Search

Background We previously reported a familial thyroid follicular cell carcinoma (FCC) in a large number of Dutch German longhaired pointers and identified two deleterious germline mutations in the TPO gene associated with disease predisposition. However, the somatic mutation profile of the FCC in dogs has not been investigated at a genome-wide scale. Results Herein, we comprehensively investigated the somatic mutations that potentially contribute to the inherited tumor formation and progression using high depth whole-genome sequencing. A GNAS p.A204D missense mutation was identified in 4 out of 7 FCC tumors by whole-genome sequencing and in 20 out of 32 dogs’ tumors by targeted sequencing. In contrast to this, in the human TC, mutations in GNAS gene have lower prevalence. Meanwhile, the homologous somatic mutation in humans has not been reported. These findings suggest a difference in the somatic mutation landscape between TC in these dogs and human TC. Moreover, tumors with the GNAS p.A204D mutation had a significantly lower somatic mutation burden in these dogs. Somatic structural variant and copy number alterations were also investigated, but no potential driver event was identified. Conclusion This study provides novel insight in the molecular mechanism of thyroid carcinoma development in dogs. German longhaired pointers carrying GNAS mutations in the tumor may be used as a disease model for the development and testing of novel therapies to kill the tumor with somatic mutations in the GNAS gene.

This case description reports an adult dog with multiple rib fractures that was initially suspected to be the victim of non-accidental trauma, but eventually was diagnosed with severe chronic interstitial nephritis and renal secondary hyperparathyroidism leading to fibrous osteodystrophy and multiple pathological fractures. The importance of the specific expertise of a dedicated expert panel on animal abuse is discussed. This case illustrates the significance of the identification of normal, pathological, and breed-related variations within an animal in addition to forensic expertise, emphasizing that the presence of multidisciplinary teams in an expert panel on animal abuse is crucial.

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.

The Dutch Kooiker dog (het Nederlandse Kooikerhondje) is one of nine Dutch dog breeds. As of 1960, a number of heritable diseases have been noted in this breed. One is an inflammatory myopathy that emerged in 1972, with numbers of affected dogs gradually increasing during the last few decades. The objective of this paper is to describe clinical signs, laboratory results, electromyography and histopathology of the muscle biopsies of the affected dogs. Method: Both retrospectively as well as prospectively affected Kooiker dogs were identified and categorized using a Tiered level of Confidence. Results: In total, 160 Kooiker dogs—40 Tier I, 33 Tier II and 87 Tier III—were included. Clinical signs were (1) locomotory problems, such as inability to walk long distances, difficulty getting up, stiff gait, walking on eggshells; (2) dysphagia signs such as drooling, difficulty eating and/or drinking; or (3) combinations of locomotory and dysphagia signs. CK activities were elevated in all except for one dog. Histopathology revealed a predominant lymphohistiocytic myositis with a usually low and variable number of eosinophils, neutrophils and plasma cells. It is concluded that, within this breed, a most likely heritable inflammatory myopathy occurs. Further studies are needed to classify this inflammatory myopathy, discuss its treatment, and unravel the genetic cause of this disease to eradicate it from this population.

The objective of the study was to describe the clinical, imaging, surgical and histological findings in a dog with Rathke's cleft cyst of the pituitary gland. A 6-year-old, female, neutered Staffordshire bull terrier was presented with an acute onset of abnormal behaviour. Magnetic resonance imaging of the skull showed a pituitary mass of 12.9 mm (height) × 8.8 mm (width) × 10.2 mm (length) with a pituitary height/brain area value of 0.73 (reference

Background Measurement of free metanephrines is recommended for screening of pheochromocytoma (PCC) but requires appropriate reference intervals (RIs). Hypothesis/Objectives To report RIs for plasma, urinary and salivary concentrations of free metanephrines and to determine the diagnostic performance of plasma free normetanephrine (pNMN) and metanephrine (pMN) concentrations in dogs with PCC, hypercortisolism (HC), and nonadrenal illness (NAI). Animals Eighty healthy dogs, 11 PCC dogs, 25 HC dogs, 6 NAI dogs. Methods Plasma, urine, and saliva were collected prospectively from healthy dogs, and free metanephrine concentrations were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, medical records of dogs that had plasma free metanephrine concentrations measured by LC-MS/MS between 2018-2021 were studied retrospectively. Results The RIs for free metanephrines in plasma, urine and saliva are reported. Dogs with PCC had significantly higher pNMN than dogs with HC (P 3.56 nmol/L) showed high sensitivity (100%, 95% confidence interval [CI]: 72-100) and specificity (94%, 95% CI: 79-99) for diagnosis of PCC, whereas pMN (>2.49 nmol/L) showed moderate sensitivity (73%, 95% CI: 39-94) and high specificity (94%, 95% CI: 79-99). Conclusions and Clinical Importance With establishment of these RIs, biochemical testing for PCC in dogs can be substantially improved. Measurement of pNMN is superior to pMN in dogs with PCC.

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.

An 11-year-old, English cocker spaniel was presented with subacute progressive signs of vestibular ataxia, tetraparesis, left-sided proprioceptive deficits, positional ventrolateral strabismus of the right eye and a right-sided menace deficit. Magnetic resonance imaging of the cranium and cranial cervical spinal cord revealed multifocal T2 hyper-/T1-hypointense intradural lesions with dural tail signs and intra-axial and intramedullary extension. Medical treatment resulted in initial improvement before deterioration was noticed. Cytological examination results of computed tomography-guided fine-needle aspiration biopsy of the C1–C2 lesion were consistent with mesenchymal neoplasia. Three days later, after progressive clinical deterioration, euthanasia was performed. Postmortem examination and subsequent histological examination of the brainstem and spinal cord revealed multifocal, strongly infiltrative growth of neoplastic cells with areas of pseudo-rosette formation by cylindrical neoplastic cells with moderately large, oval nuclei in addition to areas of spindle-shaped neoplastic cells with meningothelial whorls. The final diagnosis was a papillary (grade III) meningioma with multifocal leptomeningeal spread.

