to infect the respiratory tract of cattle and support extension of avian influenza surveillance efforts to ruminants. Furthermore, this study underscores the benefit of WD-AEC cultures for pandemic preparedness by providing a rapid and animal-free assessment of the host range of an emerging pathogen.

Mass Mortality Caused by Highly Pathogenic Influenza A(H5N1) Virus in Sandwich Terns, the Netherlands, 2022.

We collected data on mass mortality in Sandwich terns (Thalasseus sandvicensis) during the 2022 breeding season in the Netherlands. Mortality was associated with at least 2 variants of highly pathogenic avian influenza A(H5N1) virus clade 2.3.4.4b. We report on carcass removal efforts relative to survival in colonies. Mitigation strategies urgently require structured research.

Incursion of Novel Highly Pathogenic Avian Influenza A(H5N8) Virus, the Netherlands, October 2020.

Highly pathogenic avian influenza A(H5N8) virus was detected in mute swans in the Netherlands during October 2020. The virus shares a common ancestor with clade 2.3.4.4b viruses detected in Egypt during 2018-2019 and has similar genetic composition. The virus is not directly related to H5N8 viruses from Europe detected in the first half of 2020.

Highly Pathogenic Avian Influenza A(H5N1) Virus in Wild Red Foxes, the Netherlands, 2021.

We detected infection with highly pathogenic avian influenza A(H5N1) virus clade 2.3.4.4b in 2 red fox (Vulpes vulpes) cubs found in the wild with neurologic signs in the Netherlands. The virus is related to avian influenza viruses found in wild birds in the same area.

Transboundary Spread of Brucella canis through Import of Infected Dogs, the Netherlands, November 2016-December 2018.

Brucella canis had not been isolated in the Netherlands until November 2016, when it was isolated from a dog imported from Romania. Including this case, 16 suspected cases were notified to the authorities during the following 25 months. Of these 16 dogs, 10 were seropositive; tracking investigations found another 8 seropositive littermates. All seropositive animals were rescue dogs imported from Eastern Europe. B. canis was cultured from urine, blood, and other specimens collected from the dogs. Genotyping of isolates revealed clustering by litter and country. Isolating B. canis in urine indicates that shedding should be considered when assessing the risk for zoonotic transmission. This case series proves introduction of B. canis into a country to which it is not endemic through import of infected dogs from B. canis-endemic areas, posing a threat to the naive autochthonous dog population and humans.

Occupational and environmental exposure to SARS-CoV-2 in and around infected mink farms.

OBJECTIVE: Unprecedented SARS-CoV-2 infections in farmed minks raised immediate concerns regarding transmission to humans and initiated intensive environmental investigations to assess occupational and environmental exposure. METHODS: Air sampling was performed at infected Dutch mink farms, at farm premises and at nearby residential sites. A range of other environmental samples were collected from minks' housing units, including bedding materials. SARS-CoV-2 RNA was analysed in all samples by quantitative PCR. RESULTS: Inside the farms, considerable levels of SARS-CoV-2 RNA were found in airborne dust, especially in personal inhalable dust samples (approximately 1000-10 000 copies/m3). Most of the settling dust samples tested positive for SARS-CoV-2 RNA (82%, 75 of 92). SARS-CoV-2 RNA was not detected in outdoor air samples, except for those collected near the entrance of the most recently infected farm. Many samples of minks' housing units and surfaces contained SARS-CoV-2 RNA. CONCLUSIONS: Infected mink farms can be highly contaminated with SARS-CoV-2 RNA. This warns of occupational exposure, which was substantiated by considerable SARS-CoV-2 RNA concentrations in personal air samples. Dispersion of SARS-CoV-2 to outdoor air was found to be limited and SARS-CoV-2 RNA was not detected in air samples collected beyond farm premises, implying a negligible risk of environmental exposure to nearby communities. Our occupational and environmental risk assessment is in line with whole genome sequencing analyses showing mink-to-human transmission among farm workers, but no indications of direct zoonotic transmission events to nearby communities.

Predominance of Fourth Panzootic Newcastle Disease Virus Subgenotype VII.1.1 in Iran and Its Relation to the Genotypes Circulating in the Region.

