Oncolytic virotherapy with intratumoral injection of vaccinia virus TG6002 and 5-fluorocytosine administration in dogs with malignant tumors.

TG6002 is an oncolytic vaccinia virus expressing FCU1 protein, which converts 5-fluorocytosine into 5-fluorouracil. The study objectives were to assess tolerance, viral replication, 5-fluorouracil synthesis, and tumor microenvironment modifications to treatment in dogs with spontaneous malignant tumors. Thirteen dogs received one to three weekly intratumoral injections of TG6002 and 5-fluorocytosine. The viral genome was assessed in blood and tumor biopsies by qPCR. 5-Fluorouracil concentrations were measured in serum and tumor biopsies by liquid chromatography or high-resolution mass spectrometry. Histological and immunohistochemical analyses were performed. The viral genome was detected in blood (7/13) and tumor biopsies (4/11). Viral replication was suspected in 6/13 dogs. The median intratumoral concentration of 5-fluorouracil was 314 pg/mg. 5-Fluorouracil was not detected in the blood. An increase in necrosis (6/9) and a downregulation of intratumoral regulatory T lymphocytes (6/6) were observed. Viral replication, 5-fluorouracil synthesis, and tumor microenvironment changes were more frequently observed with higher TG6002 doses. This study confirmed the replicative properties, targeted chemotherapy synthesis, and reversion of the immunosuppressive tumor microenvironment in dogs with spontaneous malignant tumors treated with TG6002 and 5-fluorocytosine.

Pharmacokinetics and tolerance of repeated oral administration of 5-fluorocytosine in healthy dogs.

BACKGROUND: 5-fluorocytosine is a pyrimidine and a fluorinated cytosine analog mainly used as an antifungal agent. It is a precursor of 5-fluorouracil, which possesses anticancer properties. To reduce systemic toxicity of 5-fluorouracil during chemotherapy, 5- fluorocytosine can be used as a targeted anticancer agent. Expression of cytosine deaminase by a viral vector within a tumor allows targeted chemotherapy by converting 5-fluorocytosine into the cytotoxic chemotherapeutic agent 5-fluorouracil. However, little is known about the tolerance of 5-fluorocytosine in dogs after prolonged administration. RESULTS: In three healthy Beagle dogs receiving 100 mg/kg of 5-fluorocytosine twice daily for 14 days by oral route, non-compartmental pharmacokinetics revealed a terminal elimination half-life of 164.5 ± 22.5 min at day 1 and of 179.2 ± 11.5 min, after 7 days of administration. Clearance was significantly decreased between day 1 and day 7 with 0.386 ± 0.031 and 0.322 ± 0.027 ml/min/kg, respectively. Maximal plasma concentration values were below 100 µg/ml, which is considered within the therapeutic margin for human patients. 5-fluorouracil plasma concentration was below the limit of detection at all time points. The main adverse events consisted of depigmented, ulcerated, exudative, and crusty cutaneous lesions 10 to 13 days after beginning 5-fluorocytosine administration. The lesions were localized to the nasal planum, the lips, the eyelids, and the scrotum. Histological analyses were consistent with a cutaneous lupoid drug reaction. Complete healing was observed 15 to 21 days after cessation of 5-fluorocytosine. No biochemical or hematological adverse events were noticed. CONCLUSIONS: Long term administration of 5-fluorocytosine was associated with cutaneous toxicity in healthy dogs. It suggests that pharmacotherapy should be adjusted to reduce the toxicity of 5-fluorocytosine in targeted chemotherapy.

Endoscopic assessment of presumed acquired pyloric narrowing in cats: A retrospective study of 27 cases.