Avian infectious bronchitis (IB) is a highly contagious disease caused by infectious bronchitis virus (IBV), a coronavirus of domestic fowl. IB is a major concern in the poultry industry, causing worldwide economic losses through decreased egg production and quality and by increasing the chicken's susceptibility for secondary bacterial infections, particularly Escherichia coli. In this study, the anti-IBV activity of cathelicidins, small antimicrobial peptides of the innate immune system was investigated. The cell culture adapted (nonvirulent) IBV strain Beaudette was effectively inhibited by the human cathelicidin LL-37 in bovine hamster kidney-21 cells at nontoxic concentrations. The peptide needed to be present during virus inoculation to effectively inhibit the infection of IBV-Beaudette, indicating that LL-37 likely bound viral particles. However, no clear morphological changes in the IBV virion upon binding were observed by electron microscopy. In this cell culture model, chicken cathelicidins (CATH1-3) were inactive against IBV-Beaudette. In contrast, in multicellular infection models using the virulent IBV-M41 strain the activities of human and chicken cathelicidins were different. In particular, upon inoculation of 10-day-old embryonic eggs with IBV-M41, CATH-2 reduced the viral load to a higher extend than LL-37. Similarly, viral infection of chicken tracheal organ cultures with IBV-M41 was significantly reduced in the presence of CATH-2 but not LL-37. These results indicate a potential antiviral role for CATH-2 upon IBV infection in vivo.

IntroductionMinimally invasive microbrachytherapy is in development to treat solid tumors by intratumoral injection of (radioactive) holmium-166 (166Ho) microspheres (MS). A high local dose can be administered with minimal damage to surrounding tissue because of the short soft tissue penetration depth of 166Ho beta radiation. We aimed to prospectively evaluate the safety and efficacy of 166Ho microbrachytherapy in client-owned canine patients with soft tissue sarcomas (STS).MethodsWe included seven dogs with STS not suitable for local excision due to tumor size and/or location. 166HoMS were suspended in a carrier fluid and multiple needle-injections were performed in predetermined tumor segments to maximize tumor coverage. Tumor response was evaluated using 3D caliper and CT measurements. Follow-up further included monitoring for potential side effects and registration of subsequent treatments and survival, until at least two years after treatment.ResultsDelivered radioactive doses ranged from 70 to 969 Gy resulting in a mean tumor volume reduction of 49.0 ± 21.3% after 33 ± 25 days. Treatment-related side effects consisted of local necrosis (n = 1) and ulceration of the skin covering the tumor (n = 1), which resolved with basic wound care, and surgical excision of residual tumor, respectively. Residual tumor was surgically resected in six patients after 22–93 days. After a mean follow-up of 1,005 days, four patients were alive, two patients were euthanized because of unrelated causes, and one patient was euthanized because of disease progression after the owner(s) declined subsequent surgical treatment.Conclusion166Ho microbrachytherapy was a safe and effective neoadjuvant treatment option for canine patients with STS.

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.

Perineuronal or neuronal satellitosis is the term describing the presence of glial cells in the satellite space surrounding the neuronal perikaryon. Confusingly, this finding has been described both as a physiologic and pathologic condition in humans and animals. In animals, neuronal satellitosis has been described in mammals, as well as in avian species. For the latter, the authors wondered whether neuronal satellitosis is expressed in the normal telencephalon of different avian orders and families and whether this pattern in different species shows a specific brain-region association. For these aims, this study explored the presence of neuronal satellitosis in the major areas of the healthy telencephalon in wild and domestic avian species of different orders and families, evaluating its grade in different brain regions. Neuronal satellitosis was seen in the hyperpallium and mesopallium as areas with the highest grade. Passeriformes showed the highest grade of neuronal satellitosis compared to diurnal or nocturnal raptors, and Charadriiformes. To clarify the exact role of neuronal satellitosis in animals without neurological disease, further studies are needed. RESEARCH HIGHLIGHTS Neuronal satellitosis is a common finding in the healthy avian telencephalon. Neuronal satellitosis is a species- and brain-region-associated finding in birds. Passeriformes have the highest grade of neuronal satellitosis.

Metabolic and toxic liver disorders, such as fatty liver disease (steatosis) and drug-induced liver injury, are highly prevalent and potentially life-threatening. To allow for the study of these disorders from the early stages onward, without using experimental animals, we collected porcine livers in a slaughterhouse and perfused these livers normothermically. With our simplified protocol, the perfused slaughterhouse livers remained viable and functional over five hours of perfusion, as shown by hemodynamics, bile production, indocyanine green clearance, ammonia metabolism, gene expression and histology. As a proof-of-concept to study liver disorders, we show that an infusion of free fatty acids and acetaminophen results in early biochemical signs of liver damage, including reduced functionality. In conclusion, the present platform offers an accessible system to perform research in a functional, relevant large animal model while avoiding using experimental animals. With further improvements to the model, prolonged exposure could make this model a versatile tool for studying liver diseases and potential treatments.

Introduction: In this case study, a client-owned dog with a large pituitary tumor was experimentally treated by intratumoral injection of radioactive holmium-166 microspheres (166HoMS), named 166Ho microbrachytherapy. To our knowledge, this is the first intracranial intratumoral treatment through needle injection of radioactive microspheres.Materials and Methods: A 10-year-old Jack Russell Terrier was referred to the Clinic for Companion Animal Health (Faculty of Veterinary Medicine, Utrecht University, The Netherlands) with behavioral changes, restlessness, stiff gait, and compulsive circling. MRI and CT showed a pituitary tumor with basisphenoid bone invasion and marked mass effect. The tumor measured 8.8 cm3 with a pituitary height-to-brain area (P/B) ratio of 1.86 cm−1 [pituitary height (cm) ×10/brain area (cm2)]. To reduce tumor volume and neurological signs, 166HoMS were administered in the tumor center by transsphenoidal CT-guided needle injections.Results: Two manual CT-guided injections were performed containing 0.6 ml of 166HoMS suspension in total. A total of 1097 MBq was delivered, resulting in a calculated average tumor dose of 1866 Gy. At 138 days after treatment, the tumor volume measured 5.3 cm3 with a P/B ratio of 1.41 cm−1, revealing a total tumor volume reduction of 40%. Debulking surgery was performed five months after 166HoMS treatment due to recurrent neurological signs. The patient was euthanized two weeks later at request of the owners. Histopathological analysis indicated a pituitary adenoma at time of treatment, with more malignant characteristics during debulking surgery.Conclusion: The 40% tumor volume reduction without evident severe periprocedural side effects demonstrated the feasibility of intracranial intratumoral 166HoMS treatment in this single dog.