Following recent Newcastle disease virus (NDV) outbreaks in Iranian poultry farms which were mostly associated with lesions of the avian gastrointestinal tract, it was speculated that the scale of the outbreaks could be attributed in part to co-circulating infectious agents or a new NDV genotype/subgenotype. This speculation was due to the isolation of a few 5th panzootic subgenotype VII.2 viruses from Iranian poultry farms in 2017. Samples from different species of commercial and domestic birds were collected from different provinces of Iran, 19 of which were selected for the current study. Phylogenetic analyses showed that the recent outbreaks have been caused by only one agent, i.e. the distinctive NDV subgenotype VII.1.1 (previously known VIIl) viruses that seem to be circulating predominantly in Iran, but have also been sporadically reported from Iraq among neighbouring countries. At most, 96.3-96.7% BLAST identity to non-Iranian VII.1.1 isolates was observed. Genetic distance values of <1% were indicative of high similarity between the isolates, but the values were approaximately 2% when the current isolates were compared to the earliest recorded Iranian VII.1.1 viruses isolated in 2010. Using Bayesian analysis, annual mutation rates of 1.7156E-3 (strict) and 1.9902E-3 (relaxed) over 11 years were obtained. In addition, we report that our laboratories have not detected any genotype XIII strains since 2011. Following up on previous reports, we concluded that currently, and except in Columbiforms, subgenotype VII.1.1 may likely be the predominant subgenotype in many bird species in Iran despite the subgenotype VII.2 being predominant in neighbouring countries.

High prevalences of disseminated neoplasia in the Baltic tellin Limecola balthica in the Wadden Sea.

The Baltic tellin Limecola balthica is one of the most common bivalves in intertidal areas in the Northern Hemisphere. Over the last 2 decades, the species has been suffering from a decrease in adult survival in the European Wadden Sea. While several factors such as global warming and fisheries have been suggested to influence the population dynamics of this bivalve mollusc, the potential role of diseases has never been investigated. In this study, we investigated whether disseminated neoplasia, a common proliferative disorder in bivalve molluscs, could play a potential role in the recent population decline of Baltic tellins in the Wadden Sea. We conducted a field survey in the Dutch Wadden Sea to (1) determine whether the disease occurs in Baltic tellins in the Wadden Sea and (2) quantify the occurrence and severity of the disease via histology. Disseminated neoplasia occurred in L. balthica at each of the 10 sampled locations with very high prevalences (21-89%) compared to those reported elsewhere for this species. The highest severity category was found in 8 to 87% of affected individuals, with severity generally increasing with prevalence. Disseminated neoplasia usually increases mortality among affected individuals and may also be associated with important sub-lethal effects, especially regarding gametogenesis. Thus, we suggest that disseminated neoplasia may play a key role in the population dynamics of the Baltic tellin, the extent of which remains to be investigated in future studies.

SARS-CoV-2 infection in farmed minks, the Netherlands, April and May 2020.

Respiratory disease and increased mortality occurred in minks on two farms in the Netherlands, with interstitial pneumonia and SARS-CoV-2 RNA in organ and swab samples. On both farms, at least one worker had coronavirus disease-associated symptoms before the outbreak. Variations in mink-derived viral genomes showed between-mink transmission and no infection link between the farms. Inhalable dust contained viral RNA, indicating possible exposure of workers. One worker is assumed to have attracted the virus from mink.

Brucella suis Infection in Dog Fed Raw Meat, the Netherlands.

A Brucella suis biovar 1 infection was diagnosed in a dog without typical exposure risks, but the dog had been fed a raw meat-based diet (hare carcasses imported from Argentina). Track and trace investigations revealed that the most likely source of infection was the dog's raw meat diet.

Presence of Coxiella burnetii DNA in inflamed bovine cardiac valves.

