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.     

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.     

RADIOGRAPHIC ANATOMY OF THE CRIBRIFORM PLATE (LAMINA CRIBROSA)

The radiographic appearance of the cribriform plate was investigated in 16 canine cadaver heads. The cribriform plate appeared as a "V"-shaped multilinear bone-opaque stripe in the caudal nasal region in projections perpendicular to the hard palate in 6 dogs with a skull index between 50.00 and 54.00. In 9 dogs with a skull index between 55.40 and 74.40, the cribriform plate had a more "C"-shaped and sharp appearance. In vertically oblique projections with an obliquity greater than 20 degrees, the cribriform plate lost its sharp outline and finally (40 degrees) disappeared. In lateral projections the cribriform plate appeared as a "C"-shaped interrupted bone-opaque stripe in all 16 dogs. In more brachycephalic dogs frontal bone structures superimposed on the cribriform plate on ventrodorsal and dorsoventral views and accentuated the radiographic appearance of the plate. Vertically oblique views separated both structures to produce two lines.     

Rhizoctonia spp. Recovered from Strawberry Roots in Central Coastal California

ABSTRACT Rhizoctonia spp. were commonly recovered from the roots of strawberry plants growing in nonfumigated soil in the central coastal region of California. With the exception of one multinucleate isolate of R. solani (frequency of recovery of 0.8%), all other isolates were binucleate and were in anastomosis groups (AG) A, G, or I. AGs-A and -I were recovered from all five collection sites, whereas AG-G was recovered from only two sites. AG-A was the most commonly isolated AG, followed by AGs-I and -G. Similar levels of virulence were observed among the different AGs, but differences in virulence were observed among isolates in the same AG. Evaluating anastomosis grouping by pairing isolates recovered from strawberry with known tester isolates did not always yield a positive anastomosis reaction, even though both isolates anastomosed with other members of the same AG. Subsequent investigations with multiple isolates in the same AG from the same collection location confirmed that there was a lack of anastomosis or weak anastomosis reactions for some combinations of pairings, highlighting the need for to use multiple tester isolates or molecular techniques for AG determination. Restriction fragment length polymorphism (RFLP) analysis of a polymerase chain reaction-amplified region of the rDNA was effective for differentiating AGs. Sixteen RFLP groups were observed after cluster analysis with data for the size of the amplified products and fragment sizes after digestion with four restriction enzymes. Although each AG had isolates in multiple RFLP groups, any one individual RFLP group contained isolates of only a single AG. There was no consistent correlation between RFLP group and location of isolate collection.     

The effect of exogenous purine supply on the endogenous excretion of purine derivatives in the urine of growing lambs

An experiment was made to determine the absorption of purine metabolites in dietary nucleic acids through the digestive tract, and also to determine the utilization of nucleic acids absorbed in the body, using growing lambs. Two pairs of 120‐ and 180‐day‐old twin female lambs with a bodyweight of 18.2–19.0 kg were kept in metabolism crates and fed on purine‐free milk replacement (MR) with supplements of exogenous purine (purine base or purine nucleoside) at a level of 0.2 mmol/BW^0.75/d for 5 consecutive days, and thereafter they were maintained in the crates for 4 days. The daily amount of exogenous purine supply was calculated based on the urinary excreted purine derivatives (PD) in lambs fed on milk replacement alone. A urine sample was collected daily for 9 consecutive days, and the urinary excretion of PD was determined daily. Urinary PD excretion opened to increase within 24 h after the dose of purine bases, and the level was recovered on 3 days after ceasing the exogenous purine supply. The recovery of PD in the urine was about 70% of the purine supplement. When purine nucleosides were added to the feed, urinary PD excretion was initiated within 24 h after dosing, and the values were recovered after ending the purine nucleoside supply. The recovery rate of PD in the urine was only 30% of the supplemented purine. The plasma allantoin levels were almost similar after feeding purine bases and purine nucleosides, and the values were mostly in the range (40–60 µmol/L). These findings indicate that an exogenous purine can be directly incorporated into the body, and the purine as nucleoside is more effectively utilized for the synthesis of nucleic acids than as a purine base in the body of growing lambs.     

Observations on the cooling and cryopreservation of pig oocytes at the germinal vesicle stage

This study examined the viability of pig oocytes at the germinal vesicle stage following cooling or cryopreservation. Cumulus-intact oocytes (n = 641) were collected from slaughterhouse pig ovaries and used in two experiments. In Exp. I the viability of 1) control, 2) cryoprotectant control (CC, 1.5 M glycerol/.5 M sucrose), 3) cooled (0 degrees C) and 4) cryopreserved (-196 degrees C) oocytes was assessed after no incubation or a 24-h incubation. Survivability was judged by morphological appearance, trypan blue exclusion and fluorescein diacetate staining. Survival rate of control oocytes (90%; based primarily on morphological appearance of the cumulus) incubated 0 h was greater (P less than .05) than that of all other groups, whereas survival rate of -196 degrees C oocytes (57%) was less (P less than .05) than that of all other groups. However, vital staining of 0 degrees C and -196 degrees C oocytes showed 0% survival rate as evidenced by trypan blue uptake and lack of fluorescence. The cumulus cells surrounding oocytes that were stored at 0 degrees C or -196 degrees C survived freezing as evidenced by trypan blue exclusion and intense fluorescence. Similar differences among treatment groups were found for oocytes incubated 24 h. Exp. 2 examined the temperature at which oocytes became sensitive to cooling. Oocyte death occurred when oocytes were cooled to 15 degrees C or lower. These results demonstrate that pig oocytes at the germinal vesicle stage did not survive cooling to 15 degrees C or below. When assessing the viability of cryopreserved cumulus enclosed oocytes it is important to use vital stains in conjunction with morphological appearance.