Comparison of the local safety of two multi-component feline vaccines, adjuvanted (1 mL) versus non-adjuvanted at reduced volume (0.5 mL), using computed tomography imaging.

In 2020, a new 0.5 mL presentation of PUREVAX® RCP FeLV was registered and introduced in Europe. The objectives of this study were to investigate the local safety of this non-adjuvanted vaccine at reduced volume by classical methods (clinical examination, histopathology) and to evaluate the suitability of an alternative non-invasive methodology, the computed tomography (CT). For this purpose, the course of local reactions was assessed for 3 months after subcutaneous injection of PUREVAX® RCP FeLV 0.5 mL and compared to an adjuvanted vaccine, LEUCOFELIGEN® FeLV/RCP 1.0 mL. Injection site reactions consisted mainly of swelling reactions, which were more frequent, more pronounced and long-lasting in the adjuvanted vaccine group. Microscopically, in this group, moderate to severe inflammatory reactions were observed on day 7 (D7) and D21 post-injection and still present on D84, while mild inflammatory lesions were observed in the non-adjuvanted vaccine group only on D7 and D21. With the adjuvanted vaccine, inflamed areas were measurable by CT scan in all cats on D7 and D21, whereas they were detected only on D7 and only in 20 % of cats from the non-adjuvanted vaccine group. Besides the higher frequency, the mean inflamed volume was nearly 300 times larger in adjuvanted vaccine group on D7. Using different methodologies, the favorable safety profile of PUREVAX® RCP FeLV 0.5 mL was confirmed. Furthermore, the vaccine is aligned with current vaccination guidelines by inducing less inflammatory reactions, being adjuvant-free and injectable under a reduced volume, thus improving the convenience of administration in recommended sites (eg, legs). CT scan proved to be a suitable non-invasive method for the experimental follow-up of injection site reactions, yielding results consistent with clinical assessment and histopathology on D7 and D21. CT scan substantiated large differences between the investigated vaccines with a more prominent inflammatory reaction after injection of an adjuvanted vaccine.

A novel subunit vaccine based on the viral protein 2 of porcine parvovirus: safety profile in bred pigs at different stages of the reproduction cycle and in offspring.

Porcine parvovirus 1 (PPV1) viral protein (VP) 2 is the primary antigen responsible for inducing specific protective immunity, so it is a desirable target for development of recombinant subunit vaccines to prevent PPV1 disease. The objective of this study was to evaluate repeated doses of a novel VP2-based PPV1 subunit vaccine, namely ReproCyc® ParvoFLEX, for safety in bred pigs and in offspring under experimental settings. Therefore, the investigation of safety at all breeding stages was evaluated in four independent studies involving: pre-breeding gilts (study A), breeding-age gilts and boars (study B), early and late gestating sows and offspring (study C) and lactating sows and offspring (study D). In all four studies, animals were free from PPV1 based on serology and PCR prior to inclusion. All studies comprised one or two vaccinated groups that received the PPV1 subunit vaccine and a negative control group. Thus, safety was established due to the lack of significant differences between the vaccinated groups and the corresponding unvaccinated (negative control) groups. Gilts, sows and boars were evaluated for local and systemic reactions after vaccination as well as for reproductive performance. The survival rate and average daily weight gain (ADWG) from birth to weaning in offspring was evaluated in studies C and D. Additionally, serology was determined in studies A, C and D. The vaccine was shown to be safe with no relevant significant differences between vaccinated and unvaccinated groups in any experiment. Therefore, repeated doses of ReproCyc® ParvoFLEX were safe in target animals at different stages of the reproductive cycle and in offspring, placing this vaccine as a suitable candidate for mass vaccination programs in breeding herds.

Accumulation of Extracellular Matrix in Advanced Lesions of Canine Distemper Demyelinating Encephalitis.

