Decrease of Salmonella and Escherichia coli O157:H7 counts during dry-aging of beef but potential growth of Listeria monocytogenes under certain dry-aging conditions.

Dry-aging of beef comprises the storage of carcasses and (sub)primal cuts at a low temperature and relative humidity for a prolonged period, aiming to increase the sensory quality of meat. Limited data are available on the survival and potential growth of pathogens on the surface of beef during dry-aging. Therefore, this study evaluates the changes in Salmonella, Shiga toxin-producing Escherichia coli O157:H7 and Listeria monocytogenes counts during dry-aging. A mixture of pathogenic strains was inoculated on the surface of beef loins, which were stored under four different process conditions (2 °C and 6 °C × relative humidity 75 and 85% during 42 days). Salmonella and E. coli O157:H7 counts significantly decreased during dry-aging. The daily reductions varied from -0.07 to -0.14 log10 CFU and from -0.09 to -0.14 log10 CFU, respectively, depending on the loin, matrix and condition. The reduction of L. monocytogenes was slower, with a maximum of -0.07 log10 CFU/day. L. monocytogenes counts increased with 1.0 log10 CFU on the lean meat of one loin with pH > 6.0 at the end of dry-aging, indicating that this pathogen can potentially grow under certain dry-aging conditions.

Influence of aging time, temperature and relative humidity on the sensory quality of dry-aged Belgian Blue beef.

The objective of this study was to evaluate the effect of aging period (0, 3, 6 or 9 weeks), aging temperature (2 versus 6 °C at 75% relative humidity, experiment 1) and relative humidity (70 versus 90% at 2 °C, experiment 2) on the sensory traits, oxidative stability and proteolysis of Belgian Blue beef. For each experiment, eight loins (M. longissimus thoracis et lumborum) from four animals (left and right side) were assigned to one of the two treatments (n = 4). Results showed no further tenderization after three weeks of aging, whereas metmyoglobin formation and lipid oxidation increased until nine weeks of aging (P < 0.05). During the nine weeks of aging, atypical flavor, odor and flavor intensity was affected (P < 0.05). This was accompanied by an increase of small peptides and other nitrogenous compounds. Aging temperature and relative humidity had only a very limited effect on the quality traits.

Effect of terminal sire line and timing second vaccination on effectiveness of immunocastration, performance, and carcass and meat quality.

Performance (from 10 weeks until slaughter), carcass and meat quality, and effectiveness of immunocastration was compared in crossbred offspring of stress positive (BP+) and negative (BP-) Belgian Piétrain and Canadian Duroc (CD) given the second vaccination of Improvac® at different times (4, 6, 8 weeks before slaughter). CD offspring had a significantly higher daily gain (DG) and feed intake (DFI), and lower predicted lean meat percentage (LMP) and dressing yield compared to BP+ and BP-, while feed conversion ratio (FCR) did not differ. CD offspring had significantly lower drip loss and higher pHi, intramuscular fat content than BP+ and BP- (except for pHi). No significant effect of vaccination time on DG nor FCR was observed. Predicted LMP tended to increase as time-post injection decreased, while meat quality was minor affected. Earlier vaccination had no effect on the effectiveness of immunocastration based on testosterone and GnRH-binding.

Quality characteristics of fresh loin and cooked ham muscles as affected by genetic background of commercial pigs.

This study was performed to investigate meat quality traits of loin and ham of commercial pigs as affected by genetic differences in carcass and growth traits of the parent lines. Three hybrid sow lines were crossbred with two types of Belgian Piétrain with different breeding goals (BPgrowth and BPcarcass emphasizing daily growth and carcass conformation, respectively). Pig live performance and carcass quality of 270 offspring were measured, and meat quality of the loin and (cooked) ham was evaluated on 216 animals. Despite the differences in pig live performance and carcass quality for sow line, little effect on meat quality was observed. Only a lower (p < 0.05) intramuscular fat content of ham and a tendency (p < 0.1) toward lower cooking yield was observed in offspring of the sow line with the highest versus the lowest carcass lean content. Loin traits were only weakly associated with fresh and cooked ham quality traits.

Performance and carcass, loin and ham quality in crossbreds from three terminal sire lines.