BACKGROUND: Bacterial endocarditis is a recognised disease in humans and animals. In humans, infection with Coxiella burnetii can cause endocarditis, but this has not been investigated thoroughly in animals. Endocarditis in cattle is a common post-mortem finding in abattoirs and studies have identified Trueperella pyogenes as a major cause. Despite exposure of cattle to C. burnetii, the significance of this particular bacterium for development and progression of endocarditis has not been studied in detail. Cardiac valves of cattle affected with endocarditis (n = 100) were examined by histology, fluorescence in situ hybridization (FISH) and real time quantitative polymerase chain reaction (PCR). Serum was examined for anti-C. burnetii antibodies by enzyme-linked immunosorbent assay (ELISA). RESULTS: Serology revealed that 70% of the cattle were positive for antibodies to C. burnetii, while PCR analysis identified 25% of endocarditis valve samples as being positive. C. burnetii was not detected by FISH, probably due to the low infection levels. Most cattle had chronic valvular vegetative endocarditis with lesions being characterised by a core of fibrous tissue covered by significant amounts of fibrin, sometimes with areas of liquefaction, and with a coagulum covering the surface. In a few cases, including the case with the highest infection level, lesions were characterized by extensive fibrosis and calcification. Histologically, bacteria other than C. burnetii were observed in most cases. CONCLUSIONS: The presence of C. burnetii DNA is relatively common in cattle affected with valvular endocarditis. The role of C. burnetii remains however unknown as lesions did not differ between C. burnetii infected and non-infected cattle and because T. pyogenes-like bacteria were present in the inflamed valves; a bacterium able to induce the observed lesions. Heart valves of normal cattle should be investigated to assess if C. burnetii may be present without preexisting lesions.

Application of a competitive real time PCR for detection of Marteilia refringens genotype "O" and "M" in two geographical locations: The Ebro Delta, Spain and the Rhine-Meuse Delta, the Netherlands.

Species belonging to the genus Marteilia are protozoan parasites of bivalves. The species Marteilia refringens, jeopardizing the health of European bivalves, is included on the list of OIE notifiable pathogens. Two genotypes of Marteilia refringens are distinguished: type "O" affecting mainly oysters, and type "M" affecting mainly mussels. Historically, detection of Marteilia species is primarily carried out by histology. In recent years molecular assays are more frequently used for the detection of mollusc pathogens, also in routine monitoring. In the present work, a competitive real-time PCR assay was developed for rapid and sensitive detection of M. refringens and discrimination between "M" and "O" genotypes of M. refringens. The real-time PCR assay was shown to be analytically sensitive and specific and has a high repeatability and efficiency. Subsequent application of the assay on collected bivalves from two geographical locations, the Ebro Delta in Mediterranean Spain and the Rhine-Meuse Delta in the Netherlands resulted in detection of M. refringens type M in Mytilus galloprovincialis and M. refringens type O in Ostrea edulis from Spain. In two O. edulis specimen both M. refringens type O and type M were detected. In the Netherlands M. refringens was not observed in any of the tested Mytilus edulis and O. edulis. The results obtained by real time PCR were in correspondence with the results obtained by histopathology and a substantial agreement with the results obtained by conventional PCR. In conclusion, the developed real time PCR assay facilitates rapid detection and subtyping of M. refringens and could be applied for further studies on epidemiology of the parasite, geographical distribution and host specificity.

Environmental surveillance during an outbreak of tularaemia in hares, the Netherlands, 2015.

Tularaemia, a disease caused by the bacterium Francisella tularensis, is a re-emerging zoonosis in the Netherlands. After sporadic human and hare cases occurred in the period 2011 to 2014, a cluster of F. tularensis-infected hares was recognised in a region in the north of the Netherlands from February to May 2015. No human cases were identified, including after active case finding. Presence of F. tularensis was investigated in potential reservoirs and transmission routes, including common voles, arthropod vectors and surface waters. F. tularensis was not detected in common voles, mosquito larvae or adults, tabanids or ticks. However, the bacterium was detected in water and sediment samples collected in a limited geographical area where infected hares had also been found. These results demonstrate that water monitoring could provide valuable information regarding F. tularensis spread and persistence, and should be used in addition to disease surveillance in wildlife.

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.

Microcell parasites of molluscs: introduction to DAO Special 7.

