Jolianne M. Rijks

High-throughput sequencing reveals inbreeding depression in a natural population

Significance Many studies of wild populations reveal links between heterozygosity and fitness, with relatively heterozygous individuals carrying fewer parasites, living longer and being more attractive to mates. These patterns appear ubiquitous and are often highly significant, but heterozygosity usually accounts for very little of the total variation in fitness. However, most studies analyze only around 10 loci, representing a tiny fraction of the genome. We therefore used high-throughput DNA sequencing to estimate genome-wide heterozygosity based on over 10,000 loci and found it to accurately reflect inbreeding. Applied to wild harbor seals, genome-wide heterozygosity explained almost half of the variation in parasite infection. By implication, a greater proportion of fitness variation could be linked to genotype than previously thought. Proxy measures of genome-wide heterozygosity based on approximately 10 microsatellites have been used to uncover heterozygosity fitness correlations (HFCs) for a wealth of important fitness traits in natural populations. However, effect sizes are typically very small and the underlying mechanisms remain contentious, as a handful of markers usually provides little power to detect inbreeding. We therefore used restriction site associated DNA (RAD) sequencing to accurately estimate genome-wide heterozygosity, an approach transferrable to any organism. As a proof of concept, we first RAD sequenced oldfield mice (Peromyscus polionotus) from a known pedigree, finding strong concordance between the inbreeding coefficient and heterozygosity measured at 13,198 single-nucleotide polymorphisms (SNPs). When applied to a natural population of harbor seals (Phoca vitulina), a weak HFC for parasite infection based on 27 microsatellites strengthened considerably with 14,585 SNPs, the deviance explained by heterozygosity increasing almost fivefold to a remarkable 49%. These findings arguably provide the strongest evidence to date of an HFC being due to inbreeding depression in a natural population lacking a pedigree. They also suggest that under some circumstances heterozygosity may explain far more variation in fitness than previously envisaged.

Circulation of four Anaplasma phagocytophilum ecotypes in Europe

BackgroundAnaplasma phagocytophilum is the etiological agent of granulocytic anaplasmosis in humans and animals. Wild animals and ticks play key roles in the enzootic cycles of the pathogen. Potential ecotypes of A. phagocytophilum have been characterized genetically, but their host range, zoonotic potential and transmission dynamics has only incompletely been resolved.MethodsThe presence of A. phagocytophilum DNA was determined in more than 6000 ixodid ticks collected from the vegetation and wildlife, in 289 tissue samples from wild and domestic animals, and 69 keds collected from deer, originating from various geographic locations in The Netherlands and Belgium. From the qPCR-positive lysates, a fragment of the groEL-gene was amplified and sequenced. Additional groEL sequences from ticks and animals from Europe were obtained from GenBank, and sequences from human cases were obtained through literature searches. Statistical analyses were performed to identify A. phagocytophilum ecotypes, to assess their host range and their zoonotic potential. The population dynamics of A. phagocytophilum ecotypes was investigated using population genetic analyses.ResultsDNA of A. phagocytophilum was present in all stages of questing and feeding Ixodes ricinus, feeding I. hexagonus, I. frontalis, I. trianguliceps, and deer keds, but was absent in questing I. arboricola and Dermacentor reticulatus. DNA of A. phagocytophilum was present in feeding ticks and tissues from many vertebrates, including roe deer, mouflon, red foxes, wild boar, sheep and hedgehogs but was rarely found in rodents and birds and was absent in badgers and lizards. Four geographically dispersed A. phagocytophilum ecotypes were identified, that had significantly different host ranges. All sequences from human cases belonged to only one of these ecotypes. Based on population genetic parameters, the potentially zoonotic ecotype showed significant expansion.ConclusionFour ecotypes of A. phagocytophilum with differential enzootic cycles were identified. So far, all human cases clustered in only one of these ecotypes. The zoonotic ecotype has the broadest range of wildlife hosts. The expansion of the zoonotic A. phagocytophilum ecotype indicates a recent increase of the acarological risk of exposure of humans and animals.

