Comparison of beef traceability in serial and parallel fabrication systems using RFID and two-dimensional barcodes.

Traceability of beef attributes from small- and mid-sized farms through supply chains is a market barrier. The objective of this trial was to determine the influence of fabrication method on beef traceability system requirements. Individual identities of 54 animals were maintained through harvest, processing, packaging, and distribution. At harvest, each animal's unique radio frequency identification (RFID) animal identification number was transferred to a harvest label on each carcass quarter. Following transportation to a processor, nine carcasses were processed on alternating days by one of the two methods. Carcasses were fabricated, using a serial fabrication method (SFM), into wholesale cuts one at a time or fabricated using a parallel fabrication method (PFM), by processing multiple hindquarters or forequarters simultaneously into wholesale cuts. In-process labels were generated by scanning the two-dimensional (2D) barcode on the harvest label with a handheld mobile computer and printed from a wireless mobile printer. Tracking of SFM and PFM carcass quarters was accomplished by creating in-process labels for lugs and individual wholesale cuts, respectively. The process was recorded and the data was captured from video analysis. The mean number of in-process labels generated per carcass for SFM was 3.7 and for PFM was 30.9 (P < 0.01). The amount of time required for generating in-process labels for SFM (2 min 16 s) was less than PFM (8 min 45 s) (P = 0.01). The amount of time required to label each carcass was less (P < 0.01) for SFM (18 s) than for PFM (3 min 10 s) with in-process labels. Total cost of traceability, including fixed and consumable cost per carcass, was nearly twice as much for PFM ($17.98) than SFM ($9.02). Traceability, within both processing methods, was found to have 100% fidelity, as verified using DNA marker genotyping. Overall, the number of labels generated for traceability was less for SFM than that for PFM. The overall time spent on generating, applying, and removing labels was less for SFM than that for PFM. The total cost of traceability was approximately half for SFM compared with that for PFM; however both methods were able to track product accurately. Tracking of beef from individual animals, using RFID ear tags and 2D barcodes, appears to be feasible for the fabrication methods used in this study.

The herpes simplex virus type 1 (HSV-1) glycoprotein K(gK) is essential for viral corneal spread and neuroinvasiveness.

PURPOSE: To determine the role of herpes simplex virus-1 (HSV-1) glycoprotein K(gK) in corneal infection, neuroinvasion, and virus latency in trigeminal ganglia of mice. METHODS: The recombinant virus HSV-1 (McKrae) Delta gK (MKDelta gK) carrying a deletion of the gK gene was constructed by insertional/deletion mutagenesis and replaced by a gene cassette constitutively expressing the enhanced green fluorescence protein. The gK deletion of the MKDelta gK virus was rescued to produce the wild-type-like virus MKgK. Balb/c mice were infected ocularly with either virus, and the infection pattern in the eye, clinical disease progression, and establishment of viral latency was monitored. RESULTS: Mice infected with the MKDelta gK strain produced in a gK complementing cell line did not exhibit clinical signs when compared with mice infected with the MKgK virus. Direct visualization of infected eyes revealed that the MKDelta gK virus was unable to spread in mouse corneas, while the MKgK rescued virus spread efficiently. Nineteen of 20 scarified and 5/12 unscarified mice infected with the MKgK virus produced infectious virus after coculture with permissive cells, while 0/20 scarified and 0/12 unscarified mice infected with the MKDelta gK virus produced infectious virus. HSV DNA was detected in trigeminal ganglia by PCR in 19/20 scarified and 9/12 unscarified mice inoculated with MKgK, while HSV DNA was detected in the trigeminal ganglia of 3/20 scarified and 0/12 unscarified mice inoculated with MKDelta gK. CONCLUSIONS: The results show that HSV-1 gK is essential for efficient replication and spread in the corneal epithelium and trigeminal ganglia neuroinvasion in MKDelta gK inoculated mice.

Cloning, expression and in vitro evaluation of recombinant canine Tum5, an angiostatic domain of mammalian type IV collagen.

The process of new blood vessel formation within and around neoplastic tissue, termed angiogenesis, is a significant factor in the development, progression and metastasis of malignant tumours in all species. A major cause of death in cancer patients is the development of treatment-resistant metastatic disease, which may be avoided by therapies that target the genetically stable population of vascular endothelial cells within tumours. Tumstatin is a small protein formed by the cleavage of the alpha-3 subunit of the non-collagenous domain of mammalian type IV collagen. Recombinant human Tumstatin has been shown to have potent angiostatic properties in vitro and in vivo. Tumstatin is a potent initiator of apoptosis and inhibits the proliferation and migration of vascular endothelial cells in cell culture. Recently, a fragment of Tumstatin, termed Tum5, has been shown to have biologic activity similar to the parent compound. The systemic administration of angiostatic proteins like Tum5 may result in the remission of established tumours, while preventing or delaying the onset of clinically detectable metastasis. Recombinant canine Tum5 (cTum5) was cloned and its protein expression induced in a prokaryotic vector. The resulting cTum5 protein caused dose-dependent inhibition of vascular endothelial cells in vitro, which appears to be mediated through apoptosis.