Acquired pyloric narrowing is a rare and poorly-documented condition in cats, but the endoscopic appearance of pyloric narrowing has never previously been reported. The objectives of this study were to describe the clinical, endoscopic and histological features in cats with gastrointestinal signs where the pylorus could not be passed during endoscopy, and to compare these data with a control group. Medical files of cats that underwent upper GI endoscopy by the same operator between 2006 and 2015 were reviewed. Cats for which the pylorus could not be passed were assigned to the case group, whilst those with an easily-passable pylorus were assigned to the control group. The case group comprised 27 cats and control group comprised 35 cats. Median age and weight were not different between groups, but there were more Siamese cats in the case group (6/27) compared with the control group (1/35; P = 0.04). Chronic vomiting was the main clinical sign in both groups, but the vomitus was more likely to contain food in case group (23/25) than in cats in control group (17/30; P < 0.01). Endoscopic findings confirmed gastric inflammation in both groups, whilst histological findings revealed similar lymphoplasmacytic infiltration of the gastric mucosa and the duodenum in most cases, neoplastic features being infrequent. Acquired pyloric narrowing is probably an underdiagnosed condition in adult cats. A possible association between pyloric narrowing and gastrointestinal inflammatory disease requires further study but, for now, it is recommended that multiple gastric, pyloric, and duodenal biopsies be acquired during the endoscopy.

Cutaneous and systemic granulomatous disease associated with hairy vetch toxicosis in a French Holstein dairy herd.

This report describes an outbreak of hairy vetch toxicosis afflicting a herd of cattle with a fatal cutaneous and systemic granulomatous disease. It highlights how this condition remains poorly recognized by cattle production professionals in Europe and the need for communication about vetch-associated diseases.

Cet article décrit une épidémie de toxicose due à la vesce velue touchant un troupeau de bovins se manifestant par une maladie granulomateuse systémique et cutanée fatale. Ceci illustre comment cette atteinte reste peu connue par les professionnels de l'élevage en Europe et le besoin de communiquer sur les maladies associées à la vesce velue.

Este artículo describe un brote de toxicosis de veza vellosa (Vicia villosa) que afectó a un rebaño de ganado con una enfermedad granulomatosa cutánea y sistémica mortal. Destaca cómo esta condición sigue siendo poco reconocida por los profesionales de la producción ganadera en Europa y la necesidad de comunicación sobre las enfermedades asociadas al consumo de veza.

Este relato descreve um surto de intoxicação por ervilhaca peluda em um rebanho de gado com uma doença granulomatosa cutânea e sistêmica. Destaca-se como essa enfermidade continua pouco reconhecida pelos profissionais de bovinocultura na Europa, e a necessidade de comunicação sobre doenças associadas à ervilhaca.

Preclinical Evaluation of the Oncolytic Vaccinia Virus TG6002 by Translational Research on Canine Breast Cancer.

Oncolytic virotherapy is a promising therapeutic approach for the treatment of cancer. TG6002 is a recombinant oncolytic vaccinia virus deleted in the thymidine kinase and ribonucleotide reductase genes and armed with the suicide gene FCU1, which encodes a bifunctional chimeric protein that efficiently catalyzes the direct conversion of the nontoxic 5-fluorocytosine into the toxic metabolite 5-fluorouracil. In translational research, canine tumors and especially mammary cancers are relevant surrogates for human cancers and can be used as preclinical models. Here, we report that TG6002 is able to replicate in canine tumor cell lines and is oncolytic in such cells cultured in 2D or 3D as well as canine mammary tumor explants. Furthermore, intratumoral injections of TG6002 lead to inhibition of the proliferation of canine tumor cells grafted into mice. 5-fluorocytosine treatment of mice significantly improves the anti-tumoral activity of TG6002 infection, a finding that can be correlated with its conversion into 5-fluorouracil within infected fresh canine tumor biopsies. In conclusion, our study suggests that TG6002 associated with 5-fluorocytosine is a promising therapy for human and canine cancers.

Safety studies and viral shedding of intramuscular administration of oncolytic vaccinia virus TG6002 in healthy beagle dogs.

BACKGROUND: Cancer is a leading cause of mortality for both humans and dogs. As spontaneous canine cancers appear to be relevant models of human cancers, developing new therapeutic approaches could benefit both species. Oncolytic virotherapy is a promising therapeutic approach in cancer treatment. TG6002 is a recombinant oncolytic vaccinia virus deleted in the thymidine kinase and ribonucleotide reductase genes and armed with the suicide gene FCU1 that encodes a protein which catalyses the conversion of the non-toxic 5-fluorocytosine into the toxic metabolite 5-fluorouracil. Previous studies have shown the ability of TG6002 to infect and replicate in canine tumor cell lines, and demonstrated its oncolytic potency in cell lines, xenograft models and canine mammary adenocarcinoma explants. Moreover, 5-fluorouracil synthesis has been confirmed in fresh canine mammary adenocarcinoma explants infected with TG6002 with 5-fluorocytosine. This study aims at assessing the safety profile and viral shedding after unique or repeated intramuscular injections of TG6002 in seven healthy Beagle dogs. RESULTS: Repeated intramuscular administrations of TG6002 at the dose of 5 × 107 PFU/kg resulted in no clinical or biological adverse effects. Residual TG6002 in blood, saliva, urine and feces of treated dogs was not detected by infectious titer assay nor by qPCR, ensuring the safety of the virus in the dogs and their environment. CONCLUSIONS: These results establish the good tolerability of TG6002 in healthy dogs with undetectable viral shedding after multiple injections. This study supports the initiation of further studies in canine cancer patients to evaluate the oncolytic potential of TG6002 and provides critical data for clinical development of TG6002 as a human cancer therapy.

