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Background Equine grass sickness (EGS) has been reported in several European and extra-European countries. Despite this, no scientific paper about clinical cases of EGS in Italy has been published. EGS is a disease affecting almost exclusively horses kept on pasture, characterized by clinical signs related to lesions in autonomic nervous system (ANS), particularly in the enteric nervous system (ENS). According to clinical presentation, acute, subacute and chornic syndromes can be observed, with various sympthoms including dullness, anorexia, dysphagia, drooling of saliva, tachycardia, ptosis, patchy sweating and muscle fasciculations. In horses affected by acute forms, mild to moderate abdominal pain and large volumes of nasogastric reflux can be observed. The etiology is still speculative and many hypothesis have been proposed to explain the pathogenesis. Case presentation The present study describes four cases of EGS (one subacute and three chronic forms) occurred in Central Italy during early spring. In all the cases included in the study, the prognosis was poor and the horses were euthanized. The diagnosis was confirmed by histological examination of ANS or ENS. In two cases, in vivo diagnosis was obtained by histological examination of enteric bioptic samples collected during laparoscopy. Conclusions EGS in Italy could be underdiagnosed and incidence understimated. Greater awareness should be applied in Italy for the inclusion of EGS in differential diagnosis for horses presenting clinical signs of abdominal pain associated or not with gastric reflux and muscular fasciculation. All the cases in this study concerned horses kept in the same pasture, confirming a possible premise-linked and management-linked factors on the ethiopathogenesis of EGS. The age of horses ranged from 2 to 6 years, that is consistent with the risk factor age for EGS (from 2 to 7 years of age). Previous suspected EGS diagnosis in the same livestock and recent cool dry weather were considered additional potential risk factors.

This study aimed to describe the specific localization and anatomical pattern of 24 feline lymphomas of the nervous system for which the immunophenotype was identified by immunohistochemistry investigations to support the potential specific correlation between subtypes and anatomical patterns. In total 10 tumors affected the spinal cord, eight the brain, four the peripheral nerves, one involved both the brain and the spinal cord, and one simultaneously the brain and the optic nerves. Twenty two tumors were primary lymphomas. The affected animals were 8 years of mean age. Tumors developed as an extra-axial mass (11 cases), intra-axial mass (six cases), leptomeningeal lymphomatosis (three cases), and neurolymphomatosis (five cases). One of them expressed both leptomeningeal lymphomatosis and neurolymphomatosis patterns. Two intra-axial brain lymphomas showed an angiotropic pattern. The optic chiasm was the most involved site for neurolymphomatosis. Immunolabeling was performed using anti-CD3, CD20, CD79a, PAX5, MUM-1, CD56, and anti-CD44 antibodies. In total, 12 tumors consisted of B cell lymphomas, and six of T cell lymphomas, two cases were double-reactive lymphomas while two cases consisted of non-B non-T lymphomas. B cell lymphoma affected animals of 6.4 years of mean age, while the T cell lymphoma affected older animals (mean age of 11.1 years). Extra-axial tumors mainly consisted of B cell lymphomas (8/11). Neurolymphomatosis expressed different immunophenotypes, and the B cell phenotype was the most prevalent in the optic chiasm. Two leptomeningeal lymphomatoses expressed T cell immunophenotype. For the first time, plasmacytoid differentiation was found for angiotropic lymphoma and neurolymphomatosis. All the cases, except one, were CD56-negative. CD44-expression confirmed a common malignant potential for all the anatomical patterns of the nervous system lymphoma in cats. Immunophenotype of feline lymphoma of the nervous system and its potential association with specific anatomical patterns should be strongly required in the diagnostic workup and clinical approach to this tumor especially when its primary origin is confirmed.

West Nile virus (WNV) and Usutu virus (USUV) are mosquito-borne flaviviruses that have been associated with neurological diseases in humans and wild birds. Wild bird rescue centers are potential significant hot spots for avian infection surveillance, as recognized in the Italian Integrate National Surveillance Plan for Arboviruses. Here we report the results of a post-mortem active monitoring study conducted from November 2017 to October 2020 on animals hosted in five wild bird rescue centers of Central Italy. Five hundred seventy-six (n = 576) wild birds were tested by real-time polymerase chain reaction (RT-PCR) for the presence of WNV or USUV RNA fragments. No birds tested positive for USUV RNA (n = 0; 0.00%). Evidence of WNV RNA (Ct value = 34.36) was found in one bird (n = 1; 0.17%), an adult little grebe (Tachybaptus ruficollis subsp. ruficollis), that tested WNV positive in December 2019. This study highlights the strategic role of wildlife rescue centers in monitoring both the introduction and circulation of avian emerging zoonotic diseases. In addition, the presence of WNV during the cold season evidences the possible role of birds in overwintering mechanisms in the Italian territory and requires further investigations.

Figure 2 Supplementary. Case 2. Caudal mesenteric ganglion. A number of ganglion cells contain abnormal accumulation of yellowish pigments consistent with lipofuscins (FFPE, H&E, x40).

Figure 3 Supplementary. Case 4. Cranial mesenteric ganglion. Many ganglion cells show complete loss of Nissl���s substance replaced by a diffuse eosinophilic cytoplasm. In these cells the nucleus is not apparent. A marked gliosis is associated with neuronal degeneration (FFPE, H&E, x10)

Figure 1 Supplementary. Case 2. Cranial cervical ganglion. Severe neuronal chromatolysis and satellitosis, along with occasional lymphocytic cells (arrow head). (FFPE, Cresil violet, x20).

Hemifacial microsomia (HFM) was diagnosed in a 9‐day‐old Romagnola calf. The condition was characterized by microtia of the left ear, anotia of the right ear, asymmetry of the face, and deafness. Magnetic resonance imaging revealed agenesis of the right pinna and both tympanic bullae, asymmetry of the temporal bones and temporomandibular joints, and right pontine meningocele. Brainstem auditory evoked responses confirmed the impaired auditory capacity. At gross post mortem examination, there was agenesis and hypoplasia of the right and the left external ear, respectively. No histological abnormalities were detected in the inner ears. A trio whole‐genome sequencing approach was carried out and identified a private homozygous missense variant in LAMB1 affecting a conserved residue (p.Arg668Cys). Genotyping of 221 Romagnola bulls revealed a carrier prevalence

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.

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.

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.