In demyelinating diseases, changes in the quality and quantity of the extracellular matrix (ECM) may contribute to demyelination and failure of myelin repair and axonal sprouting, especially in chronic lesions. To characterize changes in the ECM in canine distemper demyelinating leukoencephalitis (DL), histochemical and immunohistochemical investigations of formalin-fixed paraffin-embedded cerebella using azan, picrosirius red and Gomori`s silver stain as well as antibodies directed against aggrecan, type I and IV collagen, fibronectin, laminin and phosphacan showed alterations of the ECM in CDV-infected dogs. A significantly increased amount of aggrecan was detected in early and late white matter lesions. In addition, the positive signal for collagens I and IV as well as fibronectin was significantly increased in late lesions. Conversely, the expression of phosphacan was significantly decreased in early and more pronounced in late lesions compared to controls. Furthermore, a set of genes involved in ECM was extracted from a publically available microarray data set and was analyzed for differential gene expression. Gene expression of ECM molecules, their biosynthesis pathways, and pro-fibrotic factors was mildly up-regulated whereas expression of matrix remodeling enzymes was up-regulated to a relatively higher extent. Summarized, the observed findings indicate that changes in the quality and content of ECM molecules represent important, mainly post-transcriptional features in advanced canine distemper lesions. Considering the insufficiency of morphological regeneration in chronic distemper lesions, the accumulated ECM seems to play a crucial role upon regenerative processes and may explain the relatively small regenerative potential in late stages of this disease.

Brain inflammation, neurodegeneration and seizure development following picornavirus infection markedly differ among virus and mouse strains and substrains.

Infections, particularly those caused by viruses, are among the main causes of acquired epilepsy, but the mechanisms causing epileptogenesis are only poorly understood. As a consequence, no treatment exists for preventing epilepsy in patients at risk. Animal models are useful to study epileptogenesis after virus-induced encephalitis and how to interfere with this process, but most viruses that cause encephalitis in rodents are associated with high mortality, so that the processes leading to epilepsy cannot be investigated. Recently, intracerebral infection with Theiler's murine encephalomyelitis virus (TMEV) in C57BL/6 (B6) mice was reported to induce early seizures and epilepsy and it was proposed that the TMEV mouse model represents the first virus infection-driven animal model of epilepsy. In the present study, we characterized this model in two B6 substrains and seizure-resistant SJL/J mice by using three TMEV (sub)strains (BeAn-1, BeAn-2, DA). The idea behind this approach was to study what is and what is not necessary for development of acute and late seizures after brain infection in mice. Receiver operating characteristic (ROC) curve analysis was used to determine which virus-induced brain alterations are associated with seizure development. In B6 mice infected with different TMEV virus (sub)strains, the severity of hippocampal neurodegeneration, amount of MAC3-positive microglia/macrophages, and expression of the interferon-inducible antiviral effector ISG15 were almost perfect at discriminating seizing from non-seizing B6 mice, whereas T-lymphocyte brain infiltration was not found to be a crucial factor. However, intense microglia/macrophage activation and some hippocampal damage were also observed in SJL/J mice. Overall, the TMEV model provides a unique platform to study virus and host factors in ictogenesis and epileptogenesis.

Limited role of regulatory T cells during acute Theiler virus-induced encephalitis in resistant C57BL/6 mice.