This study was designed to compare performance, carcass and meat quality of crossbred of a hybrid sow x three sire lines, i.e. stress positive Belgian Piétrain (BP), stress negative French Piétrain (FP) and Canadian Duroc (CD). BP offspring had a significantly higher carcass yield (p < .001) and lean meat content (p < .001) in comparison with FP, which was higher than CD. BP offspring had significantly lower pH (p < .05), water-holding capacity (WHC) (p < .001) and intramuscular fat (IMF) (p < .001) content in the loin compared to FP and CD, but these meat quality parameters, with the exception of pH, were superior for CD as compared to FP. In accordance with loin quality, pHi, pHu, WHC and IMF of BP were significantly lower (p < .05) compared to CD in the fresh and cooked ham. Most often, FP offspring could not be differentiated from the other offspring, with the exception of cooking loss of the cooked ham. Trained and consumer taste panels resulted in no significant differences (p > .1) in sensory attributes, however, consumers preffered CD based on ranking.

Protection of polyunsaturated oils against ruminal biohydrogenation and oxidation during storage using a polyphenol oxidase containing extract from red clover.

Polyunsaturated fatty acid (PUFA) are to a large extent subject to biohydrogenation in a ruminal environment, which results to the healthy value of these PUFA being lost upon dietary addition to ruminants. PUFA are also prone to lipid oxidation upon storage. Therefore, it was tested whether emulsions could be protected against in vitro ruminal biohydrogenation and oxidation during storage by using protein extracts rich in polyphenol oxidase, an enzyme responsible for browning of plant tissues. PUFA rich emulsions were made with a protein extract from red clover (Trifolium pratense L.) before adding a synthetic diphenol (4-methylcatechol) to induce protection. Results after in vitro incubation confirmed the hypothesis and indicated the potential to prevent PUFA in linseed or fish oil from ruminal biohydrogenation and oxidation during storage through addition of 4-methylcatechol to the emulsions. Protection depended on the amount of oil present and protein concentrations in the emulsions. Protection efficiency increased with increasing the amounts of diphenol present in the emulsion per unit interfacial surface area. It is suggested that protection is caused by an effective encapsulation by cross-linking of the protein layer at the emulsion interface. For the first time, a method is described to protect PUFA using an enzyme abundantly available in nature, polyphenol oxidase, in combination with 4-methylcatechol.

Effect of dietary antioxidant supplementation on the oxidative status of plasma in broilers.

In this study, the effect of dietary antioxidants on the plasma oxidative status of growing birds fed a diet rich in polyunsaturated fatty acids was investigated. One-day-old broilers were fed for 42 days a diet containing 4% linseed oil and supplemented with single plant extracts rich in antioxidants (natural tocopherols, rosemary, grape seed, green tea, tomato) or a combination of some of these plant extracts, in two different total doses (100 and 200 mg product/kg feed). A diet with synthetic antioxidants with and without α-tocopheryl acetate (200 mg/kg feed) were also included. The plasma oxidative status was evaluated measuring the ferric reducing ability of plasma (FRAP), the superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity. Lipid peroxidation was measured by thiobarbituric acid-reactive substances (TBARS). No significant effect of the dietary treatments was observed for FRAP as well as for TBARS. However, diet affected GSH-Px activity (p = 0.002) and a trend for an effect on SOD activity was observed (p=0.084). A higher GSH-Px activity was found for 200 mg/kg tomato extract and natural α-tocopherol in relation to the corresponding 100 mg/kg treatment, and the lowest GSH-Px activity was measured for the synthetic antioxidants treatment. The lowest and highest SOD activity were found for the 200. and 100 mg/kg treatment with tomato extract respectively. In conclusion, the oxidative status and lipid oxidation of plasma in broilers was not affected by feeding natural antioxidant extracts at the doses in the present study, but some changes in antioxidant enzyme activities were observed, of which the implication remains to be elucidated.

Characterization and high-resolution mapping of a late blight resistance locus similar to R2 in potato.

Identification of resistance (R) genes to Phytophthora infestans is an essential step in molecular breeding of potato. We identified three specific R genes segregating in a diploid mapping population. One of the R genes is located on chromosome 4 and proved phenotypically indistinguishable from the Solanum demissum-derived R2, although S. demissum is not directly involved in the pedigree of the population. By bulked segregant analysis combined with a resistance assay, a genetic linkage map of the R2-like locus was constructed with 30 coupling and 23 repulsion phase AFLP markers. Two markers flanking the R2-like locus were applied to screen an extended population of 1,586 offspring. About 103 recombinants were selected, and an accurate high-resolution map was constructed. The R2-like resistance was localized in a 0.4 cM interval and was found co-segregating with four AFLP markers, which can be used to isolate the R2-like gene by map-based gene cloning. By analyzing race-specificity and R gene-specific molecular markers, we also found that an R1-like gene and an additional unknown R gene are segregating in the population.

