European Forensic Veterinary Pathology Comes of Age.

During the mid-1700s, development of the veterinary profession was largely focussed on equine medicine and surgery. Subsequently, rather erratic development encompassed other species and eventually led to specialization in different disciplines. Teaching of veterinary pathology was well established in Europe and North America by the late 19th century. Specialization in this discipline was boosted in the 1940s by the formation, in the USA, of the Register of Veterinary Pathology and American College of Veterinary Pathologists. National societies followed soon afterwards in Europe. The European Society of Veterinary Pathology evolved during this period and the European College of Veterinary Pathologists (ECVP) was created in 1995 to promote high standards in the discipline. As an accrediting body, its emphasis is on training and harmonization across Europe. There is an increasing demand for high-grade forensic veterinary pathology reports which address the requirements of the legal system, but so far only a few countries have defined protocols for these reports. In recognition of the need for a specific qualification that benchmarks the competences and experience expected of forensic veterinary pathologists, the ECVP recently launched the Certificate in Forensic Veterinary Pathology.

Development of a sprayable hydrogel formulation for the skin application of therapeutic antibodies.

A formulation of an antibody with antibacterial properties for topical use on Staphylococcal skin infections was developed and characterized. The best formulation was obtained with 1.5% (w/v) sodium carboxymethylcellulose containing 10 mg/ml immunoglobulin. Spraying forces and rheological behavior were measured in order to characterize the hydrogel formulation. The percentage of antibody aggregates in gel as well as the antibody release, folding and target binding properties of the released antibody were analyzed to proof an acceptable shelf life and no significant changes in the activity of the antibody over time. No microbial contamination was observed in the chosen non-airless application container. Functional testing of the topical skin formulation was performed with an ex vivo biopsy culture model of dog skin. Histological analysis indicated efficacy in protection from Staphylococcus mediated skin damage and antibody delivery restricted to the epidermal surface. The results demonstrate that this hydrogel is suitable for cutaneous antibody applications in the medical field.

Testicular morphology and spermatogenesis in harbour porpoises (Phocoena phocoena).

Knowledge about reproductive parameters in male harbour porpoises such as testicular histology and germ cell maturation as well as seasonal changes in spermatogenesis is scarce. Thus, the aim of the present study was to report changes in the histological appearance of the testicular morphology of neonatal and juvenile harbour porpoises during maturation, to identify stages of spermatogenesis in adult males and to detect seasonal modifications. The identification of these stages can be used to assess the developmental profile of gene expression during spermatogenesis and to identify defects in spermatogenesis arising in pathological conditions. Testes of adult male harbour porpoises from the North and Baltic Sea that became stranded or by-caught in the years 1998-2016 were histologically examined using Haematoxylin and Eosin - staining. The Periodic Acid Schiff (PAS) staining was used for spermatogenic staging and the evaluation of the development of the acrosomic cap. For the identification of changes in testes morphology and morphometry during the course of the year, histological characteristics like germ cell associations and diameter of the convoluted seminiferous tubules were noted for each month. The analysis showed that in adult males more than one stage of spermatogenesis could be found per cross section of the convoluted seminiferous tubules similar to findings in men and some ape species. This rare phenomenon is called multi-stage-arrangement. In sexually active males from the peak breeding season (June and July) eight stages of spermatogenesis were identified and all stages occurred simultaneously, while during the low breeding season (August to May) only residual spermatogenesis or constituent germ cell populations were found. Missing germ cell generations were recorded in specimens from July to September. Our investigations provide a detailed staging of spermatogenesis and give new insight into the reproductive biology of male harbour porpoises. With these new basic parameters, indicators for endocrine disruptors can be developed in the future, aiming to detect how environmental factors could affect male fertility in wildlife.

Differences in extracellular matrix proteins between Friesian horses with aortic rupture, unaffected Friesians and Warmblood horses.

