Metastatic cornual melanoma in a Valesian copperneck goat

A 10-year-old, neutered, male Valesian copperneck goat was presented to the Clinic for Ruminants, Vetsuisse Faculty, University of Bern for a chronic wound at the base of the right horn first noticed 3 weeks before presentation. Clinical examination revealed a firm mass with ulcerations and malodorous discharge at the base of the right horn, and a duller sound on percussion of the right frontal sinus. Radiography revealed a soft tissue opacity filling the right cornual diverticulum of the frontal sinus. Upon wound debridement, tissue of grey to black discolorationwas noticed and sent in for histopathological examination, which confirmed the presence of a melanoma. Subsequent ultrasonography of the liver and radiography of the thorax revealed no sign of metastatic disease. Given the poor prognosis, the goat was euthanased, and postmortem examination revealed the presence of multiple metastatic lesions in the regional lymph nodes

Characterization of the immune response against Testudinid herpesvirus 3: new insight

Testudinid herpesvirus 3 (TeHV3) is one of the most lethal viral agents in tortoises worldwide. Although TeHV3 have been extensively studied, only little information is available about host-pathogen interaction. TeHV3 infections in different species of the genus Testudo correlate with various lesions profiles, disease severity and clinical outcome, suggesting the existence of a complex host-pathogen interaction. This might reflect a possible viral-host coevolution (Origgi, 2012).To study the host-pathogen interaction, we previously screened 5.000 clones from a bacteriophage library obtained from the TeHV3 genomic DNA using Testudo graeca seropositive sera. Of the six detected positive clones, only one was confirmed by F.A.C.S. Selected clone was determined to be a concatamer of different TeHV3 genomic fragments including the partial sequence of TE17, UL15, Major capsid protein (MCP), and Glycoprotein B (gB) genes. After complete sequencing of the selected clone, the MCP and the gB were antisenses compared to the phagemid promoter.In order to assess which of the gene fragments among TE17 and UL15 was encoding for the antigenic determinant that was recognized by the anti-TeHV3 tortoise sera, distinct approaches were followed.TE-17 and UL15 fragments were knock out from the original phagemid using the following approaches: a) directed-site mutagenesis, b) molecular cloning, and c) restriction enzymes cloning. All the modified constructs were cloned in two different E. coli cloning vectors (D5α and XL 1-Blue).Transformation of competent cells with the constructs described above did not yield any viable bacteria.Among the different aspects might have influenced transformation success rate, construct size was probably the most relevant (about 9Kb). Furthermore, we could not entirely exclude that genomic DNA editing might have induced mutations in the construct sequence causing toxic effects on the host bacterial cell. Cloning of TE-17 and UL15 gene fragments into different prokaryotic expression vectors is currently under way

Characterization of the immune response against Testudinid herpesvirus 3.

Numerous infectious diseases have been documented in reptiles, however minimal information isavailable concerning their immunological response. One of the most diffuse and lethal reptile pathogenis Testudinid herpesvirus 3 (TeHV3), a Alphaherpesvirinae. All species of tortoises (Testudinide) areconsidered susceptible to TeHV3, however the virus is over represented in the genus Testudo, whichincludes, among others, T. graeca, T. hermanni, T. marginata, and T. horsfieldii, that are popular pets inEurope. Incidence of TeHV3-associated disease is highest right after hibernation (Origgi, 2012).The aim of this work is to partially characterize the immunological response of T. graeca against TeHV3.A bacteriophage library composed of about 5.000 clones containing genomic DNA fragments of TeHV3was produced. Bacteriophages were amplified in a specific strain of E. coli and were screened withTeHV3-seropositive sera from T. graeca. Phagemids were excised from the positive bacteriophages,sequenced, and compare with the TeHV3 genome to identify the encoding genes. Six differentstructural and non-structural proteins have identified as immune relevant. Vero cells where transfectedwith phagemids of the positive clones, to confirm previous results. TeHV3’s proteins expression wasassessed by F.A.C.S using T. graeca seropositive sera. Of all the six selected clones, only that expressingthe partial sequence of the glycoprotein B (gB) showed a positive signal in the F.A.C.S. analysis. Thisresult is consistent with the well-known immunogenicity of gB of other herpesviruses including thoseinfecting humans and with the highly conserved role that gB plays in host-pathogen interaction acrossspecies and evolution (Beals et al., 2016)

TeHV3 outbreak characterization in captive Testudo spp.