First discovered decades ago, microcell protistan parasites of the genera Bonamia and Mikrocytos remain relevant today for their economic impacts on growing molluscan aquaculture industries and fisheries. Bonamia parasites have received more attention over the years in part because they are more widespread and thus of wider concern, but there has been renewed interest in Mikrocytos recently with the generation of important new findings. Among these has been the surprising observation that Mikrocytos has phylogenetic affinities to the Rhizaria, which includes the haplosporidian protists and the genus Bonamia. This Diseases of Aquatic Organisms Special, emerging from the 5th Meeting of the Microcell Working Group held at the Central Veterinary Institute, Lelystad, the Netherlands, in February 2012, presents new insights into Mikrocytos and Bonamia diversity, distributions, diagnostics, ultrastructure, and infection dynamics, and captures major developments in the field since the last review of these genera in 2004.

Bonamia parasites: a rapidly changing perspective on a genus of important mollusc pathogens.

Organisms of the genus Bonamia are intracellular protistan parasites of oysters. To date, 4 species have been described (B. ostreae, B. exitiosa, B. perspora and B. roughleyi), although the status of B. roughleyi is controversial. Introduction especially of B. ostreae and B. exitiosa to naïve host populations has been shown to cause mass mortalities in the past and has had a dramatic impact on oyster production. Both B. ostreae and B. exitiosa are pathogens notifiable to the World Organisation for Animal Health (OIE) and the European Union. Effective management of the disease caused by these pathogens is complicated by the extensive nature of the oyster production process and limited options for disease control of the cultured stocks in open water. This review focuses on the recent advances in research on genetic relationships between Bonamia isolates, geographical distribution, susceptible host species, diagnostics, epizootiology, host-parasite interactions, and disease resistance and control of this globally important genus of oyster pathogens.

Genome-wide gene expression analysis of anguillid herpesvirus 1.

BACKGROUND: Whereas temporal gene expression in mammalian herpesviruses has been studied extensively, little is known about gene expression in fish herpesviruses. Here we report a genome-wide transcription analysis of a fish herpesvirus, anguillid herpesvirus 1, in cell culture, studied during the first 6 hours of infection using reverse transcription quantitative PCR. RESULTS: Four immediate-early genes - open reading frames 1, 6A, 127 and 131 - were identified on the basis of expression in the presence of a protein synthesis inhibitor and unique expression profiles during infection in the absence of inhibitor. All of these genes are located within or near the terminal direct repeats. The remaining 122 open reading frames were clustered into groups on the basis of transcription profiles during infection. Expression of these genes was also studied in the presence of a viral DNA polymerase inhibitor, enabling classification into early, early-late and late genes. In general, clustering by expression profile and classification by inhibitor studies corresponded well. Most early genes encode enzymes and proteins involved in DNA replication, most late genes encode structural proteins, and early-late genes encode non-structural as well as structural proteins. CONCLUSIONS: Overall, anguillid herpesvirus 1 gene expression was shown to be regulated in a temporal fashion, comparable to that of mammalian herpesviruses.

Anguillid herpesvirus 1 transcriptome.

We used deep sequencing of poly(A) RNA to characterize the transcriptome of an economically important eel virus, anguillid herpesvirus 1 (AngHV1), at a stage during the lytic life cycle when infectious virus was being produced. In contrast to the transcription of mammalian herpesviruses, the overall level of antisense transcription from the 248,526-bp genome was low, amounting to only 1.5% of transcription in predicted protein-coding regions, and no abundant, nonoverlapping, noncoding RNAs were identified. RNA splicing was found to be more common than had been anticipated previously. Counting the 10,634-bp terminal direct repeat once, 100 splice junctions were identified, of which 58 were considered likely to be involved in the expression of functional proteins because they represent splicing between protein-coding exons or between 5' untranslated regions and protein-coding exons. Each of the 30 most highly represented of these 58 splice junctions was confirmed by RT-PCR. We also used deep sequencing to identify numerous putative 5' and 3' ends of AngHV1 transcripts, confirming some and adding others by rapid amplification of cDNA ends (RACE). The findings prompted a revision of the AngHV1 genome map to include a total of 129 protein-coding genes, 5 of which are duplicated in the terminal direct repeat. Not counting duplicates, 11 genes contain integral, spliced protein-coding exons, and 9 contain 5' untranslated exons or, because of alternative splicing, 5' untranslated and 5' translated exons. The results of this study sharpen our understanding of AngHV1 genomics and provide the first detailed view of a fish herpesvirus transcriptome.