Genetic and physical mapping of homologues of the virus resistance gene Rx1 and the cyst nematode resistance gene Gpa2 in potato.

Nine resistance gene homologues (RGHs) were identified in two diploid potato clones (SH and RH), with a specific primer pair based on conserved motifs in the LRR domain of the potato cyst nematode resistance gene Gpa2 and the potato virus X resistance gene Rx1. A modified AFLP method was used to facilitate the genetic mapping of the RGHs in the four haplotypes under investigation. All nine RGHs appeared to be located in the Gpa2/ Rx1 cluster on chromosome XII. Construction of a physical map using bacterial artificial chromosome (BAC) clones for both the Solanum tuberosum ssp. tuberosum and the S. tuberosum ssp. andigena haplotype of SH showed that the RGHs are located within a stretch of less than 200 kb. Sequence analysis of the RGHs revealed that they are highly similar (93 to 95%) to Gpa2 and Rx1. The sequence identities among all RGHs range from 85 to 100%. Two pairs of RGHs are identical, or nearly so (100 and 99.9%), with each member located in a different genotype. Southern-blot analysis on genomic DNA revealed no evidence for additional homologues outside the Gpa2/ Rx1 cluster on chromosome XII.

Homologues of a single resistance-gene cluster in potato confer resistance to distinct pathogens: a virus and a nematode.

The isolation of the nematode-resistance gene Gpa2 in potato is described, and it is demonstrated that highly homologous resistance genes of a single resistance-gene cluster can confer resistance to distinct pathogen species. Molecular analysis of the Gpa2 locus resulted in the identification of an R-gene cluster of four highly homologous genes in a region of approximately 115 kb. At least two of these genes are active: one corresponds to the previously isolated Rx1 gene that confers resistance to potato virus X, while the other corresponds to the Gpa2 gene that confers resistance to the potato cyst nematode Globodera pallida. The proteins encoded by the Gpa2 and the Rx1 genes share an overall homology of over 88% (amino-acid identity) and belong to the leucine-zipper, nucleotide-binding site, leucine-rich repeat (LZ-NBS-LRR)-containing class of plant resistance genes. From the sequence conservation between Gpa2 and Rx1 it is clear that there is a direct evolutionary relationship between the two proteins. Sequence diversity is concentrated in the LRR region and in the C-terminus. The putative effector domains are more conserved suggesting that, at least in this case, nematode and virus resistance cascades could share common components. These findings underline the potential of protein breeding for engineering new resistance specificities against plant pathogens in vitro.

The 5' terminal sequence of alfalfa mosaic virus RNA 3 is dispensable for replication and contains a determinant for symptom formation.

Transgenic P12 tobacco plants, transformed with the replicase genes P1 and P2 of alfalfa mosaic virus (AIMV), can be infected with RNA 3 of the tripartitite AIMV genome or with a DNA copy of RNA 3 fused to the CaMV 35S promoter and nos terminator. The effect of various modifications on the infectivity of the 35S/cDNA 3 construct to P12 plants was studied. When nonviral sequences ranging from 11 to 200 bp were inserted between the 35S promoter and cDNA 3, the infection became dependent on addition of coat protein (CP) to the inoculum. About 80% of the progeny RNAs resulting from these infections were full-length and had lost the nonviral sequence, whereas 20% were truncated by a deletion of the 5' terminal 79 nucleotides (nt). When the sequence corresponding to the 5' terminal 22 nt of RNA 3 was deleted from the 35S/cDNA 3 construct, the clone was as infectious as the wild type (wt), provided that CP was added to the inoculum, but only progeny RNA with a 5' terminal deletion of 79 nt was produced. The 5' truncated RNA 3 molecules induced necrotic ringspot-like symptoms on P12 tobacco plants, whereas wt RNA 3 did not induce detectable symptoms on these plants. It is proposed that in the infections with the modified 35S/cDNA 3 clones, CP is required in the inoculum to permit internal initiation of plus-strand RNA 3 synthesis on 3'-extended or 3'-truncated minus-strand RNA templates. Evidence was obtained that minus-strand RNA 3 synthesized under the control of the 35S promoter was not infectious to P12 plants.