Italian Tortoises species are considered either endangered or near threatened according to International Union for Conservation of Nature. When pet tortoises are abandoned or found injured or seized following illegal detention, they are sent to wildlife rehabilitation centers. From 2008, the Testudo spp. population housed in the WWF Vanzago’s oasis exhibited clinical signs compatible with Testudinid herpesviurs 3 (TeHV3) infection.  By the end of 2012 all Testudo had died. The presence of TeHV3 was investigated by molecular biology and pathology. All the tortoises housed in Vanzago resulted ELISA positive for the presence of anti-TeHV3 antibodies except one T. hermanni. Of these, 12 animals died and were all necropsied. Lesion frequency distribution was evaluate by histology. PCR was positive in 8/12 tortoises. To better complement the epidemiological evaluation of the virus in northern Italy, 20 retrospective cases were selected from the archive of the University of Milan. Of these, 5 were TeHV3 PCR positive. Lesions closely resembled those of the Vanzago’s population. These results are consistent with a high prevalence of TeHV3 in northern Italy. The finding of intranuclear inclusion bodies demonstrated to be specific but not sensitive. TeHV3 diagnostic pathological lesions have been reported to vary according with host immune response and by the viral replicative status. Molecular techniques were often necessary to confirm the infection. According to the literature and to our findings, T. hermanni spp. seems the species with higher mortality and lower antibody concentrations when infected with TeHV3.Italian Tortoises species are considered either endangered or near threatened according to International Union for Conservation of Nature. When pet tortoises are abandoned or found injured or seized following illegal detention, they are sent to wildlife rehabilitation centers. From 2008, the Testudo spp. population housed in the WWF Vanzago’s oasis exhibited clinical signs compatible with Testudinid herpesviurs 3 (TeHV3) infection.  By the end of 2012 all Testudo had died. The presence of TeHV3 was investigated by molecular biology and pathology. All the tortoises housed in Vanzago resulted ELISA positive for the presence of anti-TeHV3 antibodies except one T. hermanni. Of these, 12 animals died and were all necropsied. Lesion frequency distribution was evaluate by histology. PCR was positive in 8/12 tortoises. To better complement the epidemiological evaluation of the virus in northern Italy, 20 retrospective cases were selected from the archive of the University of Milan. Of these, 5 were TeHV3 PCR positive. Lesions closely resembled those of the Vanzago’s population. These results are consistent with a high prevalence of TeHV3 in northern Italy. The finding of intranuclear inclusion bodies demonstrated to be specific but not sensitive. TeHV3 diagnostic pathological lesions have been reported to vary according with host immune response and by the viral replicative status. Molecular techniques were often necessary to confirm the infection. According to the literature and to our findings, T. hermanni spp. seems the species with higher mortality and lower antibody concentrations when infected with TeHV3

Oligomerization and Cell Egress Controlled by Two Microdomains of Canine Distemper Virus Matrix Protein.