Model of persistent foot-and-mouth disease virus infection in multilayered cells derived from bovine dorsal soft palate.

Foot-and-mouth disease virus (FMDV) causes a highly contagious vesicular disease in livestock, with serious consequences for international trade. The virus persists in the nasopharynx of cattle and this slows down the process to obtain an FMDV-free status after an outbreak. To study biological mechanisms, or to identify molecules that can be targeted to diagnose or interfere with persistence, we developed a model of persistent FMDV infection in bovine dorsal soft palate (DSP). Primary DSP cells were isolated after commercial slaughter and were cultured in multilayers at the air-liquid interface. After 5 weeks of culture without further passage, the cells were infected with FMDV strain O/FRA/1/2001. Approximately, 20% of cells still had a polygonal morphology and displayed tight junctions as in stratified squamous epithelia. Subsets of cells expressed cytokeratin and most or all cells expressed vimentin. In contrast to monolayers in medium, multilayers in air demonstrated only a limited cytopathic effect. Integrin αV ß6 expression was observed in mono- but not in multilayers. FMDV antigen, FMDV RNA and live virus were detected from day 1 to 28, with peaks at day 1 and 2. The proportion of infected cells was highest at 24 hr (3% and 36% of cells at an MOI of 0.01 and 1, respectively). At day 28 after infection, at a time when animals that still harbour FMDV are considered carriers, FMDV antigen was detected in 0.2%-2.1% of cells, in all layers, and live virus was isolated from supernatants of 6/8 cultures. On the consensus level, the viral genome did not change within the first 24 hr after infection. Only a few minor single nucleotide variants were detected, giving no indication of the presence of a viral quasispecies. The air-liquid interface model of DSP brings new possibilities to investigate FMDV persistence in a controlled manner.

Seroprevalence and molecular characterization of foot-and-mouth disease virus in Chad.

This study aimed at determining the seroprevalence of foot-and-mouth disease (FMD) in domestic ruminants and at characterizing the virus strains circulating in four areas of Chad (East Batha, West Batha, Wadi Fira and West Ennedi). The study was carried out between October and November 2016. A total of 1,520 sera samples (928 cattle, 216 goats, 254 sheep and 122 dromedaries) were collected randomly for FMD serological analyses. Nine epithelial tissue samples were also collected from cattle showing clinical signs, for FMDV isolation and characterization. Serological results showed an overall NSP seroprevalence of 40% (375/928) in cattle in our sample (95% CrI [19-63]). However, seroprevalences of 84% (27/32), 78% (35/45) and 84% (21/25) were estimated in cattle over 5 years of age in East Batha, West Batha and Wadi Fira, respectively. In cattle under 1 year of age, 67% (18/27) seroprevalence was estimated in Wadi Fira, 64% (14/22) in East Batha and 59% (13/22) in West Batha. It was found that the high seroprevalences have been obtained in areas where pastures are shared by several different herds but also in farms where two to three species (bovine, caprine and ovine) are raised together. ELISA PrioCHECK® FMDV types O and A and in-house solid phase competition ELISA serotyping results showed that the four O, A, SAT1 and SAT2 serotypes have circulated in Chad in 2016. However, the type SAT2 dominated with an overall seroprevalence of 43% (29/67) and was present in the four areas investigated. The phylogenetic analyses of the VP1 coding sequence allowed determining the serotype SAT2 topotype VII, close to viral strains found in Cameroon in 2015 with a similarity of 98.60%.

Primary hyperparathyroidism due to a cystic parathyroid adenoma in a cat.

