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.</p

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

AbstractEel 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

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

Phylogeny of the Viral Hemorrhagic Septicemia Virus in European Aquaculture

<p>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.</p

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

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

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.</p

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

Observations on recent mass mortality events of marine mussels in the Oosterschelde, the Netherlands

Two mass mortality events (MMEs) of marine mussels that took place in the Oosterschelde, the Netherlands—the first in 2015/2016 and the second in 2019—both severely affected mussel production. The current study presents our observations on the onset and course of both MMEs and discusses probable putative causes. The two MMEs displayed a distinct course of events. The first event started in November 2015 with high mortality rates on culture plots, which remained elevated until the autumn of 2016. Approximately 40–50% of mussels from all age classes were lost on culture plots and 100% were lost from wild seed beds. The second event started in April–May 2019 and continued until the end of July, with mortality ranging from 20 to 100%, again from all age classes. Culture areas other than the Oosterschelde and other shellfish species were not affected. Histological and bacteriological screening produced no evidence for common pathogens or pollution as a primary mortality factor and there is no indication of abnormal environmental conditions preceding or during the events. We hypothesize that a cumulation of stressors results in weakening of the mussels and in elevated mortality rates. In 2019, this cumulation of stressors could be high spawning activities (an unusual high concentration of mussel larvae was found in April) that resulted in very low condition from April to June, a Phaeocystis bloom in April to May that prevented a quick recovery, and the development of granulocytomas that were found in up to 60 to 70% of live mussels as a consequence of cumulative stress. Although no (single) putative causes could be identified, this study contributes to the knowledge on MMEs in mussels and fits in a wider and disturbing trend on mortality events in shellfish

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.</p