Proteomic features linked to tenderness of aged pork loins.

There is considerable evidence that the protein component of fresh pork makes a major contribution to tenderness. In particular, the proteomic profile can be linked to postmortem events including pH decline, tissue oxidation, and protein degradation. The objectives for this study were to determine differences in sarcoplasmic proteomes that contribute to tenderness variation in aged pork longissimus dorsi muscles (LM). A defined set of pork loins selected to be similar in pH, color, and lipid yet different in tenderness were used. Pork loins were assigned to tenderness groups based on their star probe values; a high star probe group (HSP; n=12 mean star probe 7.75 kg) and low star probe group (LPS; n=12 star probe 4.95 kg) Samples were selected for proteomic experiments based on star probe values, and selected samples were within specified ranges for ultimate pH (5.54-5.86), marbling score (1.0-3.0), and percent total lipid (1.61-3.37%). Two-dimensional difference in gel electrophoresis (2D-DIGE) and mass spectrometry were used to examine sarcoplasmic protein abundance and potential modifications. Proteins spots that were significantly different across groups were selected for identification. Results from 2D-DIGE showed that HSP samples had significantly more abundant metabolic, stress response, and regulatory proteins in the sarcoplasmic fraction compared with LSP samples. The stress response protein peroxiredoxin-2 was more abundant in HSP samples as determined by 2D-DIGE ( ≤ 0.01; 2 spots) and western blot assay ( = 0.02). Low star probe samples showed significantly more degradation of the structural protein desmin in 2D-DIGE ( < 0.01) and western blot assay ( < 0.01). These results demonstrate that extreme proteolytic differences influenced measured tenderness of LSP and HSP samples and that soluble desmin and peroxiredoxin-2 may be used as biomarkers to differentiate between tough and tender aged pork products.

Postmortem protein degradation is a key contributor to fresh pork loin tenderness.

The objective of this study was to determine factors that influence tenderness independent of variation in pH, color, or marbling. To achieve the objective, 2 sample groups were chosen from a population of 159 pork loins aged 11 to 16 d. Predetermined ranges (ultimate pH, 5.54 to 5.86; marbling score, 1.0 to 3.0; percent total lipid, 1.61 to 3.37%) were defined for inclusion of individual loins in the study. The pork loins with the greatest ( = 12) and least ( = 12) Instron star probe values were assigned to 2 classification groups. The high star probe group had an average star probe that was 2.8 kg greater than the low star probe group (7.75 vs. 4.95 kg). Pork quality and sensory characteristics of pH, subjective and instrumental color values, cook loss, sensory tenderness, chewiness, juiciness, pork flavor, and off flavor were determined on fresh, never frozen pork chops. Lipid content, sarcomere length, myosin heavy-chain profile, and calpain autolysis were determined. Degradation of troponin-T, desmin, filamin, and titin were evaluated on the protein extracts from each sample. Pork loin pH, subjective color scores, Minolta L values, sarcomere length, and myosin heavy-chain composition were not different across groups. Chops from the low star probe group had a significantly greater marbling score (2.3 vs. 1.9) and lipid content (2.61 vs. 2.23%). Calpain-1 was completely autolyzed in both high and low star probe samples, demonstrating that calpain-1 potentially had been active in all samples. Low star probe whole-muscle protein extracts had more troponin-T ( < 0.01), desmin ( < 0.01), and filamin degradation ( < 0.01) than high star probe samples. Both classification groups showed degradation of titin. Remarkably, some high star probe samples still had observable intact bands of titin on SDS-PAGE gels. These results demonstrate that significant variation in instrumental tenderness is observed within a moderate pH range. Lipid content and proteolysis both appear to contribute to this variation.

Opsonization effects on Mycobacterium avium subsp. paratuberculosis--macrophage interactions.

High antibody titers in ruminants infected with Mycobacterium avium subsp. paratuberculosis correlates with disease progression. Effects of humoral responses during mycobacterial infection are not completely understood. This study suggests that activation status may be an important factor in determining macrophage ability to limit proliferation of opsonized M. avium subsp. paratuberculosis.

Inducible nitric oxide synthase immunoreactivity in the granulomatous intestinal lesions of naturally occurring bovine Johne's disease.