A 15-year-old neutered female domestic shorthair cat was presented for weight loss, polydipsia/polyuria, and lethargy. A large fluctuant mass was palpated in the ventral right cervical region. Biochemistry results were consistent with primary hyperparathyroidism. Parathyroid hormone level in the fluid was higher to that observed in the plasma, consistent with a cystic parathyroid lesion. Right parathyroidectomy and thyroidectomy were performed without complications. Ionized calcium normalized within a few hours. Histopathology yielded a diagnosis of cystic parathyroid adenoma. Follow-up showed complete recovery of clinical signs and normalization of ionized calcium. This case shows an uncommon presentation of feline primary hyperparathyroidism secondary to a cystic parathyroid adenoma and is, to our knowledge, the first case presented with a large palpable mass in which parathyroid hormone concentration was measured. This report highlights the value of selective hormonal analyses of the cystic fluid to confirm the origin of the cystic lesion pre-operatively.

Resumeq: A Novel Way of Monitoring Equine Diseases Through the Centralization of Necropsy Data.

The French surveillance network for causes of equine mortality (Resumeq) was created in 2015 for the qualitative surveillance of equine mortality through the centralization in a national database of necropsy data and their subsequent epidemiological analysis. It was designed to identify the causes of equine mortality, monitor their evolution over time and space, and detect emerging diseases as early as possible. Resumeq is an event-based surveillance system involving various players and structures. It is organized around a steering body, a scientific and technical support committee and a coordination unit. Different tools have been developed specifically for Resumeq. These include standardized necropsy protocols, a thesaurus for the anatomopathological terms and the causes of equine death, and an interactive web application so that network contributors can display data analysis results. The four French veterinary schools, seventeen veterinary laboratories, and ten veterinary clinics already contribute to the production and centralization of standardized data. To date, the data from around 1,000 equine necropsies have been centralized. While most deaths were located in western France, the geographic coverage is gradually improving. Data analysis allows the main causes of death to be ranked and major threats identified on a local, regional or national level. Initial results demonstrate the feasibility and benefits of this national surveillance tool. Moreover, in the future, this surveillance could take an international dimension if several countries decided to jointly capitalize on their necropsy data.

Proteogenomics Uncovers Critical Elements of Host Response in Bovine Soft Palate Epithelial Cells Following In Vitro Infection with Foot-And-Mouth Disease Virus.

Foot-and-mouth disease (FMD) is the most devastating disease of cloven-hoofed livestock, with a crippling economic burden in endemic areas and immense costs associated with outbreaks in free countries. Foot-and-mouth disease virus (FMDV), a picornavirus, will spread rapidly in naïve populations, reaching morbidity rates of up to 100% in cattle. Even after recovery, over 50% of cattle remain subclinically infected and infectious virus can be recovered from the nasopharynx. The pathogen and host factors that contribute to FMDV persistence are currently not understood. Using for the first time primary bovine soft palate multilayers in combination with proteogenomics, we analyzed the transcriptional responses during acute and persistent FMDV infection. During the acute phase viral RNA and protein was detectable in large quantities and in response hundreds of interferon-stimulated genes (ISG) were overexpressed, mediating antiviral activity and apoptosis. Although the number of pro-apoptotic ISGs and the extent of their regulation decreased during persistence, some ISGs with antiviral activity were still highly expressed at that stage. This indicates a long-lasting but ultimately ineffective stimulation of ISGs during FMDV persistence. Furthermore, downregulation of relevant genes suggests an interference with the extracellular matrix that may contribute to the skewed virus-host equilibrium in soft palate epithelial cells.

Coccygeal chordoma in a degu: case report and review of the literature.

An 8-y-old, intact female degu ( Octodon degus) was presented with a slow-growing mass on the tail tip. The mass was completely removed by partial caudectomy. Histologically, the last coccygeal vertebra was replaced by a lobulated neoplasm composed of large clear polygonal cells embedded in a myxoid alcian blue-positive matrix with highly vacuolated cytoplasm (physaliferous cells) and intracytoplasmic periodic acid-Schiff-positive granules. The neoplasm exhibited the morphologic features of a "classic" chordoma of humans, which is 1 of 3 distinct chordoma subtypes. Immunohistochemistry revealed dual expression of cytokeratin AE1/AE3 and vimentin, consistent with a diagnosis of chordoma. Chordomas are uncommon slow-growing neoplasms in humans and animals, arising from notochordal remnants. Depending on their subtype and location, they can have a high local recurrence rate and metastatic risk. Chordoma should be included in the differential diagnosis of a soft tissue mass on the tail of a degu, similar to the clinical situation in ferrets.