BACKGROUND: Unlike in Warmblood horses, aortic rupture is quite common in Friesian horses, in which a hereditary trait is suspected. The aortic connective tissue in affected Friesians shows histological changes such as medial necrosis, elastic fibre fragmentation, mucoid material accumulation and fibrosis with aberrant collagen morphology. However, ultrastructural examination of the collagen fibres of the mid-thoracic aorta has been inconclusive in further elucidating the pathogenesis of the disease. OBJECTIVES: To assess several extracellular matrix (ECM) components biochemically in order to explore a possible underlying breed-related systemic ECM defect in Friesians with aortic rupture. STUDY DESIGN: Cadaver study. METHODS: Tissues from affected Friesians (n = 18), unaffected Friesians (n = 10) and Warmblood horses (n = 30) were compared. Samples were taken from the thoracic aorta at the level of the rupture site, from two locations caudal to the rupture and from the deep digital flexor tendon. Total collagen content, post-translational modifications of collagen formation including lysine hydroxylation, and hydroxylysylpyridinoline (HP), lysylpyridinoline (LP) and pyrrole cross-links were analysed. Additionally, elastin cross-links, glycosaminoglycan content and matrix metalloproteinase (MMP) activity were assessed. RESULTS: Significantly increased MMP activity and increased LP and HP cross-linking, lysine hydroxylation and elastin cross-linking were found at the site of rupture in affected Friesians. These changes may reflect processes involved in healing and aneurysm formation. Unaffected Friesians had less lysine hydroxylation and pyrrole cross-linking within the tendons compared with Warmblood horses. No differences in the matrix of the aorta were found between normal Warmbloods and Friesian horses. MAIN LIMITATIONS: Small sample size. CONCLUSIONS: The differences in collagen parameters in tendon tissue may reflect differences in connective tissue metabolism between Friesians and Warmblood horses.

Widespread Usutu virus outbreak in birds in the Netherlands, 2016.

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

Friesian horses as a possible model for human acquired aortopulmonary fistulation.

BACKGROUND: Acquired aortopulmonary fistulation is a rare condition in humans. It usually results as a late complication of a true or pseudoaneurysm of the thoracic aorta. It is most commonly associated with trauma or surgery, less commonly with atherosclerosis, inflammation, hypertension or Marfan's syndrome. Aortopulmonary fistulation is also seen as a rare complication of acute aortic dissection. On rare occasions, acquired aortopulmonary fistulation is reported in aged patients without any of the above mentioned triggering factors. Thus, these cases should be considered as idiopathic aortopulmonary fistulation. Clearly, the pathogenesis of this condition is not yet completely understood. Friesian horses are highly inbred and are affected by several genetic conditions. Rupture of the thoracic aorta has a relatively high prevalence in Friesian horses and is often characterized by the formation of a pseudoaneurysm with subsequent fistulation into the pulmonary artery. Affected animals may survive for several weeks to months. FINDINGS: Here we performed vascular casting in three affected Friesian horses. In all three cases, an aortic rupture at the caudoventral side of the aorta was connected with a rupture of the main pulmonary artery just proximal to its bifurcation. CONCLUSIONS: Affected Friesians show a consistent location and configuration of the aortic rupture site, very similar to the human condition and therefore could act as a spontaneous model to study this disease.

Morphometric Properties of the Thoracic Aorta of Warmblood and Friesian Horses with and without Aortic Rupture.

Rupture of the aorta is much more common in Friesians compared with other breeds of horse. Rupture always occurs adjacent to the scar of the ligamentum arteriosum. Previous histological examination of ruptured aortic walls suggested the presence of an underlying connective tissue disorder. Therefore, the aim of the present study was to compare the structural characteristics of the tunica media of the mid-thoracic aorta, distant to the lesion, in warmblood and Friesian horses with and without thoracic aortic rupture. In unaffected Friesian horses, the thickness of the tunica media, as well as the percentage area comprised of collagen type I, were significantly higher compared with the warmblood horses, supporting the hypothesis of a primary collagen disorder in the Friesian horse breed. However, in the tunica media of the affected Friesian horses there was no significant wall thickening. Moreover, the percentage area comprised of elastin was significantly lower, while the percentage area comprised of smooth muscle was higher, compared with unaffected Friesian and warmblood horses. These lesions are suggestive of an additional mild elastin deficiency with compensatory smooth muscle cell hypertrophy in affected Friesians.

Implementing wildlife disease surveillance in the Netherlands, a One Health approach.