A variety of mammals suppress reproduction when they experience poor physical condition or environmental harshness. In many marine mammal species, reproductive impairment has been correlated to polychlorinated biphenyls (PCBs), the most frequently measured chemical pollutants, while the relative importance of other factors remains understudied. We investigate whether reproductively active females abandon investment in their foetus when conditions are poor, exemplified using an extensively studied cetacean species; the harbour porpoise (Phocoena phocoena). Data on disease, fat and muscle mass and diet obtained from necropsies in The Netherlands were used as proxies of health and nutritional status and related to pregnancy and foetal growth. This was combined with published life history parameters for 16 other areas to correlate to parameters reflecting environmental condition: mean energy density of prey constituting diets (MEDD), cumulative human impact and PCB contamination. Maternal nutritional status had significant effects on foetal size and females in poor health had lower probabilities of being pregnant and generally did not sustain pregnancy throughout gestation. Pregnancy rates across the Northern Hemisphere were best explained by MEDD. We demonstrate the importance of having undisturbed access to prey with high energy densities in determining reproductive success and ultimately population size for small cetaceans.

Two Eurasian Blackbirds (Turdus merula) from central Italy were found with severe cyclocoelid trematodosis associated with airsacculitis. The birds were submitted with severe respiratory distress; one died shortly after hospitalization, while the second bird was euthanized. At necropsy, a massive presence of cyclocoelid flukes was observed in the coelomic cavity and air sacs of both birds. The air sacs were diffusely opaque, thickened, and covered by scant fibrinous exudate mixed with numerous parasites. Histologically, the air sacs showed diffuse and severe oedema with fibrinous exudate. Diffuse mononucleated and heterophilic infiltration mixed with multiple granulomas contained degenerated trematodes. Morishitium polonicum was identified using morphologic keys and molecular analysis of extracted DNA. Infections caused by M. polonicum are poorly documented in blackbirds and the findings in these birds support the pathogenic role of this trematode as a potential cause of death in blackbirds in Italy. Extended epidemiologic surveys are required to properly assess the potential importance of M. polonicum as a life-threatening pathogen in Blackbird populations.

Members of the genus Bartonella are Gram-negative facultative intracellular bacteria that are transmitted by arthropod vectors. Bartonella alsatica was detected in the spleens and livers of 7 out of 56 wild rabbits (Oryctolagus cuniculus) and in the liver of 1 out of 87 domestic rabbits in the Netherlands. The molecular evidence of B. alsatica infection in wild as well as domestic rabbits indicates the possibility of exposure to humans when these come in close contact with rabbits and possibly their fleas with subsequent risk of Bartonella infection and disease.

A 10-year-old Koninklijk Warmbloed Paardenstamboek Nederland (KWPN, Netherlands-based organization for registration of the Dutch Warmblood horses) mare was evaluated three times over four months because of recurrent colic. At every referral, a physical examination revealed a small colon impaction, which partially responded to food deprivation and oral administration of water and magnesium sulphate. Due to the recurrent nature of the small colon impaction, several differential diagnoses were considered: inflammatory bowel disease (IBD), chronic salmonellosis and myenteric ganglionopathies. At first admission, an exploratory laparotomy was proposed, but the owner declined. On the second hospitalisation, the mare underwent a standing exploratory laparoscopy, but no abnormality related to the small colon was detected and resolved with a soap-based enema. At the third hospitalisation, the owner agreed to the exploratory laparotomy, which allowed surgical biopsies of the caecum and the large and small colon. Lymphomonocytic enteritis and mild myenteric ganglionitis were diagnosed. After laparotomy, the mare regularly fed and defecated, but a few days later, tachycardia, fever and abundant gastric reflux occurred. As the clinical condition rapidly deteriorated, the owner elected for euthanasia. A post-mortem histological examination showed severe chronic lymphocytic enterocolitis and typhlitis associated with the marked depletion of myenteric ganglion bodies. Small colon impaction is a very common disorder of the small colon in horses. In the presence of myenteric ganglionopathies, this case proposes the controversial matter of primary or secondary disorders of enteric neuromuscular function as the base of repeated small colon impactions of the horse. In this mare, recurrent small colon impaction was considered secondary to severe myenteric ganglionopathy associated with chronic intestinal pseudo-obstruction.

Histiocytic sarcoma is an aggressive hematopoietic malignancy of mature tissue histiocytes with a poorly understood etiology in humans. A histologically and clinically similar counterpart affects flat-coated retrievers (FCRs) at unusually high frequency, with 20% developing the lethal disease. The similar clinical presentation combined with the closed population structure of dogs, leading to high genetic homogeneity, makes dogs an excellent model for genetic studies of cancer susceptibility. To determine the genetic risk factors underlying histiocytic sarcoma in FCRs, we conducted multiple genome-wide association studies (GWASs), identifying two loci that confer significant risk on canine chromosomes (CFA) 5 (Pwald = 4.83x10-9) and 19 (Pwald = 2.25x10-7). We subsequently undertook a multi-omics approach that has been largely unexplored in the canine model to interrogate these regions, generating whole genome, transcriptome, and chromatin immunoprecipitation sequencing. These data highlight the PI3K pathway gene PIK3R6 on CFA5, and proximal candidate regulatory variants that are strongly associated with histiocytic sarcoma and predicted to impact transcription factor binding. The CFA5 association colocalizes with susceptibility loci for two hematopoietic malignancies, hemangiosarcoma and B-cell lymphoma, in the closely related golden retriever breed, revealing the risk contribution this single locus makes to multiple hematological cancers. By comparison, the CFA19 locus is unique to the FCR and harbors risk alleles associated with upregulation of TNFAIP6, which itself affects cell migration and metastasis. Together, these loci explain ~35% of disease risk, an exceptionally high value that demonstrates the advantages of domestic dogs for complex trait mapping and genetic studies of cancer susceptibility., Author summary We have identified two regions of the canine genome that explain a striking 35% of risk for developing histiocytic sarcoma in FCRs. The disease is uniformly lethal, affects 20% of FCRs, and parallels a cancer of the same name in humans. Both regions harbor genes involved in cell migration and cancer-related pathways. The first includes variants in regulatory regions at the tumor suppressor PIK3R6 locus that are strongly associated with histiocytic sarcoma and likely confer risk for other hematopoietic cancers. FCRs with risk alleles at the second locus demonstrate increased expression of TNFAIP6, which correlates with poor prognosis in multiple human cancers. In identifying genomic differences between affected and unaffected dogs, we advance our understanding of both canine and human health biology and set the stage for the development of diagnostic and therapeutic strategies.