Inducible nitric oxide synthase (iNOS) is important in the control of a number of intracellular pathogens, including mycobacteria, and is a marker of classic macrophage activation. In human granulomatous diseases such as leprosy, a spectrum of granulomatous lesions is described, ranging from the tuberculoid to lepromatous types. Tuberculoid granulomas are associated with enhanced iNOS production and improved clinical outcomes over the lepromatous types. The aim of this study is to determine whether an association exists between morphology of bovine Johne's disease granulomas and lesion macrophage effector functions. To accomplish this, we retrospectively evaluated 24 cases of bovine Johne's disease. In each case, we recorded the predominant granuloma morphology and evaluated iNOS immunoreactivity and bacterial burden by acid-fast stains and mycobacterial immunolabeling. The results of this study demonstrate that all cases had granulomas with features most similar to the lepromatous type. This morphology correlated with heavy bacterial burdens demonstrated by acid-fast staining and mycobacterial immunoreactivity. None of the cases had high expression of iNOS in mycobacterial-positive granulomas. When iNOS immunoreactivity was identified, it was usually located near the crypts and was distinct from the granulomatous foci.

Early induction of humoral and cellular immune responses during experimental Mycobacterium avium subsp. paratuberculosis infection of calves.

Johne's disease (paratuberculosis) of cattle is widespread and causes significant economic losses for producers due to decreased production and poor health of affected animals. The chronic nature of the disease and the lack of a reproducible model of infection hinder research efforts. In the present study, instillation of Mycobacterium avium subsp. paratuberculosis into the tonsillar crypts of neonatal calves resulted in peripheral colonization as detected by antemortem culture of feces and postmortem (320 days postchallenge) culture of intestinal tissues. Antigen-specific blastogenic, gamma interferon (IFN-gamma), and nitric oxide responses by blood mononuclear cells from infected calves exceeded prechallenge responses beginning 194 days postchallenge. Upon in vitro stimulation with paratuberculosis antigens, CD4(+) cells from infected calves proliferated, produced IFN-gamma, and increased expression of CD26 and CD45RO (indicative of an activated memory phenotype). Utilizing a lipoarabinomannan-based enzyme-linked immunosorbent assay, specific serum immunoglobulin was detected as early as 134 days postchallenge and generally increased after this time point. Two antigens of approximately 50 and approximately 60 kDa were particularly immunodominant early in infection, as shown by immunoblot with serum collected within 2 weeks postchallenge. Findings indicate that the intratonsillar inoculation route will prove useful as an experimental model for paratuberculosis infection. Additionally, this study confirms that mycobacteria-specific antibody is detectable early in the course of experimental Johne's disease, even preceding the development of specific cell-mediated responses.

Phagosomal maturation and intracellular survival of Mycobacterium avium subspecies paratuberculosis in J774 cells.

The mechanisms by which Mycobacterium avium subspecies paratuberculosis (M. a. ptb) survives within macrophages are not well characterized. One strategy for intracellular survival developed by Mycobacterium tuberculosis is inhibition of phagosomal maturation. In this study it was hypothesized that M. a. ptb is capable of survival within macrophages by residing within a phagosomal compartment that does not mature into a functional phagolysosome. To test this hypothesis the following objectives were determined. Phagosomal maturation was evaluated by comparison of stage specific markers on the membranes of phagosomes containing live M. a. ptb with those containing killed M. a. ptb, Mycobacterium smegmatis, and zymosan A using immunofluorescent labeling and confocal microscopy. Intracellular survival of live M. a. ptb within J774 macrophages was compared to that of M. smegmatis by direct determination of bacterial viability by differential live/dead staining. The results of this study show that the phagosomes containing live M. a. ptb had increased levels of an early marker (transferrin receptor [TFR]) and decreased levels of a late maturation marker (lysosome associated membrane protein one [Lamp-1]), relative to those containing killed M. a. ptb, M. smegmatis, and zymosan A. Additionally, compared to M. smegmatis, M. a. ptb has enhanced ability to survive within cultured macrophages. These data indicate that M. a. ptb resists intracellular killing by residing within a phagosomal compartment that retains the characteristics of early phagosomes and resists maturation into functional phagolysosome.

Production of a Mycobacterium avium ssp. paratuberculosis purified protein derivative (PPD) and evaluation of potency in guinea pigs.

A Mycobacterium avium ssp. paratuberculosis purified protein derivative (PPD) was produced and the biologic activity evaluated in sensitized guinea pigs. The PPD when adjusted to a protein concentration of 1mg/ml induced a delayed-type hypersensitivity response comparable to USDA Johnin OT 133-8707.

T cell-dependent inducible nitric oxide synthase production and ultrastructural morphology in BALB/c mice infected with Mycobacterium avium subspecies paratuberculosis.