BACKGROUND: Theiler's murine encephalomyelitis virus (TMEV) infection represents a commonly used infectious animal model to study various aspects of the pathogenesis of multiple sclerosis (MS). In susceptible SJL mice, dominant activity of Foxp3(+) CD4(+) regulatory T cells (Tregs) in the CNS partly contributes to viral persistence and progressive demyelination. On the other hand, resistant C57BL/6 mice rapidly clear the virus by mounting a strong antiviral immune response. However, very little is known about the role of Tregs in regulating antiviral responses during acute encephalitis in resistant mouse strains. METHODS: In this study, we used DEREG mice that express the diphtheria toxin (DT) receptor under control of the foxp3 locus to selectively deplete Foxp3(+) Tregs by injection of DT prior to infection and studied the effect of Treg depletion on the course of acute Theiler's murine encephalomyelitis (TME). RESULTS: As expected, DEREG mice that are on a C57BL/6 background were resistant to TMEV infection and cleared the virus within days of infection, regardless of the presence or absence of Tregs. Nevertheless, in the absence of Tregs we observed priming of stronger effector T cell responses in the periphery, which subsequently resulted in a transient increase in the frequency of IFNγ-producing T cells in the brain at an early stage of infection. Histological and flow cytometric analysis revealed that this transiently increased frequency of brain-infiltrating IFNγ-producing T cells in Treg-depleted mice neither led to an augmented antiviral response nor enhanced inflammation-mediated tissue damage. Intriguingly, Treg depletion did not change the expression of IL-10 in the infected brain, which might play a role for dampening the inflammatory damage caused by the increased number of effector T cells. CONCLUSION: We therefore propose that unlike susceptible mice strains, interfering with the Treg compartment of resistant mice only has negligible effects on virus-induced pathologies in the CNS. Furthermore, in the absence of Tregs, local anti-inflammatory mechanisms might limit the extent of damage caused by strong anti-viral response in the CNS.

Induced and thymus-derived Foxp3⁺ regulatory T cells share a common niche.

Foxp3⁺ regulatory T (Treg) cells, which play a central role for the maintenance of immune homeostasis and self-tolerance, are known to be both generated in the thymus (thymus-derived, tTreg cells) and in the periphery, where they are converted from conventional CD4⁺ T cells (induced Treg (iTreg) cells). Recent data suggest a division of labor between these two Treg-cell subsets since their combined action was shown to be essential for protection in inflammatory disease models. Here, using the transfer colitis model, we examined whether tTreg cells and iTreg cells fill different niches within the CD4⁺ T-cell compartment. When naive T cells were co-transferred with either pure tTreg cells or with a mixture of tTreg cells and iTreg cells, induction of Foxp3⁺ Treg cells from naive T cells was not hampered by preoccupation of the Treg-cell niche. Using neuropilin-1 (Nrp1) as a surface marker to separate tTreg cells and iTreg cells, we demonstrate that tTreg cells and iTreg cells alone can completely fill the Treg-cell niche and display comparable TCR repertoires. However, when transferred together Nrp1⁺ tTreg cells outcompeted Nrp1⁻ iTreg cells and dominated the Treg-cell compartment. Taken together, our data suggest that tTreg cells and iTreg cells share a common peripheral niche.

[Congenital cardiac anomalies (pentalogy of Fallot) in a two year old ram with brachygnathia inferior]. / Kongenitale Herzanomalien (Fallotsche Pentalogie) bei einem zweijährigen Schafbock mit Brachygnathia inferior.

In a nearly two-year-old ram, descending from a breeding trial to investigate the effects of shortness of the lower jaw (brachygnathia inferior), a congenital cardiac anomaly was observed. At the age often months the affected animal, a cross breed of Cameroon Sheep and East Friesian Milk Sheep, showed exercise-induced respiratory distress for the first time. Auscultation revealed a loud systolic heart murmur (grade 5) on both sides of the thorax, most prominent over the left third intercostal space at shoulder height. Postmortem examination of the ram's heart showed a pentalogy of Fallot, consisting of a pulmonic stenosis, a ventricular septal defect, an overriding aorta, a right ventricular hypertrophy and a patent foramen ovale. A genetic defect has to be considered as a possible reason.

Epidural myelolipoma in a Husky-cross: a case report.

Epidural spinal myelolipoma was diagnosed in an 11.5-year-old castrated male Husky-cross that was evaluated at the veterinary teaching hospital due to progressive thoracolumbar spinal hyperaesthesia and mild proprioceptive pelvic limb ataxia. A focal, ill-defined mildly inhomogenous extradural mass lesion was detected by MRI. The dog was euthanized. At necropsy an extradurally located reddish mass of about 2.5 cm in diameter was present in the vertebral canal. The mass was identified histopathologically as an epidural myelolipoma.

