Cytokine responses to EHV-1 infection in immune and non-immune ponies

Protecting equids against equine herpesvirus-1 (EHV-1) infection remains an elusive goal. Repeated infection with EHV-1 leads to protective immunity against clinical respiratory disease, and a study was conducted to measure the regulatory cytokine response (IFN-gamma and IL-4) in repeatedly infected immune ponies compared to non-immune ponies. Two groups of four ponies were established. Group 1 ponies had previously been infected on two occasions, and most recently 7 months before this study. Group 2 ponies had no history no vaccination or challenge infection prior to this study. Both groups were subjected to an intranasal challenge infection with EHV-1, and blood samples were collected pre-infection, and at 7 and 21 days post-infection for preparation of PBMCs. At each time point, the in vitro responses of PBMCs to stimulation with EHV-1 were measured, including IFN-gamma and IL-4 mRNA production, and lymphoproliferation. Group 1 ponies showed no signs of clinical disease or viral shedding after challenge infection. Group 2 ponies experienced a biphasic pyrexia, mucopurulent nasal discharge, and nasal shedding of virus after infection. Group 1 ponies had an immune response characterized both before and subsequent to challenge infection by an IFN-gamma response to EHV-1 in the absence of an IL-4 response, and demonstrated increased EHV-1-specific lymphoproliferation post-infection. Group 2 ponies had limited cytokine or lymphoproliferative responses to EHV-1 pre-challenge, and demonstrated increases in both IFN-gamma and IL-4 responses post-challenge, but without any lymphoproliferative response. Protective immunity to EHV-1 infection was therefore characterized by a polarized IFN-gamma dependent immunoregulatory cytokine response.     

Obstructive renal cyst in a dog: ultrasonography-guided treatment using puncture aspiration and injection with 95% ethanol

A 12-year-old, intact male, mixed-breed dog was evaluated after surgical removal of a mast cell tumor from the shoulder. Results of laboratory tests were within the reference ranges, but examination of urinary sediment identified epithelial cells of the upper urinary tract and microscopic hematuria. Abdominal ultrasound examination performed after a 12-hour fast identified a simple cyst in the left kidney. The cyst was approximately 26 mm in diameter, was anechoic, and had a thin, slightly hyperechoic wall with distal acoustic enhancement accompanied by dilatation of the caudal papillary duct and renal pelvis (Fig 1). Excretory urography with iopamidol^a contrast (800 mg/kg IV) confirmed the presence of dilatation of the left renal pelvis and collecting system (Fig 2).     

Review of literature and results from test matings of East Friesian milk sheep affected with brachygnathia inferior

Shortness of the lower jaw (brachygnathia inferior, underbite, overshot, parrot mouth) is an inborn and mostly hereditary malformation often seen in many sheep breeds. Chromosomal anomalies are generally not involved in brachygnathia inferior. Viral infections, teratogenic drugs and alkaloids of plants often lead to craniofacial malformations associated with brachygnathia inferior. A maternal deficiency of iron is discussed as a cause for brachygnathia inferior. We performed a three-year breeding trial using mainly East Friesian milk sheep affected by brachygnathic occlusion. Mating schemes included affected by affected and affected by unaffected matings. In the breeding trial, 60 lambs were born and from these 37 animals had variable degrees of brachygnathia inferior. The brachygnathic condition increased with rising age of the lambs. Extremely affected lambs showed palatine ulcers and growth retardation. Moreover, some animals had abnormal positions of the incisor teeth, distortion of the lower jaw and deformities of the external ear. Analysis of the pedigree did not support a monogenic inheritance pattern. An oligogenic inheritance including a dominant and recessive locus responsible for the major gene effects and possibly further modifying loci appeared much more likely. Other causes for brachygnathia inferior such as viral infections and anemia of the ewes could be ruled out. Chromosomal abnormalities were not evident and thus, large chromosomal defects were not associated with brachygnathia inferior.     

Chemotherapy and radiotherapy for treatment of cutaneous lymphoma in a ground cuscus (Phalanger gymnotis)

A 15-year-old female ground cuscus (Phalanger gymnotis) was presented with an isolated ulcerated, nonhealing lesion on the lateral thorax. Histopathology and immunohistochemistry were diagnostic for cutaneous T-cell lymphoma with incomplete excision. Oral chemotherapy with CCNU (lomustine) resulted in clinical remission that lasted 255 days, with no appreciable toxicity. Tumor recurrence was treated with radiation therapy, which resulted in 120 additional days of clinical remission. Subsequently, the tumor developed at a distant site and the cuscus was humanely euthanized. A slight decrease in appetite early in disease progression was the only adverse effect noted throughout the treatment period. Oral, minimally invasive chemotherapy, and adjunct radiation therapy were viable treatment options for this ground cuscus and should be considered for treatment of neoplasia in other nontraditional species.     