The multimeric matrix (M) protein of clinically relevant paramyxoviruses orchestrates assembly and budding activity of viral particles at the plasma membrane (PM). We identified within the canine distemper virus (CDV) M protein two microdomains, potentially assuming α-helix structures, which are essential for membrane budding activity. Remarkably, while two rationally designed microdomain M mutants (E89R, microdomain 1 and L239D, microdomain 2) preserved proper folding, dimerization, interaction with the nucleocapsid protein, localization at and deformation of the PM, the virus-like particle formation, as well as production of infectious virions (as monitored using a membrane budding-complementation system), were, in sharp contrast, strongly impaired. Of major importance, raster image correlation spectroscopy (RICS) revealed that both microdomains contributed to finely tune M protein mobility specifically at the PM. Collectively, our data highlighted the cornerstone membrane budding-priming activity of two spatially discrete M microdomains, potentially by coordinating the assembly of productive higher oligomers at the PM.IMPORTANCE Despite the availability of efficient vaccines, morbilliviruses (e.g., canine distemper virus [CDV] and measles virus [MeV]) still cause major health impairments. Although antivirals may support vaccination campaigns, approved inhibitors are to date still lacking. Targeting late stages of the viral life cycle (i.e., the cell exit system) represents a viable option to potentially counteract morbilliviral infections. The matrix (M) protein of morbillivirus is a major contributor to membrane budding activity and is assumed to assemble into dimers that further associate to form higher oligomers. Here, we rationally engineered M protein variants with modifications in two microdomains that potentially locate at dimer-dimer interfaces. Our results spotlight the cornerstone impact of both microdomains in membrane budding activity and further suggest a role of finely tuned high-order oligomer formation in regulating late stages of cell exit. Collectively, our findings highlight two microdomains in the morbilliviral M protein as novel attractive targets for drug design

Angiostrongylus dujardini infection in a coconut lorikeet (Trichoglossus haematodus) from a zoological garden in Switzerland.

Angiostrongylus spp. (Metastrongyloidea) can cause severe disease in several animal species and humans. This report describes an infection with Angiostrongylus dujardini in a captive Coconut lorikeet (Trichoglossus haematodus) from a zoo in Switzerland. The bird was reported being attacked by conspecifics, removed from the flock, and hospitalized. It showed lethargy, moderately reduced body condition, and lack of reaction to visual stimuli. Analgesic and antibiotic treatment were initiated but because of worsening of its general condition the bird was euthanized the following day. Necropsy revealed multifocal, subcutaneous hemorrhages, diffusely reddened lungs and a moderately dilated right heart with several intraluminal nematodes embedded in a coagulum. Four worms were collected and microscopically examined. They were identified as adult females, measuring 19-21 mm long x 0.4-0.5 mm wide, with general morphological and morphometric characteristics consistent with angiostrongylid nematodes. In lung sections, multifocal collection of thin-walled embryonated eggs in variable stages of development was observed along with fully developed nematode larvae within the lumina of alveoli and lung vessels. Associated granulomatous infiltrates indicated a severe, multifocal, chronic, granulomatous pneumonia. The diagnosis of A. dujardini infection was formulated by morphological examination of adult and larval stages, supported by molecular analysis (PCR-amplification and sequencing of the ITS2, 5.8S and 28S rDNA flanking regions). This is the first report of A. dujardini infection in an avian species, providing evidence that birds can serve as accidental hosts of this parasite in addition to mammals, and that the parasite can reach maturity and multiply in the avian cardiorespiratory system

First European report of Francisella tularensis subsp. holarctica isolation from a domestic cat

Francisella tularensis subsp. holarctica is a select agent causing life-threatening tularemia. It has been isolated from humans and animals, mainly lagomorphs and rodents, rarely other wild carnivore species. Increasing numbers of human tularemia cases have been reported during the last 5 years in Switzerland. Here we report the first isolation of Francisella tularensis subsp. holarctica from a domestic cat in Europe and compare its genome sequence with other Swiss isolates. The cat isolate shows a close phylogenetic relationship with a contemporary hare isolate from close geographic proximity, indicating a possible epidemiological link

Evaluation of Serological Methods and a New Real-Time Nested PCR for Small Ruminant Lentiviruses.