Validation of a new experimental model for assessing drug efficacy against infection with Trypanosoma equiperdum in horses.

Trypanosoma equiperdum, the causative agent of dourine, may affect the central nervous system, leading to neurological signs in infected horses. This location protects the parasite from most (if not all) existing chemotherapies. In this context, the OIE terrestrial code considers dourine as a non-treatable disease and imposes a stamping-out policy for affected animals before a country may achieve its dourine-free status. The use of practices as drastic as euthanasia remains controversial, but the lack of a suitable tool for studying a treatment's efficacy against dourine hampers the development of an alternative strategy for dourine infection management. The present study reports on the development of an experimental infection model for assessing drug efficacy against the nervous form of dourine. The model combines the infection of horses by Trypanosoma equiperdum and the search for trypanosomes in the cerebrospinal fluid (CSF) through an ultrasound-guided cervical sampling protocol. After a development phase involving four horses, we established an infection model that consists of inoculating 5 × 104T. equiperdum OVI parasites intravenously into adult Welsh mares (Equus caballus). To evaluate its efficacy, eight horses were infected according to this model. In all these animals, parasites were observed in the blood at 2 days post-inoculation (p.i.) and in CSF (12.5 ± 1.6 days p.i.) and seroconversion was detected (8.25 ± 0.5 days p.i.). All eight animals also developed fever (rectal temperature > 39 °C), low hematocrit (< 27%), and ventral edema (7.9 ± 2.0 days p.i.), together with other inconstant clinical signs such as edema of the vulva (six out of eight horses) or cutaneous plaques (three out of eight horses). This model provides a robust infection protocol that induces an acute trypanosome infection and that allows parasites to be detected in the CSF of infected horses within a period of time compatible with animal experimentation constraints. We conclude that this model constitutes a suitable tool for analyzing the efficacy of anti-Trypanosoma drugs and vaccines.

Bilateral bullous keratopathy secondary to melting keratitis in a Suri alpaca (Vicugna pacos).

An young alpaca was evaluated for bilateral progressive melting corneal ulcers and developped secondary bullous keratopathy during hospitalization. The tragic progression of melting ulcers in both eyes observed in our case leads us to recommend a rapid intensive medical therapy in young and debilitated alpacas presenting a corneal ulcer.

Fetopathic effects of experimental Schmallenberg virus infection in pregnant goats.

Schmallenberg virus (SBV) is an emerging virus responsible for congenital malformations in the offspring of domestic ruminants. It is speculated that infection of pregnant dams may also lead to a significant number of unrecognized fetal losses during the early period of gestation. To assess the pathogenic effects of SBV infection of goats in early pregnancy, we inoculated dams at day 28 or 42 of gestation and followed the animals until day 55 of gestation. Viremia in the absence of clinical signs was detected in all virus-inoculated goats. Fetal deaths were observed in several goats infected at day 28 or 42 of gestation and were invariably associated with the presence of viral genomic RNA in the affected fetuses. Among the viable fetuses, two displayed lesions in the central nervous system (porencephaly) in the presence of viral genome and antigen. All fetuses from goats infected at day 42 and the majority of fetuses from goats infected at day 28 of gestation contained viral genomic RNA. Viral genome was widely distributed in these fetuses and their respective placentas, and infectious virus could be isolated from several organs and placentomes of the viable fetuses. Our results show that fetuses of pregnant goats are susceptible to vertical SBV infection during early pregnancy spanning at least the period between day 28 and 42 of gestation. The outcomes of experimental SBV infection assessed at day 55 of gestation include fetal mortalities, viable fetuses displaying lesions of the central nervous system, as well as viable fetuses without any detectable lesion.

Ultrasonographic, endoscopic and histological appearance of the caecum in clinically healthy cats.

