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Background: In September 2014, there was sudden upsurge in the number of Eurasian red squirrels (Sciurus vulgaris) found dead in the Netherlands. High infection levels with the parasite Toxoplasma gondii were demonstrated, but it was unclear what had caused this increase in cases of fatal toxoplasmosis. In the present study, we aimed to gain more knowledge on the pathology and prevalence of T. gondii infections in Eurasian red squirrels in the Netherlands, on the T. gondii genotypes present, and on the determinants of the spatiotemporal variability in these T. gondii infections. The presence of the closely related parasite Hammondia hammondi was also determined. Methods: Eurasian red squirrels that were found dead in the wild or that had died in wildlife rescue centres in the Netherlands over a period of seven years (2014–2020) were examined. Quantitative real-time polymerase chain reaction was conducted to analyse tissue samples for the presence of T. gondii and H. hammondi DNA. Toxoplasma gondii-positive samples were subjected to microsatellite typing and cluster analysis. A mixed logistic regression was used to identify climatic and other environmental predictors of T. gondii infection in the squirrels. Results: A total of 178 squirrels were examined (49/178 T. gondii positive, 5/178 H. hammondi positive). Inflammation of multiple organs was the cause of death in 29 squirrels, of which 24 were also T. gondii polymerase chain reaction positive. Toxoplasma gondii infection was positively associated with pneumonia and hepatitis. Microsatellite typing revealed only T. gondii type II alleles. Toxoplasma gondii infection rates showed a positive correlation with the number of days of heavy rainfall in the previous 12 months. Conversely, they showed a negative association with the number of hot days within the 2-week period preceding the sampling date, as well as with the percentage of deciduous forest cover at the sampling site. Conclusions: Toxoplasma gondii infecti

The bacterium Treponema paraluisleporidarum causes syphilis in lagomorphs. In a set of 1,095 samples from four species—European brown hare, mountain hare, Corsican hare, and European rabbit—we tested for infection and genotyped the strains that infect wild lagomorphs. Samples originate from Sweden, the Netherlands, the United Kingdom, Germany, the Czech Republic, and Italy. The phylogenetic analyses of two informative gene targets (tp0488 and tp0548) showed high genetic diversity among the lagomorph-infecting treponemes. More specifically, we found a high number of nucleotide variants and various short repeat units in the tp0548 locus that have not been described for human syphilis and primate yaws causing Treponema pallidum. While the functional aspect of these short repeat units remains subject to ongoing investigations, it likely enables the pathogen to better survive in its lagomorph host. Our data did not support any geographic clustering, which is equally reflected in the host population genetics as shown by mitochondrial genome data corresponding to the sampled lagomorph populations. This is unexpected and in contrast with what has been shown for nonhuman primate infection with T. pallidum. In the future, the combination of multi-locus sequence typing and whole genome data from modern and ancient samples from a wide geographic range and multiple lagomorph species will contribute to a better understanding of the epidemiology and evolutionary path of lagomorph-infecting treponemes. In conclusion, our current study demonstrates widespread infection and a high genetic variation of the syphilis-causing pathogen in a higher number of positively PCR-tested European lagomorphs (n = 302/1,095).

Post-mortem research on porpoises (Phocoena phocoena) from Dutch waters, 2022. Biological data, health status and causes of death. This annual report presents the results of post-mortem examinations of beached harbour porpoises in 2022. One of the main objectives of the research is to quantify human-induced causes of death. In 2022, 57 dead harbour porpoises were examined: 31 males and 26 females, comprising 18 adults, 27 juveniles and 12 neonates. In addition two male foetuses were examined. Most of the examined harbour porpoises died as a result of infectious diseases (40.4%) and grey seal attacks (24.6%). Bycatch was the most likely cause of death for six porpoises (10.5%) and seven other porpoises died following trauma of unclear origin (12.3%)., In dit jaarrapport worden de resultaten gepresenteerd van pathologisch onderzoek aan gestrande bruinvissen in 2022. Eén van de hoofddoelen van het onderzoek is het kwantificeren van sterfte van bruinvissen door menselijk toedoen. In 2022 zijn 57 dode bruinvissen onderzocht: 31 mannelijke en 26 vrouwelijke, waarvan 18 volwassen, 27 juveniel en 12 neonaat. Er werden daarnaast twee mannelijke foetussen aangetroffen, onderzocht en bemonsterd. De meeste van de onderzochte bruinvissen stierven door infectieziekten (40,4%) en grijzezeehondaanvallen (24,6%). Bijvangst was de meest waarschijnlijke doodsoorzaak van zes bruinvissen (10,5%). Zeven andere bruinvissen stierven als gevolg van trauma door onduidelijke oorsprong (12,3%).