The trimeric hemagglutinin-esterase fusion protein (HEF) of influenza D virus (IDV) binds 9-O-acetylated sialic acid receptors, which are expressed in various host species. While cattle are the main reservoir for IDV, the viral genome has also been detected in domestic pigs. In addition, antibodies against IDV have been detected in other farm animals such as sheep, goats, and horses, and even in farmers working with IDV positive animals. Viruses belonging to various IDV clades circulate, but little is known about their differences in host and tissue tropism. Here we used recombinantly produced HEF proteins (HEF S57A) from the major clades D/Oklahoma (D/OK) and D/Oklahoma/660 (D/660) to study their host and tissue tropism and receptor interactions. To this end, we developed tissue microarrays (TMA) composed of respiratory tissues from various farm animals including cattle, domestic pigs, sheep, goats, and horses. Protein histochemical staining of farm animal respiratory tissue-microarrays with HEF proteins showed that cattle have receptors present over the entire respiratory tract while receptors are only present in the nasal and pharyngeal epithelium of pigs, sheep, goats, and horses. No differences in tropism for tissues and animals were observed between clades, while hemagglutination assays showed that D/OK has a 2-fold higher binding affinity than D/660 for receptors on red blood cells. The removal of O-acetylation from receptors via saponification treatment confirmed that receptor-binding of both clades was dependent on O-acetylated sialic acids.

In 2016, an outbreak of Chlamydia avium infection occurred among Picazuro pigeons (Patagioenas picazuro) living in an aviary in the Netherlands. Molecular typing revealed a unique strain of C. avium. Our findings show that C. avium infection, which usually causes subclinical infection, can cause fatal disease in pigeons.

Cardiac disease is a leading cause of death for both humans and dogs. Genetic cardiomyopathies, including dilated cardiomyopathy (DCM), account for a proportion of these cases in both species. Patients may suffer from ventricular enlargement and systolic dysfunction resulting in congestive heart failure and ventricular arrhythmias with high risk for sudden cardiac death. Although canine DCM has similar disease progression and subtypes as in humans, only a few candidate genes have been found to be associated with DCM while the genetic background of human DCM has been more thoroughly studied. Additionally, experimental disease models using induced pluripotent stem cells have been widely adopted in the study of human genetic cardiomyopathy but have not yet been fully adapted for the in-depth study of canine genetic cardiomyopathies. The clinical presentation of DCM is extremely heterogeneous for both species with differences occurring based on sex predisposition, age of onset, and the rate of disease progression. Both genetic predisposition and environmental factors play a role in disease development which are identical in dogs and humans in contrast to other experimental animals. Interestingly, different dog breeds have been shown to develop distinct DCM phenotypes, and this presents a unique opportunity for modeling as there are multiple breed-specific models for DCM with less genetic variance than human DCM. A better understanding of DCM in dogs has the potential for improved selection for breeding and could lead to better overall care and treatment for human and canine DCM patients. At the same time, progress in research made for human DCM can have a positive impact on the care given to dogs affected by DCM. Therefore, this review will analyze the feasibility of canines as a naturally occurring bidirectional disease model for DCM in both species. The histopathology of the myocardium in canine DCM will be evaluated in three different breeds compared to control tissue, and the known genetics that contributes to both canine and human DCM will be summarized. Lastly, the prospect of canine iPSCs as a novel method to uncover the contributions of genetic variants to the pathogenesis of canine DCM will be introduced along with the applications for disease modeling and treatment.

Scoliosis is a deformation of the spine that may have several known causes, but humans are the only mammal known to develop scoliosis without any obvious underlying cause. This is called ‘idiopathic’ scoliosis and is the most common type. Recent observations showed that human scoliosis, regardless of its cause, has a relatively uniform three-dimensional anatomy. We hypothesize that scoliosis is a universal compensatory mechanism of the spine, independent of cause and/or species. We had the opportunity to study the rare occurrence of scoliosis in a whale (Balaenoptera acutorostrata) that stranded in July 2019 in the Netherlands. A multidisciplinary team of biologists, pathologists, veterinarians, taxidermists, radiologists and orthopaedic surgeons conducted necropsy and imaging analysis. Blunt traumatic injury to two vertebrae caused an acute lateral deviation of the spine, which had initiated the development of compensatory curves in regions of the spine without anatomical abnormalities. Three-dimensional analysis of these compensatory curves showed strong resemblance with different types of human scoliosis, amongst which idiopathic. This suggests that any decompensation of spinal equilibrium can lead to a rather uniform response. The unique biomechanics of the upright human spine, with significantly decreased rotational stability, may explain why only in humans this mechanism can be induced relatively easily, without an obvious cause, and is therefore still called ‘idiopathic’.

Colibacillosis in chickens caused by avian pathogenic Escherichia coli (APEC) is known to be aggravated by preceding infections with infectious bronchitis virus (IBV), Newcastle disease virus (NDV) and avian metapneumovirus (aMPV). The mechanism behind these virus-induced predispositions for secondary bacterial infections is poorly understood. Here we set out to investigate the immunopathogenesis of enhanced respiratory colibacillosis after preceding infections with these three viruses. Broilers were inoculated intratracheally with APEC six days after oculonasal and intratracheal inoculation with IBV, NDV, aMPV or buffered saline. After euthanasia at 1 and 8 days post infection (dpi) with APEC, birds were macroscopically examined and tissue samples were taken from the trachea, lungs and air sacs. In none of the groups differences in body weight were observed during the course of infection. Macroscopic lesion scoring revealed most severe tissue changes after NDV-APEC and IBV-APEC infection. Histologically, persistent tracheitis was detected in all virus-APEC groups, but not after APEC-only infection. In the lungs, mostly APEC-associated transient pneumonia was observed. Severe and persistent airsacculitis was present after NDV-APEC and IBV-APEC infection. Bacterial antigen was detected by immunohistochemistry only at 1 dpi APEC, predominantly in NDV-APEC- and IBV-APEC-infected lungs. Higher numbers of CD4+ and CD8+ lymphocytes persisted over time in NDV-APEC- and IBV-APEC-infected tracheas, as did CD4+ lymphocytes in NBV-APEC- and IBV-APEC-infected air sacs. KUL01+ cells, which include monocytes and macrophages, and TCRγδ+ lymphocytes were observed mostly in lung tissue in all infected groups with transient higher numbers of KUL01+ cells over time and higher numbers of TCRγδ+ lymphocytes mainly at 8 dpi. qPCR analysis revealed mostly trends of transient higher levels of IL-6 and IFNγ mRNA in lung tissue after IBV-APEC and also NDV-APEC infection and persistent higher levels of IL-6 mRNA after aMPV-APEC infection. In spleens, transient higher levels of IL-17 mRNA and more persistent higher levels of IL-6 mRNA were observed after all co-infections. No changes in IL-10 mRNA expression were seen. These results demonstrate a major impact of dual infections with respiratory viruses and APEC, compared to a single infection with APEC, on the chicken respiratory tract and suggest that immunopathogenesis contributes to lesion persistence.