The surveillance of (emerging) wildlife diseases can provide important, objective evidence of the circulation of pathogens of interest for veterinary and/or public health. The involvement of multiple research institutions in wildlife disease surveillance can ensure the best use of existing knowledge and expertise, but can also complicate or add challenges to the integration of wildlife disease surveillance components into a national programme. Documenting the existing efforts in a country's surveillance of wildlife diseases, including the institutes in which it takes place, provides a basis for policy-makers and authorities to identify gaps and priorities in their current surveillance programmes. This paper describes the wildlife disease surveillance activities taking place in the Netherlands. The authors recommend that, in addition to funding these current activities, surveillance resources should be allocated with the flexibility to allow for additional targeted surveillance, to detect and adequately respond to newly introduced or emerging pathogens. Similar structured overviews of wildlife disease surveillance in other countries would be very useful to facilitate international collaboration.

La surveillance exercée sur les maladies (émergentes) de la faune sauvage permet de réunir des données déterminantes, objectives et probantes sur la présence d'agents pathogènes importants pour la santé animale et/ou publique. La participation de plusieurs instituts de recherche dans les activités de surveillance des maladies de la faune sauvage permet de tirer le meilleur parti des connaissances et de l'expertise disponibles mais, dans certains cas, elle peut aussi se traduire par une complexité ou des difficultés supplémentaires qui compromettent l'intégration des composantes axées sur les maladies de la faune sauvage dans les programmes nationaux de surveillance. La collecte d'informations sur les efforts déployés au niveau national pour surveiller les maladies des animaux sauvages ainsi que sur les institutions chargées de cette surveillance constitue une première étape essentielle pour que les responsables des politiques sanitaires et les autorités puissent identifier les lacunes et les priorités des programmes de surveillance en vigueur. Les auteurs décrivent les activités de surveillance des maladies de la faune sauvage conduites actuellement aux Pays-Bas. Ils recommandent que parallèlement au financement des activités en cours, les ressources destinées à la surveillance soient allouées de manière plus souple afin de couvrir de nouvelles activités ciblées, de détecter les agents pathogènes émergents ou d'introduction récente et de préparer une réponse adéquate. Ils préconisent de réaliser dans d'autres pays des études structurées similaires sur la surveillance des maladies de la faune sauvage afin de faciliter la collaboration internationale.

La vigilancia de enfermedades (emergentes) de la fauna silvestre puede proporcionar importantes elementos de prueba objetivos sobre la circulación de patógenos de interés para la salud pública y/o veterinaria. La participación de numerosos establecimientos de investigación en estas actividades de vigilancia puede garantizar que se haga un uso idóneo de los conocimientos teóricos y técnicos existentes, pero a veces también complica o dificulta la integración en un programa nacional de las tareas de vigilancia de las enfermedades de la fauna silvestre. El hecho de repertoriar las actividades en la materia que se llevan a cabo en un país, incluidos los establecimientos donde tienen lugar, sienta las bases para que las autoridades e instancias de planificación de políticas puedan determinar las carencias y prioridades de los programas de vigilancia que ya tengan en marcha. Tras describir las actividades de vigilancia sanitaria de la fauna silvestre que se llevan a cabo en los Países Bajos, los autores recomiendan que los recursos para fines de vigilancia se asignen de manera flexible para que, además de costear las actividades ya en curso, sirvan para financiar otras labores de vigilancia selectiva que permitan detectar patógenos emergentes o recién introducidos en el país y responder debidamente a ellos. Para facilitar la colaboración internacional sería muy útil contar con estudios estructurados similares, que ofrezcan una visión de conjunto de la vigilancia sanitaria de la fauna silvestre en otros países.

Novel Receptor Specificity of Avian Gammacoronaviruses That Cause Enteritis.