Analysis of cis-acting elements in the 5' leader sequence of alfalfa mosaic virus RNA 3.

The leader sequence of RNA 3 of the Leiden isolate of alfalfa mosaic virus strain 425 consists of 345 nucleotides (nt) and contains four putative stem-loop structures each with a motif in the loop that resembles the internal control region 2 (ICR2) of tRNA genes. The sequence of the 5' terminal 112 nt of this leader contains one of these stem-loop structures and is sufficient for a reduced accumulation of RNA 3 in protoplasts and a delayed accumulation in plants (E. A. G. van der Vossen et al., Nucleic Acids Res. 21, 1361-1367 (1993). A number of mutations were made in this 112-nt leader sequence to investigate its role in RNA 3 accumulation. Deletion of nucleotides 23-43, 44-90, or 55-112 and inversion or duplication of nucleotides 44-90 all abolished RNA 3 accumulation. Similarly, two base substitutions in the ICR2 motif (nucleotides 60-77) abolished RNA'3 accumulation. Mutations in the stem sections of the putative stem-loop structure had various effects on RNA 3 accumulation and supported the notion that this structure is important for plus-strand promoter activity.

cis-preferential stimulation of alfalfa mosaic virus RNA 3 accumulation by the viral coat protein.

RNA 3 of alfalfa mosaic virus (AIMV) encodes the movement protein P3 and the viral coat protein (CP) which is translated from the subgenomic RNA 4. RNA 3 is able to replicate in tobacco plants transformed with the AIMV replicase genes P1 and P2 (P12 plants). Frameshifts or deletions in the P3 gene have little effect on RNA 3 accumulation in P12 protoplasts whereas such mutations in the CP gene result in a 100-fold reduction of plus-strand RNA 3 accumulation. When P12 protoplasts were inoculated with a mixture of a RNA 3 mutant with a deletion in the P3 gene and a mutant with a deletion in the CP gene, CP expressed by the P3 mutant was unable to upregulate plus-strand RNA accumulation of the CP mutant. However, when a wild-type CP gene and subgenomic promoter were inserted in a RNA 3 mutant with a defective CP gene, the mutant accumulated at wild-type levels. It is concluded that the function of CP in plus-strand RNA 3 accumulation acts in cis and cannot be complemented in trans. In P12 plants, P3 and CP mutants were able to complement each other at low and variable levels. This complementation in plants appeared to be correlated with the occurrence of recombination to wild-type RNA 3.

Characterization of sequences controlling the synthesis of alfalfa mosaic virus subgenomic RNA in vivo.

RNA 3 of alfalfa mosaic virus encodes the movement protein P3 and the viral coat protein (CP). CP is translated from a subgenomic (sg) messenger, RNA 4. To characterize the sg promoter that is responsible for RNA 4 synthesis in vivo, putative sg promoter sequences were inserted in a unique Xhol site located between the initiation codon of the P3 gene and a second in-frame ATG codon in an infectious cDNA clone of RNA 3. Mutants with an active sg promoter insert expressed an N-terminally truncated P3 protein and were able to accumulate in plants. In addition, sg promoter activity was analyzed in protoplasts. When the transcription start site is taken as +1, the sequence of nucleotides -26/+1 was found to have a basal level of sg promoter activity. This activity was increased to near maximum levels when the sg promoter sequence was extended to -136/+12. The upstream positive regulatory element was mapped to nucleotides -136/-94. Engineering of point mutations and small deletions in RNA 3 around the transcription start site for RNA 4 synthesis revealed elements important for sg promoter activity with similarity to sequences conserved in sg promoters of alpha-like viruses. Some of these elements appeared to be required in cis for RNA 3 accumulation. A deletion of the C-terminal three amino acids of the P3 protein rendered this protein nonfunctional in cell-to-cell movement.

Early and late functions of alfalfa mosaic virus coat protein can be mutated separately.

