Monitoring results of wild boar (Sus scrofa) in The Netherlands: analyses of serological results and the first identification of Brucella suis biovar 2.

In Europe, wild boar populations pose an increasing risk for livestock and humans due to the transmission of animal and zoonotic infectious diseases, such as African swine fever and brucellosis. Brucella suis is widespread among wild boar in many European countries. In The Netherlands the prevalence of B. suis among wild boar has not been investigated so far, despite the high number of pig farms and the growing wild boar population. The Netherlands has a Brucella-free status for the livestock species. The objective of this study is to investigate the presence and distribution of B. suis in wild boars in The Netherlands and to assess the value of the different laboratory tests available for testing wild boars. A total of 2057 sera and 180 tonsils of wild boar were collected between 2010 and 2015. The sera were tested for Brucella antibodies and the tonsils were tested for Brucella spp. B. suis biovar 2 was detected by MLVA/MLST and culture in wild boar from the province of Limburg, while seropositive wild boar were obtained from the provinces of Limburg, Noord Brabant and Gelderland suggesting the northwards spread of B. suis biovar 2. In this paper, we describe the first isolation of B. suis biovar 2 in wild boar in The Netherlands.

Phylogeographic Distribution of Human and Hare Francisella Tularensis Subsp. Holarctica Strains in the Netherlands and Its Pathology in European Brown Hares (Lepus Europaeus).

Sequence-based typing of Francisella tularensis has led to insights in the evolutionary developments of tularemia. In Europe, two major basal clades of F. tularensis subsp. holarctica exist, with a distinct geographical distribution. Basal clade B.6 is primarily found in Western Europe, while basal clade B.12 occurs predominantly in the central and eastern parts of Europe. There are indications that tularemia is geographically expanding and that strains from the two clades might differ in pathogenicity, with basal clade B.6 strains being potentially more virulent than basal clade B.12. This study provides information on genotypes detected in the Netherlands during 2011-2017. Data are presented for seven autochthonous human cases and for 29 European brown hares (Lepus europaeus) with laboratory confirmed tularemia. Associated disease patterns are described for 25 European brown hares which underwent post-mortem examination. The basal clades B.6 and B.12 are present both in humans and in European brown hares in the Netherlands, with a patchy geographical distribution. For both genotypes the main pathological findings in hares associated with tularemia were severe (sub)acute necrotizing hepatitis and splenitis as well as necrotizing lesions and hemorrhages in several other organs. Pneumonia was significantly more common in the B.6 than in the B.12 cases. In conclusion, the two major basal clades present in different parts in Europe are both present in the Netherlands. In hares found dead, both genotypes were associated with severe acute disease affecting multiple organs. Hepatitis and splenitis were common pathological findings in hares infected with either genotype, but pneumonia occurred significantly more frequently in hares infected with the B.6 genotype compared to hares infected with the B.12 genotype.

Prevalence of Leptospira spp. and Seoul hantavirus in brown rats (Rattus norvegicus) in four regions in the Netherlands, 2011-2015.

BACKGROUND:   Brown rats (Rattus norvegicus) may carry pathogens that can be a risk for public health. Brown rats in the Netherlands were tested for the zoonotic pathogens Leptospira spp. and Seoul hantavirus (SEOV), in order to obtain insight in their prevalence. METHODS AND RESULTS:   Cross-sectional studies were performed at four locations from 2011 to 2015. The rats were tested for Leptospira spp. using real-time PCR and/or culture resulting in a prevalence ranging between 33-57%. Testing for SEOV was done through an adapted human Seoul hantavirus ELISA and real-time RT-PCR. Although at several locations the ELISA indicated presence of SEOV antibodies, none could be confirmed by focus reduction neutralization testing. CONCLUSION:   The results indicate a widespread presence of Leptospira spp. in brown rats in the Netherlands, including areas with a low leptospirosis incidence in humans. No evidence for circulation of SEOV was found in this study.

BRUCELLA PINNIPEDIALIS IN GREY SEALS ( HALICHOERUS GRYPUS) AND HARBOR SEALS ( PHOCA VITULINA) IN THE NETHERLANDS.

