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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%).

Background High-power laser therapy gained popularity recently as a regenerative treatment for tendinitis and desmitis in the horse. However, studies evaluating the effects of laser therapy on tissue repair at the histological level in large mammals are lacking. Objectives To evaluate the effects of high-power laser therapy on suspensory desmitis healing, using a model of suspensory ligament branch injury. Study design In vivo experiments. Methods Standardised lesions were surgically induced in all four lateral suspensory branches of 12 healthy Warmblood horses. Laser therapy (class 4, 15W) was applied daily on two of four induced lesions for four consecutive weeks. Horses were randomly assigned to either short-term study (horses were sacrificed after 4 weeks) or long-term study (6 months). Suspensory ligament samples were scored after staining with haematoxylin-eosin and immunostaining for collagen 1- collagen 3- and factor VIII. Results In the short-term study, significantly better (lower) scores for variation in density (17% above cut-off score in treated lesions vs. 31% above cut-off score in controls, P = .03), shape of nuclei (54% vs 92%, P = .02), fibre alignment (32% vs 75%, P = .003) and fibre structure (38% vs 71%, P = .02) were found in laser-treated lesions when compared to controls. Collagen 3 expression was significantly higher (32% vs 19%, P = .006) in control lesions. In both short- and long-term studies combined, parameters lesion size (44% vs 56%, P = .02) and shape of nuclei (53% vs 84%, P = .05) scored significantly better in treated lesions. Long-term, significantly better (lower) scores were found in the laser-treated group for lesion size (15% vs 45%, P = .008) and a higher percentage above cut-off score for density of the nuclei (27% vs 9%, P = .02), compared to controls. Main limitations The model of suspensory branch injury is not an exact representation of clinical overstrain lesions. Conclusions These results suggest that high-power laser therapy enables better lesion healing than conservative treatment.

Sialic acids are used as a receptor by several viruses and variations in the linkage type or C-5 modifications affect the binding properties. A species barrier for multiple viruses is present due to α2,3- or α2,6-linked sialic acids. The C-5 position of the sialic acid can be modified to form N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc), which acts as a determinant for host susceptibility for pathogens such as influenza A virus, rotavirus, and transmissible gastroenteritis coronavirus. Neu5Gc is present in most mammals such as pigs and horses but is absent in humans, ferrets, and dogs. However, little is known about C-5 content in wildlife species or how many C-5 modified sialic acids are present on N-linked glycans or glycolipids. Using our previously developed tissue microarray system, we investigated how 2 different lectins specific for Neu5Gc can result in varying detection levels of Neu5Gc glycans. We used these lectins to map Neu5Gc content in wild Suidae, Cervidae, tigers, and European hedgehogs. We show that Neu5Gc content is highly variable among different species. Furthermore, the removal of N-linked glycans reduces the binding of both Neu5Gc lectins while retention of glycolipids by omitting methanol treatment of tissues increases lectin binding. These findings highlight the importance of using multiple Neu5Gc lectins as the rich variety in which Neu5Gc is displayed can hardly be detected by a single lectin.

Gross morphology of healthy and degenerated intervertebral discs (IVDs) is largely similar in horses as in dogs and humans. For further comparison, the biochemical composition and the histological and biochemical changes with age and degeneration were analyzed in 41 warmblood horses. From 33 horses, 139 discs and 2 fetal vertebral columns were evaluated and scored histologically. From 13 horses, 73 IVDs were assessed for hydration, DNA, glycosaminoglycans, total collagen, hydroxyl-lysyl-pyridinoline, hydroxylysine, and advanced glycation end-product (AGE) content. From 7 horses, 20 discs were assessed for aggrecan, fibronectin, and collagen type 1 and 2 content. Histologically, tearing of the nucleus pulposus (NP) and cervical annulus fibrosus (AF), and total histological score (tearing and vascular proliferation of the AF, and chondroid metaplasia, chondrocyte-like cell proliferation, presence of notochordal cells, matrix staining, and tearing of the NP) correlated with gross degeneration. Notochordal cells were not seen in IVDs of horses. Age and gross degeneration were positively correlated with AGEs and a fibrotic phenotype, explaining gross degenerative changes. In contrast to dogs and humans, there was no consistent difference in glycosaminoglycan content and hydration between AF and NP, nor decrease of these variables with age or degeneration. Hydroxylysine decrease and collagen 1 and AGEs increase were most prominent in the NP, suggesting degeneration started in the AP. In caudal cervical NPs, AGE deposition was significantly increased in grossly normal IVDs and total collagen significantly increased with age, suggesting increased biomechanical stress and likelihood for spinal disease in this part of the vertebral column.