Persistent organic pollutants (POPs), including polychlorinated biphenyls (PCBs), polybrominated diphenylethers (PBDEs) and hexachlorobenzene (HCB), bioaccumulate in marine ecosystems. Top predators contain high levels of POPs in their lipid-rich tissues, which may result in adverse effects on their reproductive, immune and endocrine functions. Harbour porpoises (Phocoena phocoena) are among the smallest of cetaceans and live under high metabolic demand, making them particularly vulnerable to environmental pressures. Using samples from individuals of all maturity classes and sexes stranded along the southern North Sea (n = 121), we show the generational transfer of PCBs, PBDEs and HCB from adults to foetuses. Porpoise placentas contained 1.3–8.2 mg/kg lipid weight (lw) Sum-17PCB, 9 mg/kg lw). This was particularly true for adult males (92.3% >9 mg/kg lw), while adult females had relatively low PCB levels (10.5% >9 mg/kg lw) due to offloading. Nutritional stress led to higher offloading in the milk, causing a greater potential for toxicity in calves of nutritionally stressed females. No correlation between PCB concentration and parasite infestation was detected, although the probability of a porpoise dying due to infectious disease or debilitation increased with increasing PCB concentrations. Despite current regulations to reduce pollution, these results provide further evidence of potential health effects of POPs on harbour porpoises of the southern North Sea, which may consequently increase their susceptibility to other pressures. yes

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 immuno-histochemistry, 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 de-generated neutrophils, lymphocytes and macrophages, and perivascular lymphocytic cuffing. The diagnosis of cerebral toxoplasmosis was confirmed by positive staining of protozoa with anti‐Toxo-plasma gondii antibodies. Tachyzoites were not observed histologically in any of the examined tis-sues. 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 influenza D virus (IDV) uses a trimeric hemagglutinin-esterase fusion protein (HEF) for attachment to 9-O-acetylated sialic acid receptors on the cell surface of host species. So far research has revealed that farm animals such as cattle, domestic pigs, goats, sheep and horses contain the necessary receptors on the epithelial surface of the respiratory tract to accommodate binding of the IDV HEF protein of both worldwide clades D/Oklahoma (D/OK) and D/Oklahoma/660 (D/660). More recently, seroprevalence studies have identified IDV-seropositive wildlife such as wild boar, deer, dromedaries, and small ruminants. However, no research has thus far been conducted in wildlife to reveal the distribution of acetylated sialic acid receptors that accommodate binding of IDV. Using our previously developed tissue microarray (TMA) system, we developed TMAs containing respiratory tissues of various wild and domestic species including wild boar, deer, dromedary, springbok, water buffalo, tiger, hedgehog, and Asian elephant. Protein histochemical staining of these TMAs with HEF proteins showed no receptor binding for wild Suidae, Cervidae and tiger. However, receptors were present in dromedary, springbok, water buffalo, Asian elephant, and hedgehog. In contrast to previously tested farm animals, a difference in host tropism was observed between the D/OK and D/660 clade HEF proteins in Asian elephant, and water buffalo. These results show that IDV can attach to the respiratory tract of wildlife which might facilitate transmission of IDV between wildlife and domestic animals.

Recent studies have identified a new genus of the Orthomyxoviridae family, Influenza D virus (IDV). This virus was shown to infect farm animals including swine and cattle, and to efficiently replicate and transmit in ferrets, the animal model of choice for transmission of influenza A virus to humans. This partnering grant on IDV addressed the need for capacity building at EU level to improve the EU's scientific assessment capacity and international competitiveness. We have promoted cross‐disciplinary cooperation between the partner institutes representing six Member States (BE, FR, IT, LU, NL and SE). We have shown that the available antigen and genome test systems allow reliable influenza D diagnostics in partners laboratories, while for a few of the applied antibody testing methods adjustments are recommended. Tools were developed to study virus‐host range, with a gain of knowledge on host and tissue tropism of IDV in farm animals but also in wild life and very preliminary data was generated on human tissues. Serological results in European cattle suggest that influenza D virus is enzootic. Virus diversity is still unfolding: new virus introductions were identified, as well as new reassortants whose differential clinical impact or cross‐protection levels are still poorly understood. Considering drivers of emergence, IDV was in the top five in comparison with 29 other diseases. The main risk factors of IDV in cattle are related to the animal density, presence of respiratory clinical signs in cattle and contact rates between animals. Simplified quantitative IDV risk assessment exposure model indicated a possible infection of human by IDV through aerosols in cattle farms. Further studies are warranted to fully assess the risk of IDV for both animal and Human health in Europe.

International audience; Ducks usually show little or no clinical signs following highly pathogenic avian influenza virus infection. In order to analyze whether the microbiota could contribute to the control of influenza virus replication in ducks, we used a broad-spectrum oral antibiotic treatment to deplete the microbiota before infection with a highly pathogenic H5N9 avian influenza virus. Antibiotic-treated ducks and nontreated control ducks did not show any clinical signs following H5N9 virus infection. We did not detect any significant difference in virus titers neither in the respiratory tract nor in the brain nor spleen. However, we found that antibiotic-treated H5N9 virus-infected ducks had significantly increased intestinal virus excretion at days 3 and 5 postinfection. This was associated with a significantly decreased antiviral immune response in the intestine of antibiotic-treated ducks. Our findings highlight the importance of an intact microbiota for an efficient control of avian influenza virus replication in ducks.IMPORTANCE: Ducks are frequently infected with avian influenza viruses belonging to multiple subtypes. They represent an important reservoir species of avian influenza viruses, which can occasionally be transmitted to other bird species or mammals, including humans. Ducks thus have a central role in the epidemiology of influenza virus infection. Importantly, ducks usually show little or no clinical signs even following infection with a highly pathogenic avian influenza virus. We provide evidence that the microbiota contributes to the control of influenza virus replication in ducks by modulating the antiviral immune response. Ducks are able to control influenza virus replication more efficiently when they have an intact intestinal microbiota. Therefore, maintaining a healthy microbiota by limiting perturbations to its composition should contribute to the prevention of avian influenza virus spread from the duck reservoir.