Spatio-temporal development of axonopathy in canine intervertebral disc disease as a translational large animal model for nonexperimental spinal cord injury.

Spinal cord injury (SCI) represents a devastating central nervous system disease that still lacks sufficient therapies. Here, dogs are increasingly recognized as a preclinical animal model for the development of future therapies. The aim of this study was a detailed characterization of axonopathy in canine intervertebral disc disease, which produces a mixed contusive and compressive injury and functions as a spontaneous translational animal model for human SCI. The results revealed an early occurrence of ultrastructurally distinct axonal swelling. Immunohistochemically, enhanced axonal expression of ß-amyloid precursor protein, non-phosphorylated neurofilament (n-NF) and growth-associated protein-43 was detected in the epicenter during acute canine SCI. Indicative of a progressive axonopathy, these changes showed a cranial and caudally accentuated spatial progression in the subacute disease phase. In canine spinal cord slice cultures, immunoreactivity of axons was confined to n-NF. Real-time quantitative polymerase chain reaction of naturally traumatized tissue and slice cultures revealed a temporally distinct dysregulation of the matrix metalloproteinases (MMP)-2 and MMP-9 with a dominating expression of the latter. Contrasting to early axonopathy, diminished myelin basic protein immunoreactivity and phagocytosis were delayed. The results present a basis for assessing new therapies in the canine animal model for translational research that might allow partial extrapolation to human SCI.

Matrix metalloproteinase-12 deficiency ameliorates the clinical course and demyelination in Theiler's murine encephalomyelitis.

Matrix metalloproteinases (MMPs) are a family of extracellular proteases involved in the pathogenesis of demyelinating diseases like multiple sclerosis (MS). The aim of the present study was to investigate whether MMPs induce direct myelin degradation, leukocyte infiltration, disruption of the blood-brain barrier (BBB), and/or extracellular matrix remodeling in the pathogenesis of Theiler's murine encephalomyelitis (TME), a virus-induced model of MS. During the demyelinating phase of TME, the highest transcriptional upregulation was detected for Mmp12, followed by Mmp3. Mmp12 (-/-) mice showed reduced demyelination, macrophage infiltration, and motor deficits compared with wild-type- and Mmp3 knock-out mice. However, BBB remained unaltered, and the amount of extracellular matrix deposition was similar in knock-out mice and wild-type mice. Furthermore, stereotaxic injection of activated MMP-3, -9, and -12 into the caudal cerebellar peduncle of adult mice induced a focally extensive primary demyelination prior to infiltration of inflammatory cells, as well as a reduction in the number of oligodendrocytes and a leakage of BBB. All these results demonstrate that MMP-12 plays an essential role in the pathogenesis of TME, most likely due to its primary myelin- or oligodendrocyte-toxic potential and its role in macrophage extravasation, whereas there was no sign of BBB damage or alterations to extracellular matrix remodeling/deposition. Thus, interrupting the MMP-12 cascade may be a relevant therapeutic approach for preventing chronic progressive demyelination.

Distinct spatio-temporal extracellular matrix accumulation within demyelinated spinal cord lesions in Theiler's murine encephalomyelitis.

The accumulation of extracellular matrix (ECM) and glial scar formation are considered important factors for the failure of regeneration in central nervous system (CNS) injury and multiple sclerosis. Theiler's murine encephalomyelitis (TME) as a model of multiple sclerosis served to evaluate the spatio-temporal course of ECM alterations in demyelinating conditions. Microarray analysis revealed only mildly upregulated gene expression of ECM molecules, their biosynthesis pathways and pro-fibrotic factors, while upregulation of matrix remodeling enzymes was more prominent. Immunohistochemistry demonstrated progressive accumulation of chondroitin sulfate proteoglycans, glycoproteins and collagens within demyelinated TME lesions, paralleling the development of astrogliosis. Deposition of collagen IV, laminin, perlecan and tenascin-C started 28 days postinfection (dpi), collagen I, decorin, entactin and neurocan accumulated from 56 dpi on, and fibronectin from 98 dpi on. The basement membrane (BM) molecules collagen IV, entactin, fibronectin, laminin and perlecan showed perivascular and parenchymal deposition, while the non-BM components collagen I, decorin, neurocan and tenascin-C only accumulated in a nonvascular pattern in demyelinated areas. Contrary, phosphacan expression progressively decreased during TME. The immunoreactivity of aggrecan and brevican remained unchanged. The spatio-temporal association of matrix accumulation with astrogliosis suggests a mainly astrocytic origin of ECM deposits, which in turn may contribute to remyelination failure in TME.