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%).

Evidence of hearing impairment was identified in a harbour porpoise (Phocoena phocoena) on the basis of scanning electron microscopy. In addition, based on histopathology and immunohistochemistry, there were signs of unrelated cerebral toxoplasmosis. The six-year old individual live stranded on the Dutch coast at Domburg in 2016 and died a few hours later. The most significant gross lesion was multifocal necrosis and haemorrhage of the cerebrum. Histopathology of the brain revealed extensive necrosis and haemorrhage in the cerebrum with multifocal accumulations of degenerated neutrophils, lymphocytes and macrophages, and perivascular lymphocytic cuffing. The diagnosis of cerebral toxoplasmosis was confirmed by positive staining of protozoa with anti-Toxoplasma gondii antibodies. Tachyzoites were not observed histologically in any of the examined tissues. Ultrastructural evaluation of the inner ear revealed evidence of scattered loss of outer hair cells in a 290 µm long segment of the apical turn of the cochlea, and in a focal region of ~ 1.5 mm from the apex of the cochlea, which was compatible with noise-induced hearing loss. This is the first case of concurrent presumptive noise-induced hearing loss and toxoplasmosis in a free-ranging harbour porpoise from the North Sea.

Introduction: Usutu virus (USUV) is an emerging mosquito-borne flavivirus. USUV-related outbreaks of disease and mortality in wild birds, as well as neuroinvasive disease in humans have raised a health concern in Europe. Although a few pathologic studies describe the morphologic features of USUV-associated encephalitis in naturally infected wild birds, data regarding the immune response in situ are still lacking. This study aims to characterize the immune-cell populations in Usutu virus-associated encephalitis, comparing uninfected birds with birds infected with two circulating virus lineages. Materials and Methods: Immunohistochemistry was performed with specific antibodies for CD3 (T cells), PAX5 (early B cells) and MUM1 (late B cells and plasma cells) on brain tissue. Cell count was performed, and results were compared between uninfected birds (n = 4) and birds infected with Europe-3 (n = 4) and Africa-3 (n = 4) USUV-lineage. Results: The numbers of T cells and early and late B cells were higher in USUV-infected birds compared with uninfected birds, the majority being CD3+ cells. The PAX5+ cell count was higher, to a small degree, in Africa-3 compared with Europe-3-infected birds. Conclusions: This study reveals a major T cell and a minor B cell involvement in the immunopathology of Usutu virus-associated encephalitis. A slightly higher number of B cells in Africa-3-infected birds suggests a possible difference in the host response to these two lineages. Further and larger scale studies are needed to characterize the T cell population.

The binding of a viral pathogen to the host cell is the first, crucial, step of infection. This first step in host-pathogen interactions is dependent on the attachment tools of the viral pathogen and the presentation of attachment factors by the host. Pathogen receptor specificity and host receptor distribution govern which cells and tissues are susceptible, known as host tropism. Using our own developed tissue microarrays to represent the host and the viral attachment proteins to represent the pathogen, we set out to study how receptor-binding interactions can improve our understanding of receptor distribution of both domestic and wild animals and how glycobiology plays a key role in driving these receptor-binding interactions. We used these tools to study and map the receptor binding of influenza A & D attachment proteins in respiratory tissues of various domesticated and wild animals. Receptor binding is the first step in infection and it is therefore of high importance to study it. The use of tissue microarrays is a great in-between system for studying the distribution of these host receptors. The system does have its shortcomings, particularly in terms of glycolipid and mucin detection. To summarize, TMAs are not a replacement for current methods used, such as glycan arrays and animal experiments, but are an excellent addition that complements those systems for studying receptor interactions.