Cloning,expression, purification,and biological activity of five feline type I interferons

Type I interferons (IFN) are important mediators of the host defense against viral infections in mammals. In humans multiple subtypes of IFN-alpha exist, most of which possess antiviral activity. Little is known about the type I IFN genes in cats and the role they may play in feline immunological responses to viruses. We have isolated cDNAs encoding five feline IFN-alpha (feIFN) subtypes that share from 95 to 99% amino acid sequence identity. FeIFN-alpha5 has five additional amino acids inserted at position 139, which are not present in the other four subtypes. Sequence identity of the feIFN proteins encoded by the five clones compared to human IFN-alpha2 is approximately 60%. Unlike most of the human subtypes, each of the five feline IFN sequences has an N-glycosylation recognition site. Expression of all five feIFN-alpha subtypes in Chinese hamster ovary (CHO) cells was confirmed by Western blot analysis, and all resulting proteins were glycosylated. The antiviral activity of each feIFN-alpha subtype produced in transiently transfected CHO cell cultures was tested in vitro. In addition, subtype feIFN-alpha6 was expressed in the yeast, Pichia pastoris. The resulting secreted mature recombinant protein was purified and demonstrated significant antiviral activity and induction of 2',5'-oligoadenylate synthetase activity in vitro.     

Evaluation of L-selectin expression and assessment of protein tyrosine phosphorylation in bovine polymorphonuclear neutrophil leukocytes around parturition

Impaired polymorphonuclear neutrophil leukocyte (PMN) function around parturition has been associated with increased clinical mastitis in dairy cows. Rolling and attachment of PMN to the endothelium is the first step in the recruitment process and is accomplished by interaction between L-selectin on PMN and its ligand on endothelial cells. Furthermore, tyrosine phosphorylation is involved in the initiation of many PMN functions. The objective of this work was to determine changes in expression of L-selectin and tyrosine phosphorylation in the perinatal period. Eight clinically healthy Holstein cows were used as PMN donors at d-21, -14, -7,0 (calving), +1, +2, +7, +14, +28. Evaluation of L-selectin expression was carried out on activated and resting PMN. Anti-bovine L-selectin monoclonal antibody (MAB) and flow cytometric analysis were used to measure the percentage of PMN fluorescing and receptor expression (log mean fluorescent channel, LMFC). Activated and resting PMN showed similar trends in % PMN fluorescence and LM FC. The percentage of PMN fluorescing tended to decrease at parturition, followed by a significant increase at d +14 and +28 (P < 0.02). For LMFC a decrease was observed on d +1 followed by an increase through d +28 (P < 0.01). Protein tyrosine phosphorylation of lysates prepared from PMN isolated throughout the study was detected by electrophoresis and western blotting using anti-phosphotyrosine MAB. Several protein bands were tyrosine phosphorylated. Two of these bands (42-44 kDa and 90 kDa) varied in intensity over time. The intensity of the 42-44 kDa band gradually increased from d -7, peaked at d +7 (P < 0.03), and steadily decreased to d +28 (P < 0.02). Antibody to activated mitogen protein kinase reacted with the 42-44 kDa band. Reduced PMN function during the periparturient period could be related to reduced L-selectin adhesion molecules on the cell surface, and to modulation in the phosphorylation of functionally important molecules.     

Effect of intravenous infusion of recombinant ovine leptin on feed intake and serum concentrations of GH, LH, insulin, IGF-1, cortisol, and thyroxine in growing prepubertal ewe lambs