UNLABELLED: Viruses exploit molecules on the target membrane as receptors for attachment and entry into host cells. Thus, receptor expression patterns can define viral tissue tropism and might to some extent predict the susceptibility of a host to a particular virus. Previously, others and we have shown that respiratory pathogens of the genus Gammacoronavirus, including chicken infectious bronchitis virus (IBV), require specific α2,3-linked sialylated glycans for attachment and entry. Here, we studied determinants of binding of enterotropic avian gammacoronaviruses, including turkey coronavirus (TCoV), guineafowl coronavirus (GfCoV), and quail coronavirus (QCoV), which are evolutionarily distant from respiratory avian coronaviruses based on the viral attachment protein spike (S1). We profiled the binding of recombinantly expressed S1 proteins of TCoV, GfCoV, and QCoV to tissues of their respective hosts. Protein histochemistry showed that the tissue binding specificity of S1 proteins of turkey, quail, and guineafowl CoVs was limited to intestinal tissues of each particular host, in accordance with the reported pathogenicity of these viruses in vivo. Glycan array analyses revealed that, in contrast to the S1 protein of IBV, S1 proteins of enteric gammacoronaviruses recognize a unique set of nonsialylated type 2 poly-N-acetyl-lactosamines. Lectin histochemistry as well as tissue binding patterns of TCoV S1 further indicated that these complex N-glycans are prominently expressed on the intestinal tract of various avian species. In conclusion, our data demonstrate not only that enteric gammacoronaviruses recognize a novel glycan receptor but also that enterotropism may be correlated with the high specificity of spike proteins for such glycans expressed in the intestines of the avian host. IMPORTANCE: Avian coronaviruses are economically important viruses for the poultry industry. While infectious bronchitis virus (IBV), a respiratory pathogen of chickens, is rather well known, other viruses of the genus Gammacoronavirus, including those causing enteric disease, are hardly studied. In turkey, guineafowl, and quail, coronaviruses have been reported to be the major causative agent of enteric diseases. Specifically, turkey coronavirus outbreaks have been reported in North America, Europe, and Australia for several decades. Recently, a gammacoronavirus was isolated from guineafowl with fulminating disease. To date, it is not clear why these avian coronaviruses are enteropathogenic, whereas other closely related avian coronaviruses like IBV cause respiratory disease. A comprehensive understanding of the tropism and pathogenicity of these viruses explained by their receptor specificity and receptor expression on tissues was therefore needed. Here, we identify a novel glycan receptor for enteric avian coronaviruses, which will further support the development of vaccines.

Esophageal Dysfunction in Friesian Horses: Morphological Features.

Megaesophagus appears to be more common in Friesian horses than in other breeds. A prevalence of approximately 2% was observed among Friesian horses presented to the Wolvega Equine Clinic and the Utrecht University Equine Clinic. In this study, morphologic changes in the esophagi of Friesian horses with megaesophagus were compared with those of 6 control horses. Of 18 horses with clinically observed megaesophagus, only 12 animals had esophageal dilation at necropsy, usually involving the thoracic portion. Muscular hypertrophy of the distal esophagus was present in only one-third of the affected horses, indicating that this change is not the most relevant cause of megaesophagus in Friesians. Increased deposition of clumped and disorganized collagen was present in these clinically affected horses mainly in the non-dilated portion of the esophagus. At necropsy, a decrease in neural elements and elastin was present principally in horses with megaesophagus. Mild degeneration and necrosis of the tunica muscularis along the entire length of the esophagus were present in clinically affected horses and encountered only rarely in control animals. There were no significant differences among affected and control horses with respect to inflammation, mineralization, or the number of cells of Cajal. The increased occurrence of megaesophagus in the Friesian breed compared with other horse breeds, together with the presence of abnormal collagen in very young foals, supports the hypothesis that megaesophagus is hereditary in Friesians.

Thoracic aortic rupture and aortopulmonary fistulation in the Friesian horse: histomorphologic characterization.

Aortic rupture in horses is a rare condition. Although it is relatively common in the Friesian breed, only limited histopathologic information is available. Twenty Friesian horses (1-10 years old) were diagnosed with aortic rupture by postmortem examination. Ruptured aortic walls were analyzed with histology and immunohistochemistry. Based on the histologic and immunohistochemical findings, these cases were divided into 3 groups: acute (n = 4, 20%), subacute (n = 8, 40%), and chronic (n = 8, 40%). Features common to samples from horses in all groups included accumulation of mucoid material; disorganization and fragmentation of the elastic laminae; aortic medial smooth muscle hypertrophy; and medial necrosis of varying degrees, ranging from mild and patchy in the acute cases to severe midzonal necrosis in the chronic cases. Inflammation, most likely secondary to medial necrosis, varied from predominantly neutrophilic infiltrates in the media and periadventitial tissue in the acute group to the presence of mainly hemosiderophages in the periadventitial tissue in the chronic group. Medial fibrosis with aberrant collagen morphology was seen in the subacute group and, more commonly, in the chronic group. Only minimal changes were seen in the aortic vasa vasorum. Smooth muscle hypertrophy and accumulation of mucoid material were not related to the age of the lesions. The findings of this study suggest that a connective tissue disorder affecting elastin or collagen in the aortic media is potentially the underlying cause of aortic rupture in Friesian horses.