Objectives The aim of the study was to describe the ultrasonographic and endoscopic appearance and characteristics of the caecum in asymptomatic cats, and to correlate these findings with histology. Methods Ex vivo ultrasonographic and histologic evaluations of a fresh caecum were initially performed. Then, 20 asymptomatic cats, privately owned or originating from a reproductive colony, were recruited. All cats had an ultrasonographic examination of the ileocaecocolic junction, where the thickness of the caecal wall, ileocolic lymph nodes and the echogenicity of the local fat were assessed. They all underwent a colonoscopy with a macroscopic assessment of the mucosa and biopsies for histology. Results An ultrasonographic hypoechoic nodular inner layer, which corresponded to the coalescence of multiple lymphoid follicles originating from the submucosa and protruding in the mucosa on histology, was visible in all parts of the caecum. The combined mucosa and submucosa was measured ultrasonographically and defined as the follicular layer. Although all cats were asymptomatic, 3/19 cats showed mild caecal inflammation on histology. The most discriminatory ultrasonographic parameter in assessing this subclinical inflammation was the thickness of the follicular layer at the entrance of the caecum, with a cut-off value of 2.0 mm. All cats (20/20) showed some degree of macroscopic 'dimpling' of the caecal mucosa on endoscopy. Conclusions and relevance Lymphoid follicles in the caecal mucosa and submucosa constitute a unique follicular layer on ultrasound. In asymptomatic cats, a subtle, non-clinically relevant inflammation may exist and this is correlated with an increased thickness of the follicular layer on ultrasound. On endoscopy, a 'dimpled aspect' to the caecal mucosa is a normal finding in the asymptomatic cat.

Scleral and corneal xanthomatous inflammation in a gray mouse lemur (Microcebus murinus).

Bilateral multifocal corneal opacity was detected in a 4.5-year-old male captive gray mouse lemur (Microcebus murinus) without other clinical ocular changes. Histopathological examination revealed a severe diffuse granulomatous scleritis and focal keratitis with intralesional cholesterol, consistent with xanthomatous inflammation. This is the first report of xanthomatous inflammation in a gray mouse lemur. This condition may be the result of systemic factors (lipid metabolism disorders) and/or local predisposing factors such as hemorrhage or inflammation. The pathogenesis in this case could not be fully determined. Further studies on lemurs are required for a better understanding of their lipid metabolism, as well as for diagnosing and evaluating the incidence of xanthomatous inflammation in these species.

Ultrasonographic, endoscopic and histological appearances of the caecum in cats presenting with chronic clinical signs of caecocolic disease.

Objectives This study aimed to describe the ultrasonographic, endoscopic and histological characteristics of the caecum and ileocaecocolic junction in cats suffering from chronic clinical signs compatible with caecocolic disease. Methods Cats presenting with clinical signs suggestive of a caecocolic disease were prospectively recruited. All cats underwent an ultrasonographic examination of the caecum, ileum, colon, ileocolic lymph nodes and local mesenteric fat, in addition to comprehensive abdominal ultrasonography. This was followed by a colonoscopy with a macroscopic assessment of the caecocolic mucosa; caecocolic tissue samples were systematically collected for histologic analysis. Results Eighteen cats were included. Eleven of 18 cats had ultrasonographic abnormalities adjacent to the ileocaecocolic junction (lymphadenopathy, local steatitis) and 13/18 cats had abnormalities directly related to the junction (wall thickening, loss of wall layering). Seventeen of 18 cats had at least one ultrasonographic abnormality. Endoscopically, hyperaemia, oedema, discoloration and/or erosions were found in all cats. Each cat was classified as having mild or moderate-to-severe lesions according to endoscopic results; no classification could be established statistically for ultrasonographic results. The accentuation of the dimpled pattern tended to be inversely related to the severity of endoscopic lesion scoring. Histologically, a large proportion of cats showed typhlitis (13/16), one had lymphoma and two were normal. All cats with typhlitis also had colitis. There was only slight agreement between endoscopic and histological caecal results regarding the severity of lesions. Loss of caecal wall layering on ultrasound was found in 7/18 cats and, surprisingly, did not appear as a reliable predictor of the severity of inflammation or of malignancy; neither did local steatitis nor lymph node size. Conclusions and relevance Ultrasonography and endoscopy should not be used as the sole methods to investigate the ileocaecocolic region in cats with clinical signs suggestive of caecocolic disease. The presence of chronic clinical signs should routinely prompt histological biopsy.