Heterozygosity and lungworm burden in harbour seals (Phoca vitulina)

In several studies, heterozygosity measured at around 10 microsatellite markers correlates with parasite load. Usually the effect size is small, but while this may reflect reality, it may also be possible that too few markers are used or the measure of fitness contains too much error to reveal what is actually a much stronger underlying effect. Here, we analysed over 200 stranded harbour seals (Phoca vitulina) for an association between lungworm burden and heterozygosity, conducting thorough necropsies on the seals and genotyping the samples obtained for 27 microsatellites. We found that homozygosity predicts higher worm burdens, but only in young animals, where the worms have the greatest impact on fitness. Testing each locus separately, we found that a significant majority reveal a weak but similar trend for heterozygosity to be protective against high lungworm burden, suggesting a genome-wide effect, that is, inbreeding. This conclusion is supported by the fact that heterozygosity is correlated among markers in young animals but not in otherwise equivalent older ones. Taken as a whole, our results support the notion that homozygosity increases susceptibility to parasitic infection and suggest that parasites can be effective in removing inbred individuals from the population.

Ranavirus-associated mass mortality in wild amphibians, the Netherlands, 2010: a first report.

In 2010, a mass die-off of over 1000 wild water frogs (Pelophylax spp.) and at least 10 common newts (Lissotriton vulgaris) occurred in a pond in The Netherlands. Haemorrhagic disease with hepatomegaly and splenomegaly was evident. Microscopically, multiple organs presented cells with multifocal intracytoplasmic inclusion bodies, in which ranavirus-like particles were demonstrated ultrastructurally. All specimens examined tested positive for ranavirus by PCR. The sequence obtained showed a 100% identity with the one deposited for common midwife toad virus (CMTV). This is the first report of ranavirus-associated mortality in wild amphibian populations in The Netherlands. It is also the first time CMTV or a CMTV-like virus has been reported in these two species in the adult stage and outside of Spain.

Widespread activity of multiple lineages of Usutu virus, western Europe, 2016.

In the summer of 2016, Belgium, France, Germany and the Netherlands reported widespread Usutu virus (USUV) activity based on live and dead bird surveillance. The causative USUV strains represented four lineages, of which two putative novel lineages were most likely recently introduced into Germany and spread to other western European countries. The spatial extent of the outbreak area corresponded with R0 values > 1. The occurrence of the outbreak, the largest USUV epizootic registered so far in Europe, allowed us to gain insight in how a recently introduced arbovirus with potential public health implications can spread and become a resident pathogen in a naïve environment. Understanding the ecological and epidemiological factors that drive the emergence or re-emergence of USUV is critical to develop and implement timely surveillance strategies for adequate preventive and control measures. Public health authorities, blood transfusion services and clinicians in countries where USUV was detected should be aware of the risk of possible USUV infection in humans, including in patients with unexplained encephalitis or other neurological impairments, especially during late summer when mosquito densities peak.

Stage-structured transmission of phocine distemper virus in the Dutch 2002 outbreak

Heterogeneities in transmission among hosts can be very important in shaping infectious disease dynamics. In mammals with strong social organization, such heterogeneities are often structured by functional stage: juveniles, subadults and adults. We investigate the importance of such stage-related heterogeneities in shaping the 2002 phocine distemper virus (PDV) outbreak in the Dutch Wadden Sea, when more than 40 per cent of the harbour seals were killed. We do this by comparing the statistical fit of a hierarchy of models with varying transmission complexity: homogeneous versus heterogeneous mixing and density- versus frequency-dependent transmission. We use the stranding data as a proxy for incidence and use Poisson likelihoods to estimate the ‘who acquires infection from whom’ (WAIFW) matrix. Statistically, the model with strong heterogeneous mixing and density-dependent transmission was found to best describe the transmission dynamics. However, patterns of incidence support a model of frequency-dependent transmission among adults and juveniles. Based on the maximum-likelihood WAIFW matrix estimates, we use the next-generation formalism to calculate an R0 between 2 and 2.5 for the Dutch 2002 PDV epidemic.