Euthymic BALB/c and athymic nude BALB/c mice aged 3-8 days were infected intraperitoneally with Mycobacterium avium subspecies paratuberculosis (ATCC strain 19698). After euthanasia at 5 months post-inoculation, hepatic granulomas were evaluated by morphometric analysis of digital images captured from light microscopy sections, by electron microscopy and by immunohistochemical methods. Euthymic mice differed from athymic mice in that (1) their hepatic granulomas were smaller, contained fewer bacteria, and produced more inducible nitric oxide synthase, and (2) their hepatic macrophages contained fewer bacteria, a higher percentage of degraded bacteria, and increased numbers of primary lysosomes. The study showed that macrophage activation was markedly less in the T cell-deficient athymic mice than in the euthymic mice.

Intracellular trafficking of Mycobacterium avium ss. paratuberculosis in macrophages.

The granulomatous enteric lesions of cattle with Johne's disease are composed of infected macrophages, and grow by accumulation, re-infection, and expansion of macrophage populations in the intestinal wall. We have examined the growth of bacteria in macrophages to define characteristics of intracellular trafficking for exocytosis, replication, and antigen presentation. Using immunocytochemical markers for light, confocal and electron microscopy, we have examined potential pathway tropisms using data for bacterial attachment, phagosomal acidification, phagolysosomal degradation and apoptosis. Our hypotheses are that pathogenic/wild-type strains block phagosomal acidification so that the phagosome fails to obtain markers of the late phagosome and phagolysosome, and this leads to the replication pathway within bacteriophorous vacuoles. Non-pathogenic strains appear to be processed to exocytosis, and avirulent mutant strains may be degraded and have preference of antigen processing pathways that involve transport vesicles bearing MHC II antigens. Pathogenicity in a nude mouse model of intestinal infection reveals lesion development and confirms pathway preferences of virulent strains for bacteriophorous vacuole formation.

Absence of protection against challenge with aspergillus fumigatus by adoptive transfer of splenocytes from convalescent turkeys.

Only limited protective immunity against aspergillosis after experimental immunization of turkeys has been previously demonstrated. No studies evaluating the efficacy of transfer of immunity in preventing aspergillosis in birds have been reported. This study consisted of two trials assessing the level of protection against Aspergillus fumigatus challenge afforded by transfer of splenocytes from convalescent turkeys. Three treatment groups of 12-to-14-wk-old Beltsville small white (BSW) turkeys comprising the splenocyte donors were prepared by one of the following: 1) intra-air sac (IA) challenge with A. fumigatus conidia 5 wk prior to transfer; 2) IA challenge and then intravenous (i.v.) injection of killed conidia 1 wk prior to transfer; or 3) sham inoculations. Splenocytes from each group were pooled, enriched for mononuclear leukocytes by density gradient centrifugation, and diluted in cell culture medium (CM). Cell viability was assessed by dye exclusion. Each splenocyte preparation was administered intravenously to one of three recipient groups consisting of 10 BSW turkeys each. A control group (n = 10) was given cell-free CM. Recipients were challenged with viable A. fumigatus conidia 16 hr after splenocyte transfer by unilateral IA (trial 1) or i.v. (trial 2) inoculation. Lesion scores postchallenge revealed no differences between turkeys given splenocytes from convalescent vs. naive (control) turkeys. IA exposure produced ipsilateral lesions in air sacs and lung, whereas i.v. exposure produced severe miliary hepatitis. Donor cell function was confirmed by mitogen blastogenesis; however, cells were nonresponsive to A. fumigatus antigens, regardless of previous exposure status.

Heat inactivation of Mycobacterium paratuberculosis in raw milk: are current pasteurization conditions effective?

Currently, it is not known whether commercial pasteurization effectively kills Mycobacterium paratuberculosis in contaminated raw milk. Results from holder test tube experiments indicated that a residual population of viable bacteria remained after treatment at 65, 72, 74, or 76 degrees C for 0 to 30 min. Use of a laboratory-scale pasteurizer unit demonstrated that treatment of raw milk at 72 degrees C for 15 s effectively killed all M. paratuberculosis.

Morphometric analysis of CD4+, CD8+, and gamma/delta+ T-lymphocytes in lymph nodes of cattle vaccinated with Brucella abortus strains RB51 and 19.