Brucellosis is a zoonotic disease with terrestrial or marine wildlife animals as potential reservoirs for the disease in livestock and human populations. The primary aim of this study was to assess the presence of Brucella pinnipedialis in marine mammals living along the Dutch coast and to observe a possible correlation between the presence of B. pinnipedialis and accompanying pathology found in infected animals. The overall prevalence of Brucella spp. antibodies in sera from healthy wild grey seals ( Halichoerus grypus; n=11) and harbor seals ( Phoca vitulina; n=40), collected between 2007 and 2013 ranged from 25% to 43%. Additionally, tissue samples of harbor seals collected along the Dutch shores between 2009 and 2012, were tested for the presence of Brucella spp. In total, 77% (30/39) seals were found to be positive for Brucella by IS 711 real-time PCR in one or more tissue samples, including pulmonary nematodes. Viable Brucella was cultured from 40% (12/30) real-time PCR-positive seals, and was isolated from liver, lung, pulmonary lymph node, pulmonary nematode, or spleen, but not from any PCR-negative seals. Tissue samples from lung and pulmonary lymph nodes were the main source of viable Brucella bacteria. All isolates were typed as B. pinnipedialis by multiple-locus variable number of tandem repeats analysis-16 clustering and matrix-assisted laser desorption ionization-time of flight mass spectrometry, and of sequence type ST25 by multilocus sequence typing analysis. No correlation was observed between Brucella infection and pathology. This report displays the isolation and identification of B. pinnipedialis in marine mammals in the Dutch part of the Atlantic Ocean.

Identification and typing of Brucella spp. in stranded harbour porpoises (Phocoena phocoena) on the Dutch coast.

The presence of Brucella (B.) spp. in harbour porpoises stranded between 2008 and 2011 along the Dutch coast was studied. A selection of 265 tissue samples from 112 animals was analysed using conventional and molecular methods. In total, 4.5% (5/112) of the animals corresponding with 2.3% (6/265) Brucella positive tissue samples were Brucella positive by culture and these were all confirmed by real-time polymerase chain reaction (real-time PCR) based on the insertion element 711 (IS711). In addition, two more Brucella-positive tissue samples from two animals collected in 2011 were identified using real-time PCR resulting in an overall Brucella prevalence of 6.3% (7/112 animals). Brucella spp. were obtained from lungs (n=3), pulmonary lymph node (n=3) and lungworms (n=2). Multi Locus Variable Number of Tandem Repeats (VNTR) Analysis (MLVA) typing based on the MLVA-16 showed that the Brucella isolates were B. ceti. Additional in silico Multi Locus Sequence typing (MLST) after whole genome sequencing of the 6 Brucella isolates confirmed B. ceti ST 23. According to the Brucella 2010 MLVA database, the isolated Brucella strains encountered were of five genotypes, in two distinct subclusters divided in two different time periods of harbour porpoises collection. This study is the first population based analyses for Brucella spp. infections in cetaceans stranded along the Dutch coast.

Characteristics of cefotaxime-resistant Escherichia coli from wild birds in the Netherlands.

Cloacal swabs from carcasses of Dutch wild birds obtained in 2010 and 2011 were selectively cultured on media with cefotaxime to screen for the presence of extended-spectrum ß-lactamase (ESBL)/AmpC-producing Escherichia coli. Subsequently, all cefotaxime-resistant E. coli isolates were tested by broth microdilution and microarray. The presence of ESBL/AmpC and coexisting plasmid-mediated quinolone resistance (PMQR) genes was confirmed by PCR and sequencing. To determine the size of plasmids and the location of ESBL and PMQR genes, S1 pulsed-field gel electrophoresis (PFGE) was performed on transformants, followed by Southern blot hybridization. The study included 414 cloacal swabs originating from 55 different bird species. Cefotaxime-resistant E. coli isolates were identified in 65 birds (15.7%) from 21 different species. In all, 65 cefotaxime-resistant E. coli ESBL/AmpC genes were detected, mainly comprising variants of blaCTX-M and blaCMY-2. Furthermore, PMQR genes [aac(6')-lb-cr, qnrB1, and qnrS1] coincided in seven cefotaxime-resistant E. coli isolates. Overall, replicon typing of the ESBL/AmpC-carrying plasmids demonstrated the predominant presence of IncI1 (n = 31) and variants of IncF (n = 18). Our results indicate a wide dissemination of ESBL and AmpC genes in wild birds from The Netherlands, especially among aquatic-associated species (waterfowl, gulls, and waders). The identified genes and plasmids reflect the genes found predominantly in livestock animals as well as in humans.

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.