Expression of p53, Ki67, EcPV2- and EcPV3 DNA, and viral genes in relation to metastasis and outcome in equine penile and preputial squamous cell carcinoma.

REASONS FOR PERFORMING STUDY: Equine penile and preputial squamous cell carcinoma (SCC) is a potentially lethal disease of which little is known regarding the relationship between tumour characteristics and prognosis. OBJECTIVES: To assess the relationship between tumour differentiation grade (tumour subtype), presence of papillomaviruses, expression of viral genes (E2, E6, L1), nuclear proteins p53 and Ki67 and metastasis in equine penile and preputial SCC and to assess the relationship of tumour subtype, presence of papillomavirus type 2, p53 and Ki67 with survival. STUDY DESIGN: Retrospective case-control study using archived material. METHODS: Samples (n = 103) from 87 horses with penile and/or preputial intraepithelial neoplasia (PIN), papilloma or SCC and corresponding case files were evaluated. Tumours were graded microscopically and p53 and Ki67 expression evaluated immunohistochemically. Equine papillomavirus (EcPV) types 2 and 3 DNA was detected by conventional PCR. Real-time PCR was used for quantification of E2, E6 and L1 mRNA. RESULTS: Equine papillomavirus type 2 DNA was detected in 89.4% and EcPV3 in 1.5% of horses. No differences in quantitative expression of E2, E6 and L1 oncogenes between subtypes were found. Expression of p53 and occurrence of metastasis were positively correlated to a less differentiated subtype (r = 0.429, P<0.001 and r = 0.769, P = 0.001, respectively). Differences in survival between subtypes were significant (log Rank P<0.001); horses with less differentiated tumours were more likely to die of the disease (papilloma 8.3%; G1 26.1%; G2 26.3%; G3 63.3%). CONCLUSIONS: In equine penile and preputial SCC, tumour grading is an important prognosticator for survival and a predictor for presence of metastases. Expression of p53 and Ki67 and presence or expression of EcPV2 and EcPV3 do not appear to be important prognosticators.

The role of osteonecrosis in canine coronoid dysplasia: arthroscopic and histopathological findings.

Coronoid dysplasia (CD) or medial coronoid disease is part of canine elbow dysplasia and eventually results in osteoarthrosis. Although CD was originally attributed to disturbed endochondral ossification, more recent data point to the subchondral bone. The objective of this study was to assess dysplastic bone and cartilage of dogs that underwent unilateral or bilateral arthroscopic subtotal coronoidectomy for the treatment of CD. Arthroscopic findings and histopathology of bone and cartilage removed from elbow joints with CD were compared. The most common arthroscopic finding was fragmentation with softening of the subchondral bone of the central part of the medial coronoid process. In dogs without obvious fragmentation, CD was characterised by bone softening and chondromalacia. During arthroscopic intervention dysplastic bone and cartilage were collected for histopathological assessment. Forty-five slices of formalin-fixed, paraffin-embedded bone and cartilage samples were stained using haematoxylin and eosin and evaluated. Histopathological findings primarily consisted of osteonecrosis of subchondral bone with necrosis within the marrow spaces. Histopathological changes in the articular cartilage were characterised by fibrillation, chondrocyte clone formation, and focal cartilage necrosis. The pathology was found primarily in the subchondral bone and not in the articular cartilage. Vascular compromise may play a role in the pathogenesis of osteonecrosis in CD.

Mapping of the receptor-binding domain and amino acids critical for attachment in the spike protein of avian coronavirus infectious bronchitis virus.

The infection of the avian coronavirus infectious bronchitis virus (IBV) is initiated by the binding of the spike glycoprotein S to sialic acids on the chicken host cell. In this study we identified the receptor-binding domain (RBD) of the spike of the prototype IBV strain M41. By analyzing the ability of recombinantly expressed chimeric and truncated spike proteins to bind to chicken tissues, we demonstrate that the N-terminal 253 amino acids of the spike are both required and sufficient for binding to chicken respiratory tract in an α-2,3-sialic acid-dependent manner. Critical amino acids for attachment of M41 spike are present within the N-terminal residues 19-69, which overlap with a hypervariable region in the S1 gene. Our results may help to understand the differences between IBV S1 genotypes and the ultimate pathogenesis of IBV in chickens.

