Polymorphonuclear neutrophils (PMN) from 4 cows were preincubated (30 minutes, 37 C) in either actinomycin D (100 micrograms/ml) or puromycin (10 micrograms/ml), inhibitors of mRNA transcription and protein translation, or in medium 199. The PMN were incubated for a further 4.5 hours in medium containing 100 U of recombinant bovine interferon-gamma (rboIfn-gamma). The PMN were then incubated with bovine IgG1, IgG2, IgM, or aggregated IgG (aIgG; 4 C, 12 hours) for flow cytometric analysis, using fluoresceinated isotype-specific antibody. The percentage of PMN binding the ligand and the logarithmic mean fluorescent channel (LMFC), an indicator of the amount of receptor (R) expression, were recorded. Competitive inhibition of ligand binding was measured by incubating PMN with fluoresceinated IgG2 in the presence or absence of 100-fold excess of IgG1, IgG2, and aIgG. Activation with rboIfn-gamma induced a 4.5-fold increase in binding of IgG1 and a fivefold increase in LMFC for IgG2. These increases were inhibited by actinomycin D and puromycin. Percentage of PMN binding aIgG decreased after activation by rboIfn-gamma. Interferon-gamma treatment did not affect binding or LMFC of IgM. However, binding of IgM was reduced by treatment with actinomycin D. Binding of fluoresceinated IgG2 was inhibited by unlabeled IgG1, IgG2, and aIgG. Results indicate that bovine PMN Fc receptors (FcR) for IgG1 and IgG2 were rboIfn-gamma inducible, that induction required de novo transcription and translation, that a heterogeneous population of FcR exist on bovine PMN, and that IgG1 and IgG2 share a common FcR. Further, bovine PMN are capable of gene activation and are responsive to changes in their environment, thus being amenable to modulation for effective pathogen destruction.

Ovine interferon-tau (oIFN-τ) is a newly discovered type I interferon. This study used biochemical techniques to transform the oIFN-τ gene into Escherichia coli to obtain the mass and soluble expression of the recombinant protein. First, total RNA was extracted from fresh sheep embryonic tissues with TRIzol reagent and then used as a template to reverse transcribe and amplify the mature oIFN-τ gene with RT-PCR. The amplified product was next digested with the HindIII and XhoI restriction enzymes and inserted into the pET-32a(+) vector to construct the prokaryotic expression plasmid. The corrected in-frame recombinant plasmid, pET-32a(+)-oIFN-τ, was transformed into E. coli Rosetta (DE3) competent cells. After induction with isopropyl-beta-D-thiogalactopyranoside (IPTG), the recombinant protein was detected in bacteria. Finally, the bacteria were lysed by sonication, and the recombinant protein was purified by nickel affinity chromatography and DEAE anion exchange chromatography. The protein was confirmed to be oIFN-τ, which mainly existed in the soluble lysate fraction, as proven by SDS-PAGE and Western blot assays. Purified IFN-τ exists mostly in a soluble form, and its anti-vesicular stomatitis virus (VSV) activity reached 7.08×10(6)IU/mL.

Recombinant bovine interferon alpha (rBoIFN-α) has been demonstrated to have antiviral activity. However, no conduct of acute or chronic toxicity tests has been reported. Specific pathogen-free Sprague Dawley rats were administered doses at different concentrations through intraperitoneal or intravenous injection. After the administration (single for an acute toxicity test over 14 days or daily for a sub-chronic toxicity test over 30 days), the rats’ behaviour and other indicators and the degree of toxic reaction were continuously monitored. Blood was collected for haematological and serum biochemical examinations. At the end of the experiments, the rats were sacrificed for necropsy and histopathological tissue analysis. The external performance, behaviour characteristics, and changes in body temperature and body weight of the rats in each subgroup were comparable to the normal control subgroup. Except for a few cases, there were no lesions in the viscera’s pathological structures, and the blood parameters and biochemical indicators were not noticeably different from those of the control subgroup. This study suggests that rBoIFN-α seems to be safe for rats, and its use may foster the development of the cattle industry in China by protecting livestock health.