Recent population growth of the harbour porpoise ( Phocoena phocoena ), grey seal ( Halichoerus grypus ) and common seal ( Phoca vitulina ) in the North Sea has increased potential interaction between these species. Grey seals are known to attack harbour porpoises. Some harbour porpoises survive initially, but succumb eventually, often showing severely infected skin lesions. Bacteria transferred from the grey seal oral cavity may be involved in these infections and eventual death of the animal. In humans, seal bites are known to cause severe infections. In this study, a 16S rRNA-based microbiome sequencing approach is used to identify the oral bacterial diversity in harbour porpoises, grey seals and common seals; detect the potential transfer of bacteria from grey seals to harbour porpoises by biting and provide insights in the bacteria with zoonotic potential present in the seal oral cavity. β-diversity analysis showed that 12.9% (4/31) of the harbour porpoise skin lesion microbiomes resembled seal oral microbiomes, while most of the other skin lesion microbiomes also showed seal-associated bacterial species, including potential pathogens. In conclusion, this study shows that bacterial transmission from grey seals to harbour porpoises by biting is highly likely and that seal oral cavities harbour many bacterial pathogens with zoonotic potential.

Avian infectious bronchitis virus (IBV) is a coronavirus with great economic impact on the poultry industry, causing an acute and highly contagious disease in chickens that primarily affects the respiratory and reproductive systems. The cellular regulation of IBV pathogenesis and the host immune responses involved remain to be fully elucidated. MicroRNAs (miRNAs) have emerged as a class of crucial regulators of numerous cellular processes, including responses to viral infections. Here, we employed a high-throughput sequencing approach to analyze the miRNA composition of the spleen and the lungs of chicken embryos upon IBV infection. Compared to healthy chicken embryos, 13 and six miRNAs were upregulated in the spleen and the lungs, respectively, all predicted to influence viral transcription, cytokine production, and lymphocyte functioning. Subsequent downregulation of NFATC3, NFAT5, SPPL3, and TGFB2 genes in particular was observed only in the spleen, demonstrating the biological functionality of the miRNAs in this lymphoid organ. This is the first study that describes the modulation of miRNAs and the related host immune factors by IBV in chicken embryos. Our data provide novel insight into complex virus-host interactions and specifically highlight components that could affect the host's immune response to IBV infection.

Studies on the occurrence of fungal communities in the marine environment are still scarce, but mycotic infections in cetaceans are increasingly reported. Fungal disease following infection with Aspergillus species is most frequently reported, with the respiratory tract commonly affected in cetaceans and other taxa, like humans and birds. Infection with Aspergillus spp. is dependent on the characteristics of the fungus as well as the hosts immune status, with dissemination into other organs being relatively common. Along the southern North Sea, harbor porpoise (Phocoena phocoena) strandings increased significantly since 2005 and necropsies to determine causes of death have been conducted since 2008. Here we describe the post-mortem findings in stranded, free-ranging harbor porpoises on the Dutch coast which were diagnosed with fungal disease, to determine the prevalence of mycotic infections, and to compare them to those described in other species. A total of 18/754 (2.4%) harbor porpoises showed lesions compatible with localized or disseminated fungal disease as confirmed by histological examination. The respiratory tract was most commonly affected (67%), followed by the central nervous system (CNS, 33%), and auditory system (AS, 17%). Aspergillosis was confirmed for 11/18, by fungal growth (as A. fumigatus species complex, n = 9) and PCR (as Aspergillus spp., n = 1, and as A. fumigatus sensu strictu by sequence analysis, n = 1). One live stranded and euthanized animal presented partial hemiplegia of the blowhole and therefore an MRI was conducted, which resulted in a unique image of the extensive, fungus-induced lesion in the left cerebellar hemisphere, deforming and displacing the brainstem, and additionally affected the AS. The gross- and histologic lesions in the 18 porpoises diagnosed with fungal disease were similar to changes described in other mammalian species. The prevalence of fungal disease in free-living harbor porpoises is lower than seen in captive and rehabilitated animals, suggesting that captivity increases the risk to develop mycotic infections. Finally, fungal infection in the CNS and AS are usually considered consequences of vascular dissemination originating from pulmonary foci. However, only 1/7 cases with otitis and/or encephalitis demonstrated pulmonary aspergillosis, suggesting a different pathogenesis.

The death and behaviour prior to death of a bottlenose dolphin (Tursiops truncatus) known by the name of “Zafar” created significant national and international public interest. The animal was first observed in the Netherlands on the 2nd of May 2020, closely following a boat from Brittany, France, all the way into the port of Amsterdam after passing the locks at IJmuiden. After a day of residency in the industrial port of Amsterdam, the animal was successfully lured back into the North Sea. The dolphin was observed lastly on the 5th of May following a fishing vessel heading north in coastal waters near Callantsoog. Seven days later, a dead bottlenose dolphin stranded at Wijk aan Zee and through photo-identification, the animal was identified to be Zafar. A post-mortem investigation was carried out with the aim of determining the animal’s most likely cause of death, its health status, and an attempt was made to unravel the animals approximate whereabouts prior to death through stomach content analysis. The post-mortem investigation revealed that this subadult, 14 year old male had a moderate to good body condition, no sign of significant disease, and had been feeding shortly prior to death. Injuries on the animal fit with an anthropogenic source causing mortality, and the nature and severity of the lesions were most consistent with vessel collision. Based on the sightings and stranding location and the stomach content, the animal probably died within the Dutch coastal waters of Noord-Holland. This animal was first sighted in 2017 as a solitary-sociable dolphin. Such individuals have particular home-ranges, typically interact with people and little with conspecifics. It has been reported that solitary-sociable dolphins have a higher chance of suffering from human interference, and the case of Zafar clearly demonstrates this risk.

In the North Sea, white-beaked dolphins (Lagenorhynchus albirostris) occur regularly and are the second most common cetacean in the area, while their close relative, the Atlantic white-sided dolphin (Lagenorhynchus acutus), prefers the deeper waters of the northern North Sea and adjacent Atlantic Ocean. Though strandings of both species have occurred regularly in the past three decades, they have decreased in the southern North Sea during the last years. Studies describing necropsy findings in stranded Lagenorhynchus spp. are, to date, still scarce, while information gained through post-mortem examinations may reveal valuable information about underlying causes of this decline, including age structure and the reproduction status. Therefore, we retrospectively assessed and compared the necropsy results from fresh Lagenorhynchus spp. stranded along the southeastern North Sea between 1990 and 2019. A full necropsy was performed on 24 white-beaked dolphins and three Atlantic white-sided dolphins from the German and Dutch coast. Samples of selected organs were taken for histopathological, bacteriological, mycological, parasitological and virological examinations. The most common post-mortem findings were emaciation, gastritis and pneumonia. Gastritis and ulceration of the stomach was often associated with an anisakid nematode infection. Pneumonia was most likely caused by bacterial infections. Encephalitis was observed in three animals and morbillivirus antigen was detected immunohistochemically in one case. Although the animal also showed pneumonic lesions, virus antigen was only found in the brain. Parasitic infections mainly affected the gastro-intestinal tract. Lungworm infections were only detected in two cases and no associations with pathological alterations were observed. Stenurus spp. were identified in two of three cases of parasitic infections of the ears. Twelve of the 26 white-beaked dolphins stranded in Germany were found between 1993 and 1994, but there was no evidence of epizootic disease events or mass strandings during the monitored period.