Expression of cyclo-oxygenases-1 and -2, and microsomal prostaglandin E synthase-1 in penile and preputial papillomas and squamous cell carcinomas in the horse.

REASONS FOR PERFORMING STUDY: Penile and preputial papilloma and squamous cell carcinoma (SCC) are commonly diagnosed in horses. Papillomas have the potential to progress to potentially lethal SCC. Knowledge of pathogenetic mechanisms may help in prevention and definition of treatment targets. STUDY DESIGN: Retrospective study using archived material. OBJECTIVES: To determine the expression of cyclo-oxygenase 1 (COX-1), cyclo-oxygenase 2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) in penile and preputial normal tissue, papilloma and SCC in horses, and whether expression of these enzymes is influenced by degree of inflammation and differentiation grade. METHODS: Tumour differentiation grade, degree of inflammation and COX-1, COX-2 and mPGES-1 expression in 75 formalin-fixed paraffin embedded samples of penile and preputial papilloma and SCC of 68 horses were investigated by histopathology and immunohistochemistry. RESULTS: Inflammation was more prominent in SCC compared with papilloma. No correlation between expression of COX-1 or COX-2 and inflammation was found. Expression of mPGES-1 was weakly correlated with inflammation. Expression of COX-1, COX-2 and mPGES-1 was found in 42.6%, 50.7% and 96.0% of lesions respectively, but less than 1% of cells were immunopositive for COX-1 and COX-2 in 59.4% and 84.2% of cases respectively. Expression of COX-1 was moderately negatively correlated with differentiation grade, COX-2 was not correlated and mPGES-1 was poorly negatively correlated. CONCLUSIONS: Expression of COX-1 and COX-2 in penile and preputial SCC in the horse is poor and COX inhibitors may thus be of little value for prevention or treatment. Microsomal PGES-1 is more prominently expressed in well-differentiated tissue compared with poorly differentiated tissue. Further research on the role of mPGES-1 in carcinogenesis is needed to assess its potential use as a treatment target. Knowledge of arachidonic pathway enzyme expression and their role in equine penile and preputial carcinogenesis may help in developing preventive and therapeutic strategies.

Tularaemia in a brown hare (Lepus europaeus) in 2013: first case in the Netherlands in 60 years.

Tularaemia has not been reported in Dutch wildlife since 1953. To enhance detection, as of July 2011, brown hares (Lepus europaeus) submitted for postmortem examination in the context of non-targeted wildlife disease surveillance, were routinely tested for tularaemia by polymerase chain reaction (PCR). Francisella tularensis subspecies holarctica infection was confirmed in a hare submitted in May 2013. The case occurred in Limburg, near the site of the 1953 case. Further surveillance should clarify the significance of this finding.

Contributions of the S2 spike ectodomain to attachment and host range of infectious bronchitis virus.

The spike protein is the major viral attachment protein of the avian coronavirus infectious bronchitis virus (IBV) and ultimately determines viral tropism. The S1 subunit of the spike is assumed to be required for virus attachment. However, we have previously shown that this domain of the embryo- and cell culture adapted Beaudette strain, in contrast to that of the virulent M41 strain, is not sufficient for binding to chicken trachea (Wickramasinghe et al., 2011). In the present study, we demonstrated that the lack of binding of Beaudette S1 was not due to absence of virus receptors on this tissue nor due to the production of S1 from mammalian cells, as S1 proteins expressed from chicken cells also lacked the ability to bind IBV-susceptible embryonic tissue. Subsequently, we addressed the contribution of the S2 subunit of the spike in IBV attachment. Recombinant IBV Beaudette spike ectodomains, comprising the entire S1 domain and the S2 ectodomain, were expressed and analyzed for binding to susceptible embryonic chorio-allantoic membrane (CAM) in our previously developed spike histochemistry assay. We observed that extension of the S1 domain with the S2 subunit of the Beaudette spike was sufficient to gain binding to CAM. A previously suggested heparin sulfate binding site in Beaudette S2 was not required for the observed binding to CAM, while sialic acids on the host tissues were essential for the attachment. To further elucidate the role of S2 the spike ectodomains of virulent IBV M41 and chimeras of M41 and Beaudette were analyzed for their binding to CAM, chicken trachea and mammalian cell lines. While the M41 spike ectodomain showed increased attachment to both CAM and chicken trachea, no binding to mammalian cells was observed. In contrast, Beaudette spike ectodomain had relatively weak ability to bind to chicken trachea, but displayed marked extended host range to mammalian cells. Binding patterns of chimeric spike ectodomains to these tissues and cells indicate that S2 subunits most likely do not contain an additional independent receptor-binding site. Rather, the interplay between S1 and S2 subunits of spikes from the same viral origin might finally determine the avidity and specificity of virus attachment and thus viral host range.