Prominent microglial activation in the early proinflammatory immune response in naturally occurring canine spinal cord injury.

Better understanding of the pathogenesis of spinal cord injury (SCI) is needed for the development of new therapeutic strategies. Spinal cord injury has been investigated in various rodent models, but extrapolation to humans requires the use of a large animal model that more closely mimics human SCI. Dogs frequently develop spontaneous SCI with features that bear a striking resemblance to the human counterpart. We investigated the temporal course of the immune response during naturally occurring canine SCI and in organotypic canine spinal cord slice cultures that are devoid of peripheral immune cells. By immunohistochemistry, the inflammatory response in subacute canine SCI was largely restricted to resident immune cells as demonstrated by activation of major histocompatibility complex class II-expressing microglia/macrophages. By quantitative polymerase chain reaction, there was parallel upregulation of proinflammatory cytokine gene expression (i.e. of interleukin 6 [IL-6] and IL-8 with a trend toward upregulation of tumor necrosis factor) in acute canine SCI. Expression of neuroprotective cytokines (e.g. IL-10) remained unchanged, and transforming growth factor ß upregulation was delayed. In organotypic spinal cord slices, there was similar activation of major histocompatibility complex class II-positive microglia and prolonged upregulation of inflammatory cytokines, indicating that resident rather than infiltrating cells play major roles in the postinjury immune response. Thus, canine SCI represents a bridge between rodent models and human SCI that may be relevant for clinical and preclinical treatment studies.

Pathologic fracture of the femur due to endometrial adenocarcinoma metastasis in a female pet rabbit (Oryctolagus cuniculi).

This case report describes the clinical, pathomorphological and histopathological findings in a 12-year-old female pet rabbit with a pathologic fracture of the femur due to a metastasising endometrial adenocarcinoma. The animal was presented due to a history of right hindlimb lameness and inappetence. A fracture of the right femur with adjacent lyric bone lesions was detected by clinical and X-ray examination. Necropsy and histopathology revealed an endometrial adenocarcinoma with metastasis to the right femoral bone, resulting in the pathologic fracture. In both locations, tumour cells were immunohistochemically positive for pan-cytokeratin. Additionally, the animal suffered from an Encephalitozoon cuniculi infection which manifested itself in a multifocal granulomatous encephalitis and bilateral chronic inflammatory lesions in the kidneys.The report shows that in case of bone fractures in female pet rabbits the possibility of a pathologic fracture due to metastasising endometrial adenocarcinoma has to be considered.

Sustained viral load and late death in Rag2-/- mice after influenza A virus infection.

The importance of the adaptive immune response for secondary influenza infections and protection from a lethal challenge after vaccination has been well documented. However, some controversy still exists concerning the specific involvement of B and T cells during a primary infection. Here, we have followed the survival, weight loss, viral load and lung pathology in Rag2-/- knock-out mice after infection with influenza A virus (H1N1). Infected wild type mice initially lost weight early after infection but then cleared the virus and recovered. Rag2-/- mice, however, showed similar weight loss kinetics in the early stages after infection but weight loss continued post infection and culminated in death. In contrast to wild type mice, Rag2-/- mice were not able to clear the virus, despite an increased inflammatory response. Furthermore, they did not recruit virus-specific lymphocytes into the lung in the later stages after infection and exhibited sustained pulmonary lesions.