To investigate the role of alfalfa mosaic virus coat protein (CP) in genome activation, asymmetric plus-strand RNA accumulation, and cell-to-cell spread of the virus, mutations were made in the CP gene and putative CP binding sites in the 3'-untranslated region (UTR) of RNA 3. Mutants that produced no CP-related peptide or CP with an N-terminal deletion of 20 amino acids were defective in all three functions. Insertion of several nonviral amino acids at position 85 of CP had little effect on genome activation and plus-strand RNA accumulation but abolished cell-to-cell spread. A mutant encoding CP with a C-terminal deletion of 21 amino acids was defective in plus-strand RNA accumulation but showed substantial levels of genome activation and cell-to-cell spread. Mutations in the 3'-UTR that interfered with CP binding affected plus-strand RNA accumulation and cell-to-cell spread. Neither CP nor CP binding sites at the 3'-end of RNA 3 were required for minus-strand RNA accumulation. The results demonstrate that early and late functions of CP can be mutated separately, indicating that different domains of CP are involved in the three functions investigated.

Infection of tobacco with alfalfa mosaic virus cDNAs sheds light on the early function of the coat protein.

Coat protein (CP) is required in the inoculum to initiate infection of plants with the three genomic RNAs of alfalfa mosaic virus. Inoculation of plants with DNA copies of RNAs 1, 2, and 3, fused to the 35S promoter, resulted in virus replication but the infection level was increased several-fold by addition of CP to the inoculum. When one of the three cDNAs was replaced by its corresponding RNA molecule there was no infection unless CP was present in the inoculum. Plants transformed with a DNA copy of RNA 1 or RNA 2 could be infected with mixtures of cDNAs 2 and 3 or cDNAs 1 and 3, respectively. Again, when one cDNA in the inoculum was replaced by its corresponding RNA, infection depended on the presence of CP in the inoculum. However, when DNA copies of both RNA 1 and RNA 2 were present in the plant genome, the plants became infected after inoculation with cDNA 3 or RNA 3 without any requirement for CP. It is concluded that the necessity of CP in the inoculum depends on the timing of the production of the replicase subunits P1 and P2, encoded by RNAs 1 and 2, respectively, and on the availability of viral template RNAs once the replication complex has been formed. Models to explain the early function of CP are discussed.

Role of the 5' leader sequence of alfalfa mosaic virus RNA 3 in replication and translation of the viral RNA.

RNA 3 of alfalfa mosaic virus (AIMV) encodes the movement protein P3 and the viral coat protein which is translated from the subgenomic RNA 4. The 5'-leader sequences of RNA 3 of AIMV strains S, A, and Y differ in length from 314 to 392 nucleotides and contain a variable number of internal control regions of type 2 (ICR2 motifs) each located in a 27 nt repeat. Infectious cDNA clones were used to exchange the leader sequences of the three strains. This revealed that the leader sequence controls the specific ratio in which RNAs 3 and 4 are synthesized for each strain. In addition, it specifies strain specific differences in the kinetics of P3 accumulation in plants. Subsequent deletion analysis revealed that a 5'-sequence of 112 nt containing one ICR2 motif was sufficient for a 10 to 20% level of RNA 3 accumulation in protoplasts and a delayed accumulation in plants. An additional leader sequence of maximally 114 nt, containing two ICR2 motifs, was required to permit wildtype levels of RNA 3 accumulation. The effect of deletions in the leader sequence on P3 synthesis in vitro and in vivo was investigated.

Defective interfering L RNA segments of tomato spotted wilt virus retain both virus genome termini and have extensive internal deletions.

Defective interfering (DI) RNA molecules derived from the genomic L RNA segment of tomato spotted wilt virus (TSWV) were generated during sequential passage of the virus at high multiplicity. Characterization of DI RNAs from four distinct isolates by Northern blot analysis and sequence determination revealed that both the 5' and 3' genomic termini were retained in these molecules. Each DI RNA contained a single internal deletion of approximately 60% to 80% of the L RNA segment. All DI RNAs studied maintain an open reading frame (ORF) which suggests that these defective molecules should be translatable by ribosomes. Detection of only defective molecules with ORFs indicates either that association with ribosomes or translation is a prerequisite for the selection and maintenance of replicating DI RNAs, or that the truncated proteins produced play a role in their selection or replication. Analysis of the junction sites in the DI RNAs showed that short nucleotide sequences are repeated, one at the release and another at the reinitiation point on the L RNA. One of these is lost during the generation of the DI molecules. The presence of repeated sequences at the junction sites seems to be unique for tospovirus DI L RNAs; they have not been described for other DI systems of either positive- or negative-strand RNA viruses. A model for TSWV DI RNA generation is proposed in which the viral polymerase can 'jump' across the internal sequences from one secondary structure to another containing the repeated sequences, during the replication of the viral complementary L RNA segment.