Investigation of amphibian mortality events in wildlife reveals an on-going ranavirus epidemic in the North of the Netherlands

In the four years following the first detection of ranavirus (genus Ranavirus, family Iridoviridae) infection in Dutch wildlife in 2010, amphibian mortality events were investigated nationwide to detect, characterize and map ranaviruses in amphibians over time, and to establish the affected host species and the clinico-pathological presentation of the disease in these hosts. The ultimate goal was to obtain more insight into ranavirus disease emergence and ecological risk. In total 155 dead amphibians from 52 sites were submitted between 2011 and 2014, and examined using histopathology, immunohistochemistry, virus isolation and molecular genetic characterization. Ranavirus-associated amphibian mortality events occurred at 18 sites (35%), initially only in proximity of the 2010 index site. Specimens belonging to approximately half of the native amphibian species were infected, including the threatened Pelobates fuscus (spadefoot toad). Clustered massive outbreaks involving dead adult specimens and ranavirus genomic identity indicated that one common midwife toad virus (CMTV)-like ranavirus strain is emerging in provinces in the north of the Netherlands. Modelling based on the spatiotemporal pattern of spread showed a high probability that this emerging virus will continue to be detected at new sites (the discrete reproductive power of this outbreak is 0.35). Phylogenetically distinct CMTV-like ranaviruses were found in the south of the Netherlands more recently. In addition to showing that CMTV-like ranaviruses threaten wild amphibian populations not only in Spain but also in the Netherlands, the current spread and risk of establishment reiterate that understanding the underlying causes of CMTV-like ranavirus emergence requires international attention.

Widespread Usutu virus outbreak in birds in the Netherlands, 2016

We report a widespread Usutu virus outbreak in birds in the Netherlands. Viral presence had been detected through targeted surveillance as early as April 2016 and increased mortality in common blackbirds and captive great grey owls was noticed from August 2016 onwards. Usutu virus infection was confirmed by post-mortem examination and RT-PCR. Extensive Usutu virus activity in the Netherlands in 2016 underlines the need to monitor mosquito activity and mosquito-borne infections in 2017 and beyond.

Phocine Distemper Outbreak, the Netherlands, 2002

During the 2002 phocine distemper epidemic, 2,284 seals, primarily harbor seals (Phoca vitulina), were found stranded along the Dutch coast. Stranding pattern varied with age, sex, state of decomposition, wind, and location. Cumulative proportion of deaths (54%) was comparable to that in the first reported epidemic in 1988.

Obliterative Endophlebitis in Mute Swans (Cygnus olor) Caused by Trichobilharzia sp. (Digenea: Schistosomatidae) Infection

Schistosome infections in mammals cause chronic proliferative vascular lesions associated with the presence of adult parasites in the lumen of mesenteric and portal veins. In birds, however, this has never been reported. In this study, we found obliterative endophlebitis associated with the presence of adult schistosomes (Trichobilharzia sp., probably Trichobilharzia filiformis) as the main pathologic finding in five of eight mute swans (Cygnus olor). On histologic examination, the intestinal and portal veins of these swans showed moderate to severe, diffuse, hyperplastic endophlebitis, characterized by myointimal hyperplasia, often with obliteration of the vascular lumen. In addition, moderate to severe lymphocytic and granulocytic enteritis occurred in all eight swans associated with the presence of schistosome eggs in the intestinal mucosa. Other findings included hepatic and splenic hemosiderosis and high hepatic copper levels. The vascular lesions associated with Trichobilharzia sp. infection may have contributed to the emaciation and death of those mute swans by obstruction of venous return in the intestinal and portal veins.