Persistent truncus arteriosus with dissecting aneurysm and subsequent cardiac tamponade in a lamb.

The present case report describes the necropsy and histopathology findings of a 3-week-old lamb with persistent truncus arteriosus (PTA). This rare cardiac malformation was characterized by the presence of a common arterial trunk arising from the right ventricle and overriding a ventricular septal defect. The pulmonary arteries originated from a short common trunk from this PTA, which subsequently continued as the thoracic aorta. The death of the lamb was attributed to a rupture of the PTA with subsequent cardiac tamponade. Histologically, a dissecting aneurysm with elastic fiber fragmentation in the wall of the PTA was identified as the underlying pathologic condition. Altered hemodynamic forces with subsequent secondary vasculopathy, as well as congenital primary vasculopathies, should be considered as an underlying pathogenetic mechanism.

Diagnostic procedures and surgical treatment of craniodorsal coxofemoral luxation in calves.

OBJECTIVE: To report use of ultrasonographic examination of the coxofemoral joint, a surgical technique for repair of craniodorsal coxofemoral dislocation, and outcome. STUDY DESIGN: Clinical case reports. ANIMALS: Calves (n=4) with coxofemoral luxation. METHODS: Craniodorsal coxofemoral luxation was diagnosed by physical examination, radiographic, and ultrasonographic findings. Open surgical reduction of the femoral head was performed using a modified caudal approach. RESULTS: Craniodorsal luxation of the femoral head and the presence of an intact femoral neck were confirmed by ultrasonography. All luxations were successfully reduced and reluxation did not occur. At follow-up, 1 heifer had calved and 1 was 5 months pregnant. One calf died of bronchopneumonia 6 days after surgery. One calf had severe coxofemoral degenerative joint disease diagnosed (ultrasonography, radiography, and arthrocentesis) 3 months after surgery and confirmed by necropsy. CONCLUSION: Ultrasonography proved to be a simple and effective non-invasive technique for diagnosis of coxofemoral luxation. Immediate surgical intervention in hip dislocation in calves is necessary to avoid unnecessary trauma to subchondral structures. In calves, open instead of closed surgical reduction appears preferable because it allows access to the acetabular cavity for removal of debris. CLINICAL RELEVANCE: Ultrasonography should be considered a supplementary but not an alternative to radiographic examination for diagnosis of coxofemoral luxation and for follow-up examinations after reduction.

MMP-12, MMP-3, and TIMP-1 are markedly upregulated in chronic demyelinating theiler murine encephalomyelitis.

Theiler murine encephalomyelitis (TME) represents a highly relevant viral model for multiple sclerosis. Matrix metalloproteinases (MMPs) degrade extracellular matrix molecules and are involved in demyelination processes. To elucidate their impact on demyelination in TME, spinal cords of TME virus (TMEV)-infected SJL/J mice were taken at 9 different time points postinfection (pi) ranging from 1 hour to 196 days pi and investigated for the expression of TMEV, MMP-2, -3, -7, -9, -10, -11, -12, -13, -14, -15, -24, and TIMP-1 to -4 by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). High TMEV RNA levels were detectable throughout the observation period using RT-qPCR. In addition, TMEV RNA was visualized within demyelinated lesions by in situ hybridization. MMP-3 mRNA was significantly upregulated at 1 day pi and again in the late phase of infection. TIMP-1 mRNA was significantly elevated throughout the observation period. MMP-12 mRNA was most prominently upregulated in the late phase of infection and MMP-12 protein was localized in intralesional microglia/macrophages and astrocytes by immunohistochemistry. In summary, in early TMEV infection, MMP-3 and TIMP-1 mRNA upregulation might be directly virus-induced, whereas persistent TMEV infection directly or indirectly stimulated MMP-12 production in microglia/macrophages and astrocytes and might account for ongoing demyelination in TME.