In sheep, serum concentrations of leptin change congruently with increases or decreases in nutritional status, while intracerebroventricular infusions of leptin dramatically suppress feed intake in well-fed lambs, and may also increase growth hormone (GH), and/or luteinizing hormone (LH) in undernourished lambs. The objective of the present study was to determine the effects of peripherally delivered ovine leptin, via intravenous infusions, on feed intake and serum concentrations of GH, LH, insulin, IGF-1, cortisol, and thyroxine. Twelve ewe lambs weighing 29.4 +/- 0.7 kg were infused intravenously with a linearly increasing dose of leptin or saline (n = 6 per group) for 10 days, reaching a maximum dose delivered of 0.5mg/h on day 10. Feed intake was assessed twice daily, and blood samples were collected every 10 min for 6 h on days 0, 2, 5, 8, and 10. Serum concentrations of leptin increased in leptin-treated lambs by day 2 (P = 0.05), and continued to increase to concentrations 9-fold greater than saline-infused lambs by day 10 (P < 0.001). Despite the substantial increase in serum leptin, feed intake did not differ between leptin and saline-infused lambs except on day 3.5 (P = 0.01). Furthermore, intravenous infusions of leptin did not significantly influence serum concentrations of insulin, cortisol, IGF-1, thyroxine, LH, or GH. Collectively, these observations contrast with the potent hypophagic effects of leptin when delivered intracerebroventricularly into well-fed lambs. The reasons for the disparate response of lambs treated intravenously with leptin, versus that reported for lambs treated intracerebroventricularly with leptin are not known, but may provide insight into the mechanism(s) of leptin resistance.     

Matrix metalloproteinase activity in tumor, stromal tissue, and serum from cats with malignancies

Matrix metalloproteinases (MMPs) are enzymes that play key roles in angiogenesis, tumor invasion, and metastasis in a wide variety of species. The purpose of this study was to evaluate pro and active MMP 2 and 9 concentrations in tumor, normal stromal tissue, and serum from tumor-bearing cats. We hypothesized that serum concentrations of pro and active forms of MMPs 2 and 9 would be predictive of MMP concentrations in tumor tissue and that these MMP concentrations would correlate with the histopathologic grade of the malignancies. Pro and active forms of MMPs 2 and 9 were determined by gelatin zymography and subsequent computerized densitometry from tumor and nearby stromal tissue and serum from 49 cats with various malignancies. The serum concentrations of MMPs from these tumor-bearing cats were compared with serum concentrations of MMPs from 44 normal cats of similar age and gender. Measurable concentrations of MMPs 2 and 9 were found within tumor, stromal, and serum samples. Mean concentrations of total pro and active MMPs 2 and 9 within tumor tissue were significantly higher (P values <.0001, .0031, <.001, and .0064, respectively) when compared with stromal tissue from the same animals. Serum MMP concentrations from tumor-bearing cats were higher than those from normal cats. Poor correlation was found between serum MMP concentrations and tissue MMP concentrations of increasing histologic grades of malignancies.     

Effects of dietary flaxseed oil supplementation on equine plasma fatty acid concentrations and whole blood platelet aggregation

An 18-week feeding trial was performed to investigate the effects of an omega-3 (n-3) fatty acid-enriched ration on plasma fatty acid concentrations and platelet aggregation in healthy horses. Flaxseed oil served as the source of the n-3 fatty acid alpha-linolenic acid (ALA). Twelve horses were fed dietary maintenance requirements using a complete pelleted ration (80%) and timothy grass hay (20%) for a 2-week acclimation period before being randomly assigned either to a treatment (group 1) or control (group 2) group. Group 2 horses (n = 6) were fed the diet described in the acclimation period, whereas group I horses (n = 6) were fed a 10% flaxseed oil-enriched complete pellet (80%) and grass hay (20%). Biological samples and physical measurements were collected at one point during the acclimation period (week 0) and every 4 weeks thereafter (weeks 4, 8, 12, and 16). Body weight, CBC (including platelet count), plasma fibrinogen. electrolyte (Na, K, and Cl) concentrations, and biochemical profile enzyme activities (aspartate aminotransferase, alkaline phosphatase, gamma-glutamyltransferase, and creatine kinase) did not change markedly with diet. Platelet aggregation was not altered by the supplementation of flaxseed oil in these healthy horses, although increases in plasma cis-polyunsaturated 18-carbon fatty acids C18:3; n-3 (ALA) and C18:2; n-6 (linoleic acid), biologically active C20:5; n-3 (eicosapentaenoic acid [EPA]), and malondialdehyde (MDA) were evident. There were no marked decreases in C20:4; n-6 (arachidonic acid [AA]) or increases in C22:6; n-3 (docosahexaenoic acid [DHA]), signifying that flaxseed oil may have had a high percentage of omega-6 (n-6) fatty acids as well as n-3 fatty acids, and this relatively high n-6: n-3 fatty acid ratio may have affected the biochemical effect of n-3 fatty acids. In healthy horses supplemented with flaxseed oil, platelet aggregation was not altered, which may have been due to the limited biologic effect in healthy subjects or the inability of flaxseed oil to induce the necessary biochemical effect of replacing n-6 fatty acids with n-3 types.     