Objective-To assess the biological response to recombinant feline interferon-omega (rFeIFN-omega) following ocular or oral administration in cats via estimation of Mx protein expression in conjunctival cells (CCs) and WBCs. Animals-10 specific pathogen-free cats. Procedures-In multiple single-dose drug experiments, each cat received various concentrations of rFeIFN-omega administered topically into both eyes (50 to 10,000 U/eye) and orally (200 to 20,000 units). The same cats received saline (0.9% NaCl) solution topically and orally as control treatments. The CCs and WBCs were collected prior to treatment (day 0), on day 1, and every third or seventh day thereafter until samples yielded negative results for Mx protein. Samples were examined for Mx protein expression via immunohistochemistry and immunoblotting procedures involving murine anti-Mx protein monoclonal antibody M143. Results-After topical application of 10,000 U of rFeIFN-omega/eye, CCs stained for Mx protein for a minimum of 7 days, whereas WBCs were positive for Mx protein for a minimum of 31 days. After topical application of lower concentrations, CCs did not express Mx protein, in contrast to WBCs, which stained for Mx protein at 1,000 units for at least 1 day. Following oral administration, Mx protein was expressed in WBCs at rFeIFN-omega concentrations as low as 200 units, whereas CCs did not stain for Mx protein at any concentration. Conclusions and Clinical Relevance-Results indicate that Mx protein expression (a marker of the biological response to rFeIFN-omega) in CCs and WBCs of rFeIFN-omega-treated cats depends on the dose of rFeIFN-omega, site of administration, and cell type.

The influence of recombinant bovine interferon gamma (rBOIFN-gamma) treatment on resistance of clinically normal and dexamethasone-treated calves to Haemophilus somnus infection was evaluated. Four groups of 6 calves each were treated with saline solution (controls), dexamethasone (0.04 mg/kg of body weight/for 3 days), rBOIFN-gamma (2 micrograms/kg for 2 days), or dexamethasone and rBOIFN-gamma (aforementioned dosages). All treatments were started 24 hours before intrabronchial challenge exposure with 5 x 10(9) colony-forming units of H somnus. Rectal temperature and WBC count were monitored daily. Two of the dexamethasone-treated calves died of pneumonia 4 days after challenge exposure and were necropsied. All other calves were euthanatized and necropsied 7 days after challenge exposure. All calves had pneumonia of variable intensity. Dexamethasone-treated calves had increased volume of pneumonic lung (P < 0.05) and increased severity of pneumonia, compared with control calves. Recombinant bovine interferon gamma treatment resulted in reduction in pneumonic lung volume and severity of pneumonia in dexamethasone-treated calves (P < 0.05), although it did not influence severity of pneumonia in nondexamethasone-treated calves.

Cell extracts that were prepared from blood mononuclear leukocytes from 66 samples obtained from 6 clinically normal calves contained mean 2',5'-oligoadenylate (2',5'-oligo[A]) synthetase activity sufficient to synthesize 186 +/- 82 pmol of 2',5'-oligo(A)/h/10(6) cells. Calves had no measurable serum interferon (IFN) activity. Five calves were given IM injections of 10(4), 10(5), 5 x 10(5), 10(6), and 10(7) U of bovine IFN-alpha 1/kg of body weight at 2-week intervals. Five dosing sequences were used with a 5 x 5 Latin square design so that each calf received each dose once. Activity of 2',5'-oligo(A) synthetase increased at 24 hours in response to all dosages of IFN and then declined following first-order kinetics, with an apparent half-life (t1/2) of 2.1 +/- 0.5 days. The area under the concentration-time curve for 2',5'-oligo(A) synthetase increased with dose of IFN more rapidly than did peak response. Serum IFN that was measured at 1-day intervals following administration of IFN was consistently measurable only at dosages above 10(6) U of IFN/kg. The t1/2 for circulating IFN was 12.4 +/- 1.0 hours. Over all dosages, increases in 2',5'-oligo(A) synthetase activity were measurable for 3.5 days longer than were increases in IFN following IM injection of IFN. None of the calves developed detectable anti-IFN antibodies.