T-lymphocyte subpopulations were examined in vivo by computer-assisted morphometry of superficial cervical lymph nodes of cattle vaccinated with Brucella abortus. Twenty-four 8-month-old Hereford heifers were injected subcutaneously in the axillary area with 1 x 10(10) live B. abortus strain RB51 (SRB51, n = 12) or strain 19 (S19, n = 6) suspended in 2 ml of saline. Six control heifers were injected with sterile saline. Lymph nodes were collected at 1, 2, 4, 6, 10 and 12 weeks postvaccination. Both SRB51 and S19 were cultured from lymph nodes, but SRB51 persisted for a longer period after vaccination (10 weeks) than S19 (6 weeks). Cryostat sections were incubated with monoclonal antibody to CD4 (IL-A11), CD8 (IL-A51), or gamma/delta (IL-A29) bovine T-cell surface antigen and processed for immunoperoxidase staining. Numbers of stained lymphocytes in randomly selected fields were calculated using image-analysis software. There were no significant differences in the number (P = 0.07) or relative proportions (P = 0.22) of CD4+, CD8+, and gamma/delta+ lymphocytes in SRB51, S19, and control lymph nodes. There was a statistically significant difference in the distribution of the three T-cell subsets (P = 0.001). The CD4+ cells were most closely grouped and the gamma/delta+ cells had the most widely scattered distribution, regardless of vaccination status. The results support other studies indicating lymphocyte depletion is not a sequela of infection with B. abortus vaccine strains given to conventionally reared cattle.

T lymphocyte reactivity to glutamic acid-alanine-tyrosine in vitro does not reflect antibody response in vivo.

Mechanisms responsible for the differences in humoral immune response to GAT (a random linear amino acid polymer) were investigated in a line of chickens consisting of four sublines homozygous for Ea-B (B1 or B19) and high or low antibody response to GAT (Ir-GATH or Ir-GATL). Previous research provided evidence of chromosomal recombination between the serologically determined regions of the MHC (encoded by B-F and B-G genes) and the gene or genes that control immune response to GAT, but immune response to GAT did not seem to be mediated through differences in B-L gene products. In the present study, proliferation of GAT-primed T lymphocytes indicated that reactivity in vitro was not associated with antibody levels produced in the animal. Cell surface markers were identified by flow cytometry. Lymphocytes from Ea-B19 chickens that were Ir-GATL had a higher percentage of suppressor T (CD8)-positive cells than did lymphocytes from Ir-GATH chickens. The Ea-B1 chickens that were Ir-GATL had a higher percentage of CD4-positive lymphocytes than did chickens that were Ir-GATH. This may indicate that low response to GAT in the Ea-B19 chickens, but not in Ea-B1 chickens, is mediated by CD8-positive cells. The ability of antigen-presenting cells (APC) to process and present GAT to antigen-primed T lymphocytes was tested in vitro. Measurements of lymphocyte proliferation indicated that, within the Ea-B1 blood type, APC from Ir-GATL chickens produced higher (P < .05) stimulation of both GAT low- and GAT high-responder lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene complementation in biological crosses for humoral immune response to glutamic acid-alanine-tyrosine.

Level of humoral immune response to GAT has been associated with the MHC of chickens. Matings between two unrelated lines of chickens with low antibody response to GAT (G-B1 and S1 Line B19L) resulted in progeny that were higher responders to GAT challenge (P < .05) than either of the parental lines. Progeny of matings between two related sublines that are low responders to GAT (S1 Lines B19L and B1L) had antibody responses to GAT that were not higher than the parental lines. Progeny of the between-line cross were backcrossed to S1 B19L and G-B1 (B13) parental lines, as well as mated inter se. These matings produced F2 progeny whose GAT response was significantly associated (P < .05) with their MHC (Ea-B) type. The progeny were of three MHC types (B19B19, B19B13, and B13B13) that bound 66.6, 71.9, and 4.6%, respectively, of the GAT in a radioimmunoassay. The results from these matings suggest that MHC or MHC-linked genes, as well as genes not linked to the MHC, contribute to control of humoral immune response to GAT in the lines of chicken tested.

Association of Marek's disease with Ea-B and immune response genes in subline and F2 populations of the Iowa State S1 Leghorn line.

Chickens from the Iowa State S1 White Leghorn line, selected for characteristics of Ea-B serotype, humoral immune response to glutamic acid-alanine-tyrosine (Ir-GAT), and response to Rous sarcoma virus (RSV)-induced tumors, were evaluated for genetic resistance to Marek's disease (MD). In the first two trials, sublines that were triple homozygous for the three traits were challenged with MD virus. Birds of the B1B1 blood type were significantly (P less than .001) more resistant to MD than chickens of the B19B19 blood type. High responders to GAT were significantly (P less than .001) more resistant to MD virus than low responders. The RSV classification had no detectable association with MD resistance. Chickens challenged with MD virus in the third trial were an F2 population produced from inter se matings of S1 chickens heterozygous for the three traits under selection. Data from this trial confirmed increased MD resistance of chickens possessing the B1B1 blood type when associated with genes encoding high immune response to GAT.