Small ruminant lentiviruses (SRLVs), i.e., CAEV and MVV, cause insidious infections with life-long persistence and a slowly progressive disease, impairing both animal welfare and productivity in affected herds. The complex diagnosis of SRLVs currently combines serological methods including whole-virus and peptide-based ELISAs and Immunoblot. To improve the current diagnostic protocol, we analyzed 290 sera of animals originating from different European countries in parallel with three commercial screening ELISAs, Immunoblot as a confirmatory assay and five SU5 peptide ELISAs for genotype differentiation. A newly developed nested real-time PCR was carried out for the detection and genotype differentiation of the virus. Using a heat-map display of the combined results, the drawbacks of the current techniques were graphically visualized and quantified. The immunoblot and the SU5-ELISAs exhibited either unsatisfactory sensitivity or insufficient reliability in the differentiation of the causative viral genotype, respectively. The new truth standard was the concordance of the results of two out of three screening ELISAs and the PCR results for serologically false negative samples along with genotype differentiation. Whole-virus antigen-based ELISA showed the highest sensitivity (92.2%) and specificity (98.9%) among the screening tests, whereas PCR exhibited a sensitivity of 75%

Case report: Intracoelomic neoplastic mass of undetermined origin in an Asia minor spur-thighed tortoise (Testudo graeca ibera)

This article describes the diagnostic, treatment and attempted characterization of a neoplasia of undetermined origin in a Asia minor spur-thighed tortoise. A 21-year-old male Asia minor spur-thighed tortoise (Testudo graeca ibera) was admitted for a 4-month history of diarrhea, and a 2-month history of anorexia and lethargy. Physical examination revealed a firm midcoelomic mass in the right prefemoral fossa. Blood biochemistry indicated hypocalcemia and mild elevation of aspartate aminotransferase. Supportive care was administered in the form of heating, baths, and calcium injections. Ultrasound examination of the coelomic cavity revealed a 6-cm diameter, highly vascularized mass with liver-like echogenicity. Neoplasia was suspected, and endoscopy was performed, revealing a brown circumscribed mass with smooth edges. Surgical removal of the mass was evaluated by CT scan and achieved via a plastrotomy; however, the patient died 1 day post-surgery. The mass was located on the dorsal right side of the coelomic cavity in the anatomic location of the right testicle. Histopathology revealed neoplastic cells organized in packets supported by fibrous septa. Neoplastic cells showed moderate and inconsistent positive immunohistochemical labeling for S100 and NSE, and negative immunohistochemical labeling for pan-cytokeratin, vimentin, CD3, CD79a, chromogranin A, and synaptophysin. The prominent histological and anatomical characteristics of the mass indicated a possible testicular or neuroendocrine (e.g., adrenal gland) origin. Due to inconclusive immunohistochemical profiles and poorly differentiated neoplastic cells, only a final diagnosis of intracoelomic malignant tumor of undetermined origin could be established. This case underscores the difficulties encountered in achieving definitive diagnoses of neoplastic diseases in reptile medicine

Fatal spirorchiidosis in European pond turtles (Emys orbicularis) in Switzerland.

Infections with intravascular digenean trematodes of the Spirorchiidae family (spirorchiidoses) are of great conservation concern both in marine and freshwater turtles due to their pathogenic potential. Between 2014 and 2021, Spirorchis sp. infections associated with granulomatous inflammation and sudden death were detected in European pond turtles (Emys orbicularis) from three conservation breeding facilities in Switzerland. Blood fluke eggs associated with lesions were found in the intestine, spleen, testis, skeletal musculature, heart, kidneys, stomach, pancreas, liver, lung, and meninges from nine pond turtles submitted for necropsy and in the intestinal content from five of these animals. Two novel polymerase chain reactions (PCRs) targeting the 28S ribosomal RNA gene and the ITS2 region and subsequent sequencing revealed 100% nucleotide identity with a Spirorchis sp. previously isolated from an Escambia map turtle (Graptemys ernsti) in the USA. Our findings suggest a spill-over event secondary to direct or indirect contact with invasive North American turtle species in Switzerland. We describe the clinical, haematological, ultrasonographical, endoscopical, parasitological, pathological, and molecular findings associated with spirorchiid blood fluke infections of the Spirorchis genus in E. orbicularis, as well as the biosecurity measures that were developed to prevent the spread of this parasite among breeding and highly endangered free-ranging E. orbicularis populations in Switzerland