In August 2021, a large-scale mortality event affected harbor porpoises (Phocoena phocoena) in the Netherlands. Pathology and ancillary testing of 22 animals indicated that the most likely cause of death was Erysipelothrix rhusiopathiae infection. This zoonotic agent poses a health hazard for cetaceans and possibly for persons handling cetacean carcasses.

In August 2021, a large-scale mortality event affected harbor porpoises (Phocoena phocoena) in the Netherlands. Pathology and ancillary testing of 22 animals indicated that the most likely cause of death was Erysipelothrix rhusiopathiae infection. This zoonotic agent poses a health hazard for cetaceans and possibly for persons handling cetacean carcasses.

Rapid changes of marine ecosystems resulting from human activities and climate change, and the subsequent reported rise of infectious diseases in marine mammals, highlight the urgency for timely detection of unusual health events negatively affecting populations. Studies reporting pathological findings in the commonly stranded harbor porpoise (Phocoena phocoena) on North Atlantic coastlines are essential to describe new and emerging causes of mortality. However, such studies often cannot be used as long-term health surveillance tools due to analytical limitations. We tested 31 variables gained from stranding-, necropsy-, dietary- and marine debris data from 405 harbor porpoises using applied supervised and unsupervised machine learning techniques to explore and analyze this large dataset. We classified and cross-correlated the variables and characterized the importance of the different variables for accurately predicting cause-of-death categories, to allow trend assessment for good conservation decision. The variable ‘age class’ seemed most influential in determining cause-of-death categories, and it became apparent that juveniles died more often due to acute causes, including bycatch, grey-seal-predation and other trauma, while adults of infectious diseases. Neonates were found in summer, and mostly without prey in their stomach and more often stranded alive. The variables assigned as part of the external examination of carcasses, such as imprints from nets and lesions induced by predators, as well as nutritional condition were most important for predicting cause-of-death categories, with a model prediction accuracy of 75%. Future porpoise monitoring, and in particular the assessment of temporal trends, should predominantly focus on influential variables as determined in this study. Pathogen- and contaminant assessment data was not available for all cases, but would be an important step to further complete the dataset. This could be vital for drawing population-inf

Humans impact natural systems at an unprecedented rate. The North Sea is one of the regions in the world with the highest levels of anthropogenic activity. Here, the harbor porpoise (Phocoena phocoena) is an abundant species and is often regarded as an ecosystem sentinel. A post-mortem surveillance program was established in the Netherlands aimed at increasing knowledge of the effects of human activities on harbor porpoises. In this study, we describe the pathological findings related to anthropogenic and natural causes of death categories in 612 harbor porpoises that stranded between 2008 and 2019, and assess their relations to age, sex, season, and location. The largest anthropogenic category was bycatch (17%), with mainly juveniles affected and peak periods in March and September–October. Other, infrequently diagnosed anthropogenic causes of death were trauma (4%), largely most likely due to ship collisions, and marine debris ingestion and entanglement (0.3%). The risk of dying from anthropogenic causes was highest for juveniles. Lesions compatible with noise-induced hearing loss were investigated in carcasses which were fresh enough to do so (n = 50), with lesions apparent in two porpoises. Non-direct human-induced threats included infectious diseases, which were by far the largest cause of death category (32%), and affected mainly adults. Also, gray seal (Halichoerus grypus) attacks were a frequently assigned cause of death category (24%). There were more acute predation cases in the earlier study years, while porpoises with lesions that suggested escape from gray seal attacks were diagnosed more recently, which could suggest that porpoises adapted to this threat. Our study contributes to understanding porpoise health in response to persisting, new, emerging, and cumulative threats. Building up such knowledge is crucial for conservation management of this protected species.