Glycoprotein K specified by herpes simplex virus type 1 is expressed on virions as a Golgi complex-dependent glycosylated species and functions in virion entry.

To facilitate detection of glycoprotein K (gK) specified by herpes simplex virus, a 12-amino-acid epitope tag was inserted within gK domain III. Recombinant virus gKprotC-DIII, expressing the tagged gK, was isolated. This virus formed wild-type plaques and replicated as efficiently as the wild-type KOS virus in Vero cells. Anti-protein C MAb detected high-mannose and Golgi complex-dependent glycosylated gK within cells as well as on purified virions. The gK-null virus DeltagK (gK(-/-)) entered Vero cells substantially more slowly than the wild-type KOS (gK(+/+)), while DeltagK virus grown in complementing VK302 cells (gK(-/+)) entered with entry kinetics similar to those of the KOS virus.

An alpha-helical domain within the carboxyl terminus of herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) is associated with cell fusion and resistance to heparin inhibition of cell fusion.

Previous studies from our laboratory indicated that a 28-amino-acid carboxyl-terminal truncation of gB caused extensive virus-induced cell fusion (Baghian et al., 1993, J Virol 67, 2396-2401). We tested the ability of additional truncations and mutations within gB to cause cell fusion in the recently established virus-free cell fusion assay (Turner et al., 1998, J. Virol. 72, 873-875). Deletion of the carboxyl-terminal 28 amino acids of gB (gBDelta28), which removed part of the predicted alpha-helical structure H17b, caused extensive cell fusion. A gB truncation specified by gBDelta36, which removed the entire H17b domain, caused as much cell fusion as the gBDelta28 truncation. Similarly, gB(A874P) containing a substitution of an Ala with Pro within H17b caused cell fusion. Heparin, a gB-specific inhibitor of virus-induced cell fusion, inhibited both wild-type gB and gB(syn3)-mediated cell fusion. In contrast, fusion of cells transfected with gB(Delta28), gB(Delta36), or gB(A874P) was resistant to heparin inhibition of cell fusion. We concluded the following: (1) The predicted alpha-helical structure of H17b within the carboxyl terminus of gB is involved in both virus-induced and virus-free cell fusion. (2) Heparin is a specific inhibitor of gB-mediated fusion in both systems. (3) Resistance to heparin inhibition of gB-mediated cell fusion is associated with the predicted alpha-helical structure H17b within the carboxyl terminus of gB.

Genetic analysis of the role of herpes simplex virus type 1 glycoprotein K in infectious virus production and egress.

Herpes simplex virus type 1 (KOS)DeltagK is a mutant virus which lacks glycoprotein K (gK) and exhibits defects in virion egress (S. Jayachandra, A. Baghian, and K. G. Kousoulas, J. Virol. 69:5401-5413, 1997). To further understand the role of gK in virus egress, we constructed recombinant viruses, DeltagKhpd-1, -2, -3, and -4, that specified gK amino-terminal portions of 139, 239, 268, and 326 amino acids, respectively, corresponding to truncations immediately after each of the four putative membrane-spanning domains of gK. DeltagKhpd-1 and DeltagKhpd-2 viruses produced lower yields and smaller plaques than DeltagK. Numerous DeltagKhpd-1 capsids accumulated predominately within large double-membrane vesicles of which the inner membrane appeared to be derived from viral envelopes while the outer membrane appeared to originate from the outer nuclear membrane. The mutant virus DeltagKhpd-3 produced higher yields and larger plaques than the DeltagK virus. The mutant virus DeltagKhpd-4 produced yields and plaques similar to those of the wild-type virus strain KOS, indicating that deletion of the carboxy-terminal 12 amino acids did not adversely affect virus replication and egress. Comparisons of the gK primary sequences specified by alphaherpesviruses revealed the presence of a cysteine-rich motif (CXXCC), located within domain III in the lumen side of gK, and a tyrosine-based motif, YTKPhi (where Phi is any bulky hydrophobic amino acid), located between the second and third hydrophobic domains (domain II) in the cytoplasmic side of gK. The mutant virus gK/Y183S, which was constructed to specify gK with a single-amino-acid change (Y to S) within the YTKPhi motif, replicated less efficiently than the DeltagK virus. The mutant virus gK/C304S-C307S, which was constructed to specify two serine instead of cysteine residues within the cysteine-rich motif (CXXCC changed to SXXSC) of gK domain III, replicated more efficiently than the DeltagK virus. Our data suggests that gK contains domains in its amino-terminal portion that promote aberrant nucleocapsid envelopment and/or membrane fusion between different virion envelopes and contains domains within its domains II and III that function in virus replication and egress.