Specificity of inhibition between Salmonella strains

A phenomenon of inhibition among Salmonella organisms colonizing the gut of young animals can be demonstrated which is important in connection with immunization using live Salmonella vaccines. For practical utilization of this inhibition phenomenon, research into its specificity is very important. In vitro broth culture and animal experiments have shown inhibition to be genus-specific, i.e. there is no inhibition between strains of different genera of Enterobacteriaceae in most cases. The strongest inhibition occurs between isogenic (identical genotype) strains (strain-specific inhibition). This applies to Salmonella and other genera of Enterobacteriaceae. There was a close correlation between the degree of the inhibitory effect and the genetic relatedness of the strains, as has been demonstrated in Salmonella strains of different epidemiological classification. The inhibitory capacity was most pronounced among closely related strains. Clonal strains of Salmonella Enteritidis phage type 4 fully inhibited caecal colonization by Salmonella Enteritidis phage type 4 strains in 65% of the combinations tested in chicks. In broth culture experiments, the share of combinations with complete inhibition was above 90%. A significant inhibitory competence of a selected strongly inhibitory Salmonella Enteritidis phage type 4 strain against the majority of Salmonella Enteritidis field isolates can be expected. However, Salmonella Enteritidis strains of all phase types showed almost no inhibitory competence against other Salmonella serotypes.     

Treatment of canine parvoviral enteritis with interferon-omega in a placebo-controlled challenge trial

Canine parvoviral enteritis continues to cause significant morbidity and mortality in dogs worldwide, and efficacious antiviral therapies are lacking. The present trial was aimed at evaluating the therapeutic efficacy of a recombinant feline interferon (type omega) preparation in the treatment of parvoviral enteritis in dogs. A double-blind, placebo-controlled challenge trial was performed in beagle pups (8-9 weeks); clinical signs, body weight, hematologic parameters, and mortality were monitored for a period of 14 days after challenge. Fourteen animals were inoculated with virulent canine parvovirus; 10 animals that developed clinical signs thereby meeting the inclusion criteria were admitted to the treatment phase in two randomly selected groups (placebo and IFN) of equal size. The IFN group received daily intravenous injections of rFeIFN-omega (2.5 MU/kg) for three consecutive days. The placebo group received daily injections of saline without IFN. Both groups of animals received individual supportive treatment consisting of adjusted diet and electrolyte solution.All five dogs in the placebo group developed fulminating enteritis with typical clinical signs and died within 10 days post-inoculation (or 6 days post-treatment). In the IFN-treated group, one animal died on day 2 after the treatment was started, whereas the other four dogs survived the challenge and gradually recovered. Our data confirm that the rFeIFN-omega can exert a significant therapeutic effect on dogs with parvoviral enteritis by improving clinical signs and reducing mortality.     

Pheasant ataxia: a new condition in pheasant poults

Between 1995 and 1997 a neurological condition in pheasant poults from 24 sites in England and Scotland was investigated. Affected birds showed varying degrees of ataxia and incoordinated movements and, in severe cases, recumbency, but generally remained alert with their heads held upright. The condition characteristically affected poults from seven weeks of age and the incidence on any one site was low. No significant bacteria were isolated consistently from brain tissue. The condition was characterised histologically by a non-suppurative meningoencephalitis, in which lesions were found predominantly in the cerebellum in 61 of 81 samples examined (75.3 per cent). A non-suppurative myelitis was recorded in 16 of 20 spinal cords examined. No lesions were recorded in peripheral neural tissue and lesions were rare in other tissues. The condition appeared not to have been recorded previously in pheasants. A viral aetiology was suspected but Newcastle disease virus was not involved.