This annual report presents the results of post-mortem examinations of stranded harbour porpoises in 2021. One of the main objectives of the research is to quantify human-induced causes of death. In 2021, 54 dead harbour porpoises were examined: 35 males and 19 females, comprising 24 adults, 23 juveniles and 7 neonates. In addition three foetuses found, examined and sampled. Most of the examined harbour porpoises died as a result of infectious diseases (39%) and grey seal attacks (20%). Bycatch was the most likely cause of death of 7 porpoises (13%) and 5 other porpoises died following trauma of unclear origin (9%)., In dit jaarrapport worden de resultaten gepresenteerd van pathologisch onderzoek aan gestrande bruinvissen in 2021. Eén van de hoofddoelen van het onderzoek is het kwantificeren van sterfte van bruinvissen door menselijk toedoen. In 2021 zijn 54 dode bruinvissen onderzocht: 35 mannelijke en 19 vrouwelijke, waarvan 24 volwassen, 23 juveniel en 7 neonaten. Er werden daarnaast 3 foetussen gevonden, onderzocht en bemonsterd. De meeste van de onderzochte bruinvissen waren gestorven door infectieziekten (39%) en grijzezeehondaanvallen (20%). Bijvangst was de meest waarschijnlijke doodsoorzaak van 7 bruinvissen (13%). 5 andere bruinvissen stierven als gevolg van trauma door onduidelijke oorsprong (9%).

Bycatch is considered one of the most significant threats affecting cetaceans worldwide. In the North Sea, bottom-set gillnets are a specific risk for harbor porpoises (Phocoena phocoena). Methods to estimate bycatch rates include on-board observers, remote electronic monitoring, and fishermen voluntarily reporting; none of these are systematically conducted. Additionally, necropsies of stranded animals can provide insights into bycatch occurrence and health status of individuals. There are, however, uncertainties when it comes to the assessment of bycatch in stranded animals, mainly due to the lack of diagnostic tools specific for underwater entrapment. We conducted a literature review to establish criteria that aid in the assessment of bycatch in small cetaceans, and we tested which of these criteria applied to harbor porpoises retrieved from gillnets in the Netherlands (n = 12). Twenty-five criteria were gathered from literature. Of these, “superficial incisions,” “encircling imprints,” and “recent ingestion of prey” were observed in the vast majority of our confirmed bycatch cases. Criteria like “pulmonary edema,” “pulmonary emphysema,” and “organ congestion” were also frequently observed, although considered unspecific as an indicator of bycatch. Notably, previously mentioned criteria as “favorable health status,” “absence of disease,” or “good nutritional condition” did not apply to the majority of our bycaught porpoises. This may reflect an overall reduced fitness of harbor porpoises inhabiting the southern North Sea or a higher chance of a debilitated porpoise being bycaught, and could result in an underestimation of bycatch rates when assessing stranded animals.

Bycatch is considered one of the most significant threats affecting cetaceans worldwide. In the North Sea, bottom-set gillnets are a specific risk for harbor porpoises (Phocoena phocoena). Methods to estimate bycatch rates include on-board observers, remote electronic monitoring, and fishermen voluntarily reporting; none of these are systematically conducted. Additionally, necropsies of stranded animals can provide insights into bycatch occurrence and health status of individuals. There are, however, uncertainties when it comes to the assessment of bycatch in stranded animals, mainly due to the lack of diagnostic tools specific for underwater entrapment. We conducted a literature review to establish criteria that aid in the assessment of bycatch in small cetaceans, and we tested which of these criteria applied to harbor porpoises retrieved from gillnets in the Netherlands (n = 12). Twenty-five criteria were gathered from literature. Of these, “superficial incisions,” “encircling imprints,” and “recent ingestion of prey” were observed in the vast majority of our confirmed bycatch cases. Criteria like “pulmonary edema,” “pulmonary emphysema,” and “organ congestion” were also frequently observed, although considered unspecific as an indicator of bycatch. Notably, previously mentioned criteria as “favorable health status,” “absence of disease,” or “good nutritional condition” did not apply to the majority of our bycaught porpoises. This may reflect an overall reduced fitness of harbor porpoises inhabiting the southern North Sea or a higher chance of a debilitated porpoise being bycaught, and could result in an underestimation of bycatch rates when assessing stranded animals.