Functional characterization of the HveA homolog specified by African green monkey kidney cells with a herpes simplex virus expressing the green fluorescence protein.

We cloned the gene specified by African monkey kidney cells (Vero) that codes for the homolog of the herpes virus entry mediator (HveA) specified by HeLa cells. The primary sequence of the monkey HveA (HveAs) differed significantly from HveA. Single amino acid differences were distributed throughout the amino and carboxyl terminal portions of the HveAs in comparison with the HveA, whereas certain regions were highly conserved. The predicted membrane spanning domains of the two receptors differed substantially due to insertions and deletions of short amino acid sequences. The ability of HveAs to mediate HSV virus entry was tested in a series of experiments using the recombinant virus KOS/EGFP, which constitutively expressed the enhanced green fluorescence protein (EGFP) and Chinese hamster ovary cells (CHO) transformed with the HveAs gene. The KOS/EGFP virus was constructed by inserting an EGFP gene cassette within the intergenic region between the UL53 (gK) and UL54 (ICP27) genes. The KOS/EGFP virus formed viral plaques and replicated as well as the wild-type KOS virus. HveAs-transformed CHO cells constitutively expressing HveAs mediated herpesvirus entry efficiently, whereas cells transformed with the HveAs gene in the noncoding orientation did not mediate virus entry. A genetically engineered protein composed of the amino-terminal portion of the HveAs protein fused to the heavy chain of mouse IgG immunoglobulin as well as mouse antibodies raised against HveAs blocked virus entry into HveAs-transformed CHO cells. Thus, HveAs is the functional homolog of HveA.

Expression of the enhanced green fluorescent protein by herpes simplex virus type 1 (HSV-1) as an in vitro or in vivo marker for virus entry and replication.

Glycoprotein K (gK) is involved in membrane fusion phenomena during infectious virus production and egress and is an important determinant for neurovirulence. To assess better the in vitro and in vivo roles of gK in virus replication, a recombinant virus was constructed expressing an engineered enhanced green fluorescent protein (EGFP) under the control of the human cytomegalovirus immediate early gene promoter (HCMV-IEP) inserted in place of the gK gene. The EGFP gene insertion was confirmed by diagnostic polymerase chain reaction (PCR), and the presence of the EGFP protein was detected by western immunoblot analysis using anti-GFP monoclonal antibody. Fluorescence microscopy revealed that virus infected cells emitted bright fluorescence when examined using filters for fluorescein. Fluorescence emission was detected as early as 4 h post-infection. Fluorescence intensity increased over time and was stable at late times after infection at which point viral plaques continued to emit bright green fluorescence. The amount of fluorescence emitted by virus infected Vero cells was monitored by fluorescence cytometry using a FACS cytometer. At an MOI of 3, all infected cells emitted strong green fluorescence as quantified by cytometry at 48 h post-infection. The deltagK-EGFP expressing recombinant virus will enable the determination of the role of gK in virus entry and egress as well as the role of gK in the molecular pathogenesis of herpes simplex virus type 1 (HSV-1).

Preparation, characterization, and in vivo evaluation of an oil suspension of a bovine growth hormone releasing factor analog.

Pharmacia & Upjohn Inc. has discovered a superior bovine growth hormone releasing factor analog, [IIe2,Ser8,28,Ala15,Leu27,Hse30] bGRF (1-30)-NH-ethyl, acetate salt (U-90699F), for enhancement of animal growth. This report delineates the preparation, characterization, and in vivo evaluation of a U-90699F oil suspension. The oil suspension formulation was selected because of its simplicity, inexpensiveness, and ability to produce sustained U-90699F release. 40% U-90699F monomers were incorporated into Miglyol oil with acceptable injectability. Both reversed-phase-high-pressure liquid chromatography (RP-HPLC) and Fourier transform infrared spectroscopy (FTIR) were utilized to evaluate its chemical and structural stability. This suspension formulation demonstrated no significant changes in concentration as determined by RP-HPLC for 10 weeks at both 25 and 39 degrees C. The U-90699F dispersed in oil also showed no signs of structural conversion from alpha-helix to beta-sheet as monitored by FTIR. However, there was an increase in alpha-helix/disordered coil structure after initial drug incorporation. The suspension was subcutaneously administered to Holstein steers. Areas under the serum somatotropin concentration curve were used to determine the duration of action. It was found that the suspension was able to effectively elevate serum somatotropin for at least 14 days.