The antiviral activity of recombinant DNA-derived bovine alpha 1-1 interferon on an established swine testicular cell line and primary testicular cell cultures derived from swine of various ages (2 days, 3 weeks, and 5 weeks) was determined. Bovine interferon induced a dose-dependent increase in 2-5A synthetase in testicular cells, regardless of the source of the cells. Furthermore, interferon inhibited replication of vesicular stomatitis virus to an equivalent extent in all testicular cell cultures. The results indicate that 2-5A synthetase is a reliable marker of interferon activity in swine testicular cell cultures and that the induction of 2-5A synthetase and antiviral effects of recombinant bovine interferon in primary testicular cell cultures are not dependent on the age of the donor animal.

Bovine fibroblast interferon (BoF-IFN), produced in bovine embryonic kidney cell cultures by priming and infection with bluetongue virus, was partially purified by controlled pore glass chromatography. The partially purified B0F-IFN then was subjected to beaded agarose affinity chromatography. The IFN eluted by affinity chromatography in 2 distinct fractions-1 after the addition of 1M NaCl and the other one after the addition of 1.5M NaCl containing 50% ethylene glycol. Analysis of fractions by sodium dodecyl sulfate/polyacrylamide-gel electrophoresis revealed a broad molecular weight range (14,900 to 27,900) for the IFN eluted by 1M NaCl, and 2 discrete molecular weight ranges (16,000 to 19,500 and 28,300 to 34,000) for IFN eluted by 1.5M NaCl containing 50% ethylene glycol. The specific activity of the IFN eluted with 1.5M NaCl containing ethylene glycol was 2.85 X 10(6) U/mg of protein, compared with 5.7 X 10(5) U/mg of protein in the controlled pore glass-purified IFN.

Bovine fibroblast interferon (BoF-IFN), produced in primary bovine embryonic kidney cell cultures after priming and infection with bluetongue virus, was purified by controlled pore glass (CPG) chromatography to a specific activity of 10(6) U/mg of protein, with 40% recovery of the original activity. The crude IFN was concentrated more than sevenfold during purification. This proved to be a relatively simple, practical method of obtaining sufficient quantities of partially purified natural BoF-IFN for further studies. The CPG-purified BoF-IFN was further concentrated by sequential ultrafiltration and was analyzed by sodium dodecyl sulfate/polyacrylamide-gel electrophoresis (SDS-PAGE). Interferon, recovered from denaturing conditions either by dialysis against phosphate-buffered saline solution or by dilution in cell culture medium containing 10% fetal bovine serum, migrated as a single stainable protein with molecular weight of 21,000 on analytic SDS-PAGE gels. Recovered IFN activity from preparative SDS-PAGE totalled 8.7% of that applied. Attempts to further pruify CPG-purified BoF-IFN by zinc chelate affinity chromatography were unsuccessful.

A study was undertaken to investigate the combined effects of fasting and different diets on interferon (IFN) production and virus replication measured in nasal secretions of calves inoculated with a vaccine strain of infectious bovine rhinotracheitis virus. Four groups of calves were inoculated intranasally with infectious bovine rhinotracheitis virus. Two groups were inoculated 24 hours after onset of a 3-day fast; upon refeeding, 1 group was fed a maintenance diet (M diet) of hay, and the other was fed a higher energy diet (HE diet) of hay and concentrate. Nonfasted control groups were fed the M diet or the HE diet. Overall IFN production was highest (P less than 0.01) in nonfasted calves fed the M diet throughtout the study and lowest in nonfasted calves fed the HE diet. Fasted calves refed the HE diet produced consistently and significantly more IFN than did nonfasted calves fed this diet. Fasted calves refed the M diet, however, produced significantly less IFN, compared with control calves fed the M diet throughout the study. Overall mean virus excretion was similar in all groups; therefore, the amount of virus replication per se did not account for the differences in IFN production, nor did greater IFN production result in less virus excretion. Serum cortisol concentrations and immune responses were not significantly affected by fasting or diet.