Aortic rupture and aorto-pulmonary fistulation in the Friesian horse: characterisation of the clinical and gross post mortem findings in 24 cases.

REASONS FOR PERFORMING STUDY: In horses, aortic sinus of Valsalva aneurysms or tears in the aortic root are well-recognised conditions in breeding stallions, often leading to sudden death. A more uncommon form of aortic rupture, located proximal to the ligamentum arteriosum has been reported in 3 Friesian horses. OBJECTIVES: The purpose of this study was to phenotypically characterise aortic rupture and aorto-pulmonary fistulation in Friesian horses in terms of clinical and post mortem data based on 24 cases. METHODS: Friesian horses that were diagnosed with aortic rupture and aorto-pulmonary fistulation over a period of 13 years (1997-2010) at the Department of Equine Sciences of Utrecht University (n = 15) and Wolvega Equine Hospital (n = 9), were included in this study. Case history, results of clinical examination and gross post mortem findings were screened and analysed. RESULTS: Some cases were found dead without prior symptoms, but in several cases signs such as recurrent colic, peripheral oedema and sustained tachycardia were present for several weeks prior to cardiac failure. Clinical examination during hospitalisation revealed increased rectal temperature, peripheral oedema and increased jugular pulse with a bounding arterial pulse. In the majority of horses an aortic rupture of the aortic arch near the ligamentum arteriosum, concurrent with a circumferential cuff of perivascular haemorrhage and aorto-pulmonary fistulation, was found at post mortem examination. CONCLUSIONS: Aorto-pulmonary fistulation in conjunction with aortic rupture is more common in Friesians than previously estimated. In some cases findings demonstrate a progressive pathology rather than acute cardiac failure and sudden death. An appropriate approach is necessary during post mortem examination of the heart in order not to overlook the diagnosis. POTENTIAL RELEVANCE: Equine practitioners should realise that in Friesian horses presented with a history of recurrent false colic, coughing, sustained tachycardia and/or peripheral oedema, aortic rupture and aorto-pulmonary fistulation should be included in the differential diagnosis.

Binding of avian coronavirus spike proteins to host factors reflects virus tropism and pathogenicity.

The binding of viruses to host cells is the first step in determining tropism and pathogenicity. While avian infectious bronchitis coronavirus (IBV) infection and avian influenza A virus (IAV) infection both depend on α2,3-linked sialic acids, the host tropism of IBV is restricted compared to that of IAV. Here we investigated whether the interaction between the viral attachment proteins and the host could explain these differences by using recombinant spike domains (S1) of IBV strains with different pathogenicities, as well as the hemagglutinin (HA) protein of IAV H5N1. Protein histochemistry showed that S1 of IBV strain M41 and HA of IAV subtype H5N1 displayed sialic acid-dependent binding to chicken respiratory tract tissue. However, while HA bound with high avidity to a broad range of α2,3-linked sialylated glycans, M41 S1 recognized only one particular α2,3-linked disialoside in a glycan array. When comparing the binding of recombinant IBV S1 proteins derived from IBV strains with known differences in tissue tropism and pathogenicity, we observed that while M41 S1 displayed binding to cilia and goblet cells of the chicken respiratory tract, S1 derived from the vaccine strain H120 or the nonvirulent Beaudette strain had reduced or no binding to chicken tissues, respectively, in agreement with the reduced abilities of these viruses to replicate in vivo. While the S1 protein derived from the nephropathogenic IBV strain B1648 also hardly displayed binding to respiratory tract cells, distinct binding to kidney cells was observed, but only after the removal of sialic acid from S1. In conclusion, our data demonstrate that the attachment patterns of the IBV S proteins correlate with the tropisms and pathogenicities of the corresponding viruses.