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.

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.

Detection of Low Pathogenic Avian Influenza Virus Subtype H10N7 in Poultry and Environmental Water Samples During a Clinical Outbreak in Commercial Free-Range Layers, Netherlands 2017.

Wild birds are the natural reservoir of the avian influenza virus (AIV) and may transmit AIV to poultry via direct contact or indirectly through the environment. In the Netherlands, a clinically suspected free-range layer flock was reported to the veterinary authorities by the farmer. Increased mortality, a decreased feed intake, and a drop in egg production were observed. Subsequently, an infection with low pathogenic avian influenza virus was detected. This study describes the diagnostic procedures used for detection and subtyping of the virus. In addition to routine diagnostics, the potential of two different environmental diagnostic methods was investigated for detecting AIV in surface water. AIV was first detected using rRT-PCR and isolated from tracheal and cloacal swabs collected from the hens. The virus was subtyped as H10N7. Antibodies against the virus were detected in 28 of the 31 sera tested. An intravenous pathogenicity index (IVPI) experiment was performed, but no clinical signs (IVPI = 0) were observed. Post-mortem examination and histology confirmed the AIV infection. Multiple water samples were collected longitudinally from the free-range area and waterway near the farm. Both environmental diagnostic methods allowed the detection of the H10N7 virus, demonstrating the potential of these methods in detection of AIV. The described methods could be a useful additional procedure for AIV surveillance in water-rich areas with large concentrations of wild birds or in areas around poultry farms. In addition, these methods could be used as a tool to test if the environment or free-range area is virus-free again, at the end of an AIV epidemic.

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.

Susceptibility of Chickens to Low Pathogenic Avian Influenza (LPAI) Viruses of Wild Bird- and Poultry-Associated Subtypes.

Analysis of low pathogenic avian influenza (LPAI) viruses circulating in the Netherlands in a previous study revealed associations of specific hemagglutinin (HA) and neuraminidase (NA) subtypes with wild bird or poultry hosts. In this study, we identified putative host associations in LPAI virus internal proteins. We show that LPAI viruses isolated from poultry more frequently carried the allele A variant of the nonstructural protein (NS) gene, compared to wild bird viruses. We determined the susceptibility of chickens to wild bird-associated subtypes H3N8 and H4N6 and poultry-associated subtypes H8N4 and H9N2, carrying either NS allele A or B, in an infection experiment. We observed variations in virus shedding and replication patterns, however, these did not correlate with the predicted wild bird- or poultry-associations of the viruses. The experiment demonstrated that LPAI viruses of wild bird-associated subtypes can replicate in chickens after experimental infection, despite their infrequent detection in poultry. Although the NS1 protein is known to play a role in immune modulation, no differences were detected in the limited innate immune response to LPAI virus infection. This study contributes to a better understanding of the infection dynamics of LPAI viruses in chickens.

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.

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.

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.

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.

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.

Detection of novel strains of cyprinid herpesvirus closely related to koi herpesvirus.

Cyprinid herpesvirus 3 (CyHV-3) or koi herpesvirus (KHV) is a devastating virus of carp. Using generic primers for the DNA polymerase and the major capsid protein genes of cyprinid herpesviruses, nucleotide sequences divergent from previously described CyHV-3 were obtained. At least 3 novel groups of putative CyHV-3-like viruses were identified, sharing 95 to 98% nucleotide identity with CyHV-3 strains. Carp carrying the CyHV-3 variants did not show clinical signs consistent with CyHV-3 infection and originated from locations with no actual CyHV-3 outbreaks. These strains might represent low- or non-pathogenic variants of CyHV-3.

Development of a real-time PCR for detection of the oyster pathogen Nocardia crassostreae based on its homogeneous 16S-23S rRNA intergenic spacer region.

Nocardia crassostreae, the causative agent of Pacific oyster nocardiosis (PON), is a Gram-positive actinomycete bacterium associated with Pacific oyster (Crassostrea gigas) mortalities. Oysters infected with this bacterium have been reported previously from the west coast of North America and Japan. More recently, N. crassostreae was reported in oyster culture areas in the Netherlands. In this study, a sensitive real-time PCR for specific detection of N. crassostreae was developed, and the intra-species divergence of N. crassostreae from different geographical locations was studied. The 16S-23S rRNA intergenic spacer (ITS) region of N. crassostreae was sequenced for a number of infected oysters originating from the Netherlands, Japan and Canada. The sequence analyses showed an absence of genetic variation in the ITS region between N. crassostreae from different geographical locations. Based on these ITS sequences a species-specific and highly sensitive SYBR Green real-time PCR assay was developed to facilitate detection of N. crassostreae in oyster tissue. To evaluate this new detection tool for N. crassostreae a preliminary validation was carried out and real-time PCR results were compared with other detection methods (histology, conventional PCR and bacterial isolation) using field samples from Lake Grevelingen, the Netherlands. The genetic homogeneity in the ITS region between N. crassostreae from different geographical locations might be explained by the recent spread of the organism via the international trade in Pacific oysters for aquaculture purposes. However, the lack of genetic variation could also suggest that N. crassostreae is a genetically monomorphic species.

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.

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.

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.

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.

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.