Introduction: Infectious bronchitis (IB) is a gammacoronavirus-induced respiratory disease in chickens. IB virus strain QX is additionally known for its detrimental nephropathogenicity. In this study we compared the phenotypic differences between an IB QX vaccine virus and its field progenitor with focus on viral replication and dissemination through the host. Materials and Methods: Specific pathogen free chickens were inoc-ulated intratracheally with either 10 3 EID 50 vaccine strain Nobilis IB Primo QX (MSD Animal Health) or field strain IBV-D388 and killed at 1 day intervals over an 8-day period. Histopathology was studied over time in the trachea, kidney, cloaca and gastrointestinal tract. Immunohistochemistry was used to demonstrate viral protein expression in these tissues. qPCR was performed on tracheal and cloacal swabs and kidney to reveal viral RNA loads. Results: IB Primo QX and IBV-D388 induced comparable tracheal le-sions, characterized by epithelial cell death and desquamation and heter-ophilic and lymphohistiocytic infiltration. Changes were earlier and more severe with IBV-D388, while tracheal viral RNA loads were comparable over time for both viruses. In contrast, degenerative and inflammatory le-sions and viral protein and RNA were only found after IBV-D388 infection in the kidneys. This renal viral presence was preceded by viral RNA detection in the cloaca and viral protein expression in the gastrointestinal tract, albeit without associated lesions in these tissues. Discussion: Dissemination to the kidneys by IBV-D388 might result from cloacal ascending infection. In contrast to its field progenitor, IB Primo QX was unable to cause renal infection, which likely considerably contributes to attenuation of its phenotype.

Merkel cell carcinoma (MCC) is in humans and cats a malignant cutaneous neuroendocrine carcinoma, whereas in dogs it possibly has a more benign behaviour. It may be cytologically confused with round cell tumours such as lymphoma because of its striking cytomorphologic similarity. Although MCC is considered to arise from Merkel cells, recent findings indicated that primitive (epi‐)dermal stem cells, early B‐cells or dermal fibroblasts were the origin of human MCC. The aim of our study was to evaluate a possible lymphoid origin in feline and canine MCCs. Specific analysis of CD3, PAX‐5, KIT and PARR assay were performed in 3 feline and 3 canine MCCs. All MCCs (6/6) were negative for CD3 and PAX‐5. KIT was expressed in all MCCs (6/6). Assessment of clonality by PARR assay exhibited a polyclonal B‐ and T‐cell receptor rearrangement in all five cases tested. In conclusion, a lymphoid origin of feline and canine MCCs could not be demonstrated. This is in contrast with human MCCs, that often express early B‐cell lineage markers.

With global increases in anthropogenic pressures on wildlife populations comes a responsibility to manage them effectively. The assessment of marine ecosystem health is challenging and often relies on monitoring indicator species, such as cetaceans. Most cetaceans are however highly mobile and spend the majority of their time hidden from direct view, resulting in uncertainty on even the most basic population metrics. Here, we discuss the value of long-term and internationally combined stranding records as a valuable source of information on the demographic and mortality trends of the harbour porpoise (Phocoena phocoena) in the North Sea. We analysed stranding records (n = 16,181) from 1990 to 2017 and demonstrate a strong heterogeneous seasonal pattern of strandings throughout the North Sea, indicative of season-specific distribution or habitat use, and season-specific mortality. The annual incidence of strandings has increased since 1990, with a notable steeper rise particularly in the southern North Sea since 2005. A high density of neonatal strandings occurred specifically in the eastern North Sea, indicative of areas important for calving, and large numbers of juvenile males stranded in the southern parts, indicative of a population sink or reflecting higher male dispersion. These findings highlight the power of stranding records to detect potentially vulnerable population groups in time and space. This knowledge is vital for managers and can guide, for example, conservation measures such as the establishment of time-area -specific limits to potentially harmful human activities, aiming to reduce the number and intensity of human -wildlife conflicts.

Nanobody-targeted photodynamic therapy (NB-PDT) has been developed as a potent and tumor-selective treatment, using nanobodies (NBs) to deliver a photosensitizer (PS) specifically to cancer cells. Upon local light application, reactive oxygen species are formed and consequent cell death occurs. NB-PDT has preclinically shown evident success and we next aim to treat cats with oral squamous cell carcinoma (OSCC), which has very limited therapeutic options and is regarded as a natural model of human head and neck SCC. Immunohistochemistry of feline OSCC tissue confirmed that the epidermal growth factor receptor (EGFR) is a relevant target with expression in cancer cells and not in the surrounding stroma. Three feline OSCC cell lines were employed together with a well-characterized human cancer cell line (HeLa), all with similar EGFR expression, and a low EGFR-expressing human cell line (MCF7), mirroring the EGFR expression level in the surrounding mucosal stroma. NBA was identified as a NB binding human and feline EGFR with comparable high affinity. This NB was developed into NiBh, a NB-PS conjugate with high PS payload able to effectively kill feline OSCC and HeLa cell lines, after illumination. Importantly, the specificity of NB-PDT was confirmed in co-cultures where only the feline OSCC cells were killed while surrounding MCF7 cells were unaffected. Altogether, NiBh can be used for NB-PDT to treat feline OSCC and further advance NB-PDT towards the human clinic.

Pheochromocytoma in cats is a rare clinical condition characterised by the development of a secretory endocrine tumour that arises from the adrenal medulla. An 8-year-old castrated male, domestic shorthair cat was referred for further investigation of a 4-month history of progressive weight loss with normal appetite, polyuria/polydipsia, generalised weakness, and severe hypertension. Sonography and computed tomography of the abdomen disclosed a mass arising from the left adrenal gland. The contralateral adrenal gland was normal in size and shape. Results from a low dose dexamethasone suppression test and measurements of plasma aldosterone concentration and plasma renin activity ruled out a cortisol-secreting tumour and aldosteronoma. The clinical presentation made a sex-steroid secreting tumour unlikely. Increased plasma metanephrine and normetanephrine concentrations prioritised the differential diagnosis of pheochromocytoma. The cat underwent adrenalectomy of the left gland and histopathological diagnosis with immunohistochemical markers confirmed the diagnosis.