Background and Purpose: Corticosteroids are intra-articularly injected to relieve pain in joints with osteoarthritis (OA) or acute tissue damage such as ligament or tendon tears, despite its unverified contraindication in unstable joints. Biomaterial-based sustained delivery may prolong reduction of inflammatory pain, while avoiding harmful peak drug concentrations. Experimental Approach: The applicability of prolonged corticosteroid exposure was examined in a rat model of anterior cruciate ligament and medial meniscus transection (ACLT + pMMx) with ensuing degenerative changes. Key Results: Intra-articular injection of a bolus of the corticosteroid triamcinolone acetonide (TAA) resulted in enhanced joint instability in 50% of the joints, but neither instability-induced OA cartilage degeneration, synovitis, nor the OA-related bone phenotype was affected. However, biomaterial microsphere-based extended TAA release enhanced instability in 94% of the animals and induced dystrophic calcification and exacerbation of cartilage degeneration. In healthy joints, injection with TAA releasing microspheres had no effect at all. In vitro, TAA inhibited cell migration out of joint tissue explants, suggesting inhibited tissue healing in vivo as mechanisms for enhanced instability and subsequent cartilage degeneration. Conclusions and Implications: We conclude that short-term TAA exposure has minor effects on surgically induced unstable joints, but its extended presence is detrimental by extending instability and associated joint degeneration through compromised healing. This supports a contraindication of prolonged corticosteroid exposure in tissue damage-associated joint instability, but not of brief exposure.

Background and Purpose: Corticosteroids are intra-articularly injected to relieve pain in joints with osteoarthritis (OA) or acute tissue damage such as ligament or tendon tears, despite its unverified contraindication in unstable joints. Biomaterial-based sustained delivery may prolong reduction of inflammatory pain, while avoiding harmful peak drug concentrations. Experimental Approach: The applicability of prolonged corticosteroid exposure was examined in a rat model of anterior cruciate ligament and medial meniscus transection (ACLT + pMMx) with ensuing degenerative changes. Key Results: Intra-articular injection of a bolus of the corticosteroid triamcinolone acetonide (TAA) resulted in enhanced joint instability in 50% of the joints, but neither instability-induced OA cartilage degeneration, synovitis, nor the OA-related bone phenotype was affected. However, biomaterial microsphere-based extended TAA release enhanced instability in 94% of the animals and induced dystrophic calcification and exacerbation of cartilage degeneration. In healthy joints, injection with TAA releasing microspheres had no effect at all. In vitro, TAA inhibited cell migration out of joint tissue explants, suggesting inhibited tissue healing in vivo as mechanisms for enhanced instability and subsequent cartilage degeneration. Conclusions and Implications: We conclude that short-term TAA exposure has minor effects on surgically induced unstable joints, but its extended presence is detrimental by extending instability and associated joint degeneration through compromised healing. This supports a contraindication of prolonged corticosteroid exposure in tissue damage-associated joint instability, but not of brief exposure., Biomaterials & Tissue Biomechanics

Neisseria animaloris is considered to be a commensal of the canine and feline oral cavities. It is able to cause systemic infections in animals as well as humans, usually after a biting trauma has occurred. We recovered N. animaloris from chronically inflamed bite wounds on pectoral fins and tailstocks, from lungs and other internal organs of eight harbour porpoises. Gross and histopathological evidence suggest that fatal disseminated N. animaloris infections had occurred due to traumatic injury from grey seals. We therefore conclude that these porpoises survived a grey seal predatory attack, with the bite lesions representing the subsequent portal of entry for bacteria to infect the animals causing abscesses in multiple tissues, and eventually death. We demonstrate that forensic microbiology provides a useful tool for linking a perpetrator to its victim. Moreover, N. animaloris should be added to the list of potential zoonotic bacteria following interactions with seals, as the finding of systemic transfer to the lungs and other tissues of the harbour porpoises may suggest a potential to do likewise in humans.

Toll-like receptors (TLR) are ancient innate immune receptors crucial for immune homeostasis and protection against infection. TLRs are present in mammals, birds, amphibians and fish but have not been functionally characterized in reptiles despite the central position of this animal class in vertebrate evolution. Here we report the cloning, characterization, and function of TLR5 of the reptile Anolis carolinensis (Green Anole lizard). The receptor (acTLR5) displays the typical TLR protein architecture with 22 extracellular leucine rich repeats flanked by a N-and C-terminal leucine rich repeat domain, a membrane-spanning region, and an intracellular TIR domain. The receptor is phylogenetically most similar to TLR5 of birds and most distant to fish TLR5. Transcript analysis revealed acTLR5 expression in multiple lizard tissues. Stimulation of acTLR5 with TLR ligands demonstrated unique responsiveness towards bacterial flagellin in both reptile and human cells. Comparison of acTLR5 and human TLR5 using purified flagellins revealed differential sensitivity to Pseudomonas but not Salmonella flagellin, indicating development of species-specific flagellin recognition during the divergent evolution of mammals and reptiles. Our discovery of reptile TLR5 fills the evolutionary gap regarding TLR conservation across vertebrates and provides novel insights in functional evolution of host-microbe interactions.