Particle size studies for subcutaneous delivery of poly(lactide-co-glycolide) microspheres containing ovalbumin as vaccine formulation.

The primary objectives of the present study were to produce poly(lactide-co-glycolide) (PLGA) microspheres with different diameters, to characterize these microspheres which were loaded with a model antigen, ovalbumin and to evaluate the effect of microsphere particle size on the serum antibody levels following administration to mice. Four kinds of ovalbumin-loaded PLGA microspheres with different diameters (1.2, 3.5, 7.0 and 14.3 microns as mean volume diameter) were manufactured by a w/o/w emulsion/solvent evaporation method. Low loading percent (0.08%-0.25% w/w) and efficiencies (8-25% w/w) were observed. Examination using scanning electron photomicrographs showed smooth spherical particles. The in-vitro release of ovalbumin from microspheres showed an expected burst release with all batches and the extent of the burst release increased with decreasing diameters of spheres; PLGA microspheres with the smallest diameter (1.2 microns) showed an 80% burst release within one day. Approximately 10-60% of ovalbumin remained unreleased 30 days later. The single subcutaneous administrations of ovalbumin-loaded PLGA microspheres with different diameters to mice induced good antibody responses above ovalbumin saline negative controls at 3, 6, 9, and 12 weeks after inoculation. Especially, 0.16% ovalbumin-loaded PLGA microspheres having mean volume diameter of 3.5 microns exhibited the best immune responses with values greater than those obtained after inoculation with adjuvants such as complete Freund's adjuvant or alum as positive control. The strong adjuvant activity of PLGA microspheres as vaccine formulation was suggested.

Dose and load studies for subcutaneous and oral delivery of poly(lactide-co-glycolide) microspheres containing ovalbumin.

Poly(lactide-co-glycolide) microspheres containing different loads of OVA (0.05, 0.1, 0.5 and 1.0% w/w) were manufactured by a w/o/w emulsion/solvent evaporation method. Low load efficiencies of less than 20% were observed. Normal size distributions with mean volume diameters ranging from 3.7 to 4.7 microns were obtained for different batches. The in vitro release of OVA from different loaded microspheres showed an expected burst release with all batches. The in vivo dose study (1, 10, 25, 50 micrograms of OVA) was performed by subcutaneous and oral inoculation in mice by single (0 week) or double (0 and 3 weeks) administration of PLGA 50/50 microspheres containing 0.1% OVA. Subcutaneous administration showed an immune response (serum Ig levels by ELISA) statistically (Fisher's paired t-test; P < 0.05) above OVA saline negative controls at 3, 6 and 12 weeks after administration. Oral administration of microspheres produced statistically higher systemic immune responses at the higher doses. Single and double inoculation orally and subcutaneously produced similar serum antibody levels. The in vivo load study was performed by subcutaneous and oral administration to mice of 25 micrograms OVA contained in various loaded (0.05, 0.1, 0.5 and 1.0% w/w) microspheres. Serum immune responses at 3, 6, and 12 weeks after inoculation were statistically above OVA saline controls and were inversely proportional to the OVA load using either route. This observation suggested a relationship between the number of microspheres delivered and the in vivo serum response. Single subcutaneous administration of 0.05 or 0.1% OVA loaded PLGA 50/50 microspheres induced larger immune responses compared with complete Freund's adjuvant.

Release of highly water-soluble medicinal compounds from inert, heterogeneous matrixes. II: Melt.

The release from matrixes compressed from melts of hydrogenated castor oil and ephedrine hydrochloride or procaine hydrochloride has been studied and is compared with the release from matrixes prepared by compression of physical mixtures. In both, the release is dependent on the square root of time; however, the release is slower from the matrix prepared by the melt process. Parameters that affect the release are compared. The independence of release from stirring speed and the greater tortuosity of the matrix prepared by the melt process indicate that the release is by a matrix diffusion mechanism. In contrast, boundary layer diffusion is operative in the release from the matrixes prepared by compression of physical mixtures. It is demonstrated that for a given formulation, the method of processing may alter the mechanism and rate of release.

Release of highly water-soluble medicinal compounds from inert, heterogeneous matrixes. I: Physical mixture.

The release from a matrix compressed from a physical mixture of hydrogenated castor oil and ephedrine hydrochloride or procaine hydrochloride has been investigated. The effects on release of the concentration of medicinal compound, particle size of medicinal compound, agitation of dissolution medium, and porosity and tortuosity of the matrix are presented. An attempt is made to fit the experimental data to an acceptable diffusion model.