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.

Merkel cell carcinoma (MCC) is in humans and cats a malignant cutaneous neuroendocrine carcinoma, whereas in dogs it possibly has a more benign behaviour. It may be cytologically confused with round cell tumours such as lymphoma because of its striking cytomorphologic similarity. Although MCC is considered to arise from Merkel cells, recent findings indicated that primitive (epi-)dermal stem cells, early B-cells or dermal fibroblasts were the origin of human MCC. The aim of our study was to evaluate a possible lymphoid origin in feline and canine MCCs. Specific analysis of CD3, PAX-5, KIT and PARR assay were performed in 3 feline and 3 canine MCCs. All MCCs (6/6) were negative for CD3 and PAX-5. KIT was expressed in all MCCs (6/6). Assessment of clonality by PARR assay exhibited a polyclonal B- and T-cell receptor rearrangement in all five cases tested. In conclusion, a lymphoid origin of feline and canine MCCs could not be demonstrated. This is in contrast with human MCCs, that often express early B-cell lineage markers.

Whale lice (Cyamidae; Amphipoda) are ectoparasitic crustaceans adapted to the marine environment with cetaceans as their host. There are few reports of cyamids occurring in odontocetes from the North Sea, and long-term studies are lacking. Marine mammal health was monitored along the German and Dutch coasts in the past decades, with extensive post mortem investigations conducted. The aim of this study was to analyse archived ectoparasite samples from stranded cetaceans from the North Sea (2010–2019), to determine species, prevalence and impact of ectoparasite infection. Ectoparasites were found on two cetacean species – harbour porpoises (Phocoena phocoena), as the most abundant cetacean species in the North Sea, and on a pilot whale (Globicephala melas), as a rare species here. Prevalence of ectoparasitic crustaceans in cetaceans was low: 7.6% in porpoises stranded in the Netherlands (n = 608) and 1.6% in porpoises stranded in Germany (n = 122). All whale lice infections were found on hosts with skin lesions characterised by ulcerations. Morphological investigations revealed characteristic differences between the cyamid species Isocyamus (I.) delphinii and I. deltobranchium identified. Isocyamus deltobranchium was determined in all infected harbour porpoises. I. delphinii was identified on only the pilot whale. Molecular analyses showed 88% similarity of mDNA COI sequences of I. delphinii with I. deltobranchium supporting them as separate species. Phylogenetic analyses of additional gene loci are required to fully assess the diversity and exchange of whale lice species between geographical regions as well as host specificity. Differing whale lice prevalences in porpoises stranded in the Netherlands and Germany could indicate a difference in severity of skin lesions between these areas. It should be further investigated if more inter- or intraspecific contact, e.g., due to a higher density of porpoises or contact with other cetaceans, or a poorer health status of porpoises in the southern North Sea could explain these differences., Graphical abstract Image 1, Highlights • First long term study of ectoparasitic crustaceans on North Sea odontocetes. • Differences in prevalence in harbour porpoises between German and Dutch waters. • Two cyamid species identified using morphological and molecular traits. • associated to skin lesions in all investigated harbour porpoises and pilot whale. • I. deltobranchium and I. delphinii closely related.

With global increases in anthropogenic pressures on wildlife populations comes a responsibility to manage them effectively. The assessment of marine ecosystem health is challenging and often relies on monitoring indicator species, such as cetaceans. Most cetaceans are however highly mobile and spend the majority of their time hidden from direct view, resulting in uncertainty on even the most basic population metrics. Here, we discuss the value of long-term and internationally combined stranding records as a valuable source of information on the demographic and mortality trends of the harbour porpoise (Phocoena phocoena) in the North Sea. We analysed stranding records (n = 16,181) from 1990 to 2017 and demonstrate a strong heterogeneous seasonal pattern of strandings throughout the North Sea, indicative of season-specific distribution or habitat use, and season-specific mortality. The annual incidence of strandings has increased since 1990, with a notable steeper rise particularly in the southern North Sea since 2005. A high density of neonatal strandings occurred specifically in the eastern North Sea, indicative of areas important for calving, and large numbers of juvenile males stranded in the southern parts, indicative of a population sink or reflecting higher male dispersion. These findings highlight the power of stranding records to detect potentially vulnerable population groups in time and space. This knowledge is vital for managers and can guide, for example, conservation measures such as the establishment of time-area-specific limits to potentially harmful human activities, aiming to reduce the number and intensity of human-wildlife conflicts.

In the North Sea, white-beaked dolphins (Lagenorhynchus albirostris) occur regularly and are the second most common cetacean in the area, while their close relative, the Atlantic white-sided dolphin (Lagenorhynchus acutus), prefers the deeper waters of the northern North Sea and adjacent Atlantic Ocean. Though strandings of both species have occurred regularly in the past three decades, they have decreased in the southern North Sea during the last years. Studies describing necropsy findings in stranded Lagenorhynchus spp. are, to date, still scarce, while information gained through post-mortem examinations may reveal valuable information about underlying causes of this decline, including age structure and the reproduction status. Therefore, we retrospectively assessed and compared the necropsy results from fresh Lagenorhynchus spp. stranded along the southeastern North Sea between 1990 and 2019. A full necropsy was performed on 24 white-beaked dolphins and three Atlantic white-sided dolphins from the German and Dutch coast. Samples of selected organs were taken for histopathological, bacteriological, mycological, parasitological and virological examinations. The most common post-mortem findings were emaciation, gastritis and pneumonia. Gastritis and ulceration of the stomach was often associated with an anisakid nematode infection. Pneumonia was most likely caused by bacterial infections. Encephalitis was observed in three animals and morbillivirus antigen was detected immunohistochemically in one case. Although the animal also showed pneumonic lesions, virus antigen was only found in the brain. Parasitic infections mainly affected the gastro-intestinal tract. Lungworm infections were only detected in two cases and no associations with pathological alterations were observed. Stenurus spp. were identified in two of three cases of parasitic infections of the ears. Twelve of the 26 white-beaked dolphins stranded in Germany were found between 1993 and 1994, but there was no evidence of epizootic disease events or mass strandings during the monitored period.