The innate antiviral immune system of the cat: molecular tools for the measurement of its state of activation

The innate immune system plays a central role in host defence against viruses. While many studies portray mechanisms in early antiviral immune responses of humans and mice, much remains to be discovered about these mechanisms in the cat. With the objective of shedding light on early host-virus interactions in felids, we have developed 12 real-time TaqMan(?) qPCR systems for feline genes relevant to innate responses to viral infection, including those encoding for various IFNα and IFNω subtypes, IFNβ, intracellular antiviral factor Mx, NK cell stimulator IL-15 and effectors perforin and granzyme B, as well as Toll-like receptors (TLRs) 3 and 8. Using these newly developed assays and others previously described, we measured the relative expression of selected markers at early time points after viral infection in vitro and in vivo. Feline embryonic fibroblasts (FEA) inoculated with feline leukemia virus (FeLV) indicated peak levels of IFNα, IFNβ and Mx expression already 6h after infection. In contrast, Crandell-Rees feline kidney (CrFK) cells inoculated with feline herpes virus (FHV) responded to infection with high levels of IFNα and IFNβ only after 24h, and no induction of Mx could be detected. In feline PBMCs challenged in vitro with feline immunodeficiency virus (FIV), maximal expression levels of IFNα, β and ω subtype genes as well as IL-15 and TLRs 3, 7 and 8 were measured between 12 and 24h after infection, whereas expression levels of proinflammatory cytokine gene IL-6 were consistently downregulated until 48h post inoculation. A marginal upregulation of granzyme B was also observed within 3h after infection. In an in vivo experiment, cats challenged with FIV exhibited a 2.4-fold increase in IFNα expression in blood 1 week post infection. We furthermore demonstrate the possibility of stimulating feline immune cells in vitro with various immune response modifiers (IRMs) already known for their immunostimulatory properties in mice and humans, namely Poly IC, Resiquimod (R-848) and dSLIM?, a synthetic oligonucleotide containing several unmethylated CpG motifs. Stimulation of feline PBMCs with dSLIM? and R-848 effectively enhanced expression of IFNα within 12h by factors of 6 and 12, respectively, and Poly IC induced an increase in Mx mRNA expression of 28-fold. Altogether, we describe new molecular tools and their successful use for the characterization of innate immune responses against viruses in the cat and provide evidence that feline cells can be stimulated by synthetic molecules to enhance their antiviral defence mechanisms.     

A new PCR followed by MboI digestion for the detection of all variants of the Clostridium perfringens cpb2 gene

Clostridium perfringens which is a causative agent of several diseases in animals and humans is capable of producing a variety of toxins. Isolates are typed into five types on the basis of the presence of one or more of the four major toxins genes, i.e. cpa, cpb, etx, and iap. A decade ago another toxin termed beta2 (beta2) and its gene (cpb2) were identified. Two alleles of cpb2 are known and a possible link between differences in gene expression and allelic variation has been reported. A correlation between the level of expression and the origin of the isolates has also been suggested. The demonstration and typing of the cpb2 gene in the genome of isolates can be seen as a vital part of research on the role of the beta2 toxin in the pathogenesis of disease. This study describes a PCR with a single primer set which in contrast to published primer sets recognizes both alleles. Subsequent restriction enzyme analysis of the PCR product enables typing of the alleles. Applying this protocol on a total of 102 isolates, a sub-variant was found which occurred only in C. perfringens isolates from pigs and appeared to be the predominant variant found in C. perfringens isolates from this species.     

Seizure activity in dogs is associated with enhanced TIMP-2 expression of microglia

In the pathogenesis of epilepsy aberrant synaptic plasticity plays an important role. Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are responsible for nervous tissue remodelling resulting in synaptic plasticity in the central nervous system (CNS) and might therefore be crucially involved in epileptogenesis. To assess the potential pathogenetic role of microglial MMPs and TIMPs in seizure induction, twenty-four dogs suffering from different intracranial diseases with and without seizure activity were comparatively examined. Microglial cells were isolated by density gradient centrifugation and their expression profiles of MMP-2, MMP-9, MMP-12, MMP-13, MMP-14, TIMP-1, TIMP-2, and RECK (reversion-inducing cysteine-rich protein with Kazal motifs) were examined via quantitative real-time PCR (qPCR). Interestingly, a significant up-regulation of TIMP-2 expression was found for the first time in dogs suffering from seizures. In conclusion, microglial TIMP expression might be involved in seizure generation.