Type C RNA tumor viruses as determinants of chemical carcinogenesis: effects of sequence of treatment.

Fischer rat embryo cells were treated with 3-methylcholanthrene before or after inoculation with Rauscher murine leukemia virus. Transformation was not observed in untreated control cultures, cultures given virus or 3-methyl-cholanthrene alone, or cultures treated first with 3-methylcholanthrene followed by inoculation with the virus after removal of the chemical. Transformation was dependent upon the presence of Rauscher murine leukemia virus at the time of chemical treatment.

Short communication: estimates of genetic variation of milk fatty acids in US Holstein cows.

Interest in changing the milk fatty acid profile is growing. However, little is known about the genetic variability of milk fatty acids in the US Holstein population. Therefore, genetic parameters for milk fatty acids were estimated using a single-trait, mixed, linear animal model on 592 individual milk samples from 233 daughters of 53 sires in a cow herd genetically representative of the US Holstein population. Heritability (h(2)) and repeatability (r) estimates +/- standard errors for yields of individual fatty acids ranged from 0.00 +/- 0.08 (C4:0) to 0.43 +/- 0.13 (C12:0) for heritabilities and from 0.21 +/- 0.05 (C18:1) to 0.43 +/- 0.05 (C12:0) for repeatabilities. Saturated (h(2) = 0.23 +/- 0.12; r = 0.36 +/- 0.05) and de novo synthesized fatty acids (C6:0 to C14:0; h(2) = 0.30 +/- 0.13; r = 0.40 +/- 0.05) had numerically higher estimates than did monounsaturated (h(2) = 0.09 +/- 0.09; r = 0.22 +/- 0.05) and polyunsaturated fatty acids (h(2) = 0.08 +/- 0.09; r = 0.27 +/- 0.05). For relative proportions of individual fatty acids, the greatest heritability and repeatability estimates were obtained for C8:0 (h(2) = 0.18 +/- 0.12; r = 0.36 +/- 0.05), C10:0 (h(2) = 0.22 +/- 0.13; r = 0.46 +/- 0.05), C12:0 (h(2) = 0.18 +/- 0.12; r = 0.46 +/- 0.05), C16:0 (h(2) = 0.09 +/- 0.12; r = 0.48 +/- 0.05), C16:1 (h(2) = 0.49 +/- 0.13; r = 0.49 +/- 0.05), and C18:0 (h(2) = 0.24 +/- 0.11; r = 0.39 +/- 0.05). Our results suggest the existence of genetic variability of milk fatty acids, in particular of medium-and long-chain fatty acids (C8:0 to C18:0), which could be used to improve the nutritional and textural properties of milk fat by selective breeding.

Short communication: Composition of milk protein and milk fatty acids is stable for cows differing in genetic merit for milk production.

Changing the composition of milk protein and of milk fatty acids alters nutritional and physical properties of dairy products and their consumer appeal. Genetic selection for milk yield decreases concentrations of milk protein and of milk fat. Little is known, however, about how the decrease affects composition of milk protein and milk fatty acids. The objective of this study was to quantify changes in composition of milk protein and of milk fatty acids in cows differing in genetic merit for milk production. Three measures of genetic merit for milk production were used for each cow: genetic line, parent average predicted transmitting ability (PTA) for milk, and cow milk PTA. Composition of milk protein and milk fatty acids were compared in 448 milk samples from 178 cows representing 2 divergent lines of Holsteins that were bred for high or average PTA for milk and combined milk protein and fat yield. High-line cows (n = 97) produced more milk that contained less fat and had higher proportions of alphaS1-casein in milk protein than did average-line cows (n = 81). We additionally obtained from 233 cows (178 cows representing the 2 genetic lines and 55 cows with ancestors from both genetic lines) the parent average milk PTA and cow milk PTA and compared composition of milk protein and of milk fatty acids in 592 milk samples. Cows whose parent average milk PTA was above or equal to the median of the 233 cows produced more milk that contained less protein and less fat and that tended to have greater proportions of alphaS1-casein in milk protein than cows whose average milk PTA was below the median. Similarly, cows with above or equal median milk PTA of the 233 cows produced more milk that contained less protein and less fat and had greater proportions of alphaS1-casein in milk protein than did cows with below-median milk PTA. Milk fatty acid composition was not consistently different between groups. Therefore, selection for milk yield decreased concentrations of milk protein and milk fat but had little effect on composition of milk protein and milk fatty acids.

Butter composition and texture from cows with different milk fatty acid compositions fed fish oil or roasted soybeans.

Changing the milk fatty acid composition can improve the nutritional and physical properties of dairy products and their acceptability to consumers. A more healthful milk fatty acid composition can be achieved by altering the cow's diet, for example, by feeding supplemental fish oil (FO) or roasted soybeans (RSB), or by selecting cows with a more unsaturated milk fatty acid composition. We examined whether feeding supplemental FO or RSB to cows that had a more unsaturated milk fatty acid composition acted additively to produce butter with improved fatty acid composition and texture. Using a 3 x 3 Latin square design with 2 replications, we fed diets to multiparous Holstein cows (60 to 200 DIM) chosen for producing either more or less unsaturated milk fatty acid composition (n = 6 for each group) for three 3-wk periods. The control diet contained 3.7% crude fat and the 2 experimental diets contained, on a dry matter basis, 0.8% of additional lipids in the form of 0.9% of FO or 5% of RSB. The milk, collected in the third week of feeding, was used to make butter, which was analyzed for its fatty acid composition and physical properties. Dry matter intake, milk yield, and milk composition were not significantly affected by cow diet or by cow selection. Cows that produced a more unsaturated and healthful milk fat prior to the feeding study, according to a "health-promoting index" [HPI = (sum of % of unsaturated fatty acids)/ (%12:0 + 4 x %14:0 + %16:0)], maintained a higher HPI in their butter during the feeding study than did cows with a low HPI. Milk from cows fed supplemental FO or RSB yielded more unsaturated butters with a higher HPI. This butter also was softer when the cows were fed RSB. Feeding RSB to cows chosen for their high milk HPI yielded the most unsaturated butter with the highest HPI and softest texture. Thus, selecting cows with a more health-promoting milk fatty acid composition and feeding supplemental RSB can be used in combination to produce butter that has a consumer-friendly texture and a healthful fatty acid profile.

Uptake, metabolism, and persistence of 3-methylcholanthrene in rat embryo cells infected with murine leukemia virus.

The uptake and persistence of 3-methylcholanthrene have been followed in both unifected rat embryo tissue culture cells and in cells infected with type C RNA virus. No significant differences in these parameters were observed as a function of viral infection or cell passage level. Moreover, neither binding of 3-methylcholanthrene to nucleic acids or proteins nor carcinogen metabolism were altered by the viral carrier state. Although transformation of rat cells by chemical carcinogens alone has been reported by us and other authors, the low-passage rat embryo cells used in this study will not transform unless cells are carrying exogenous type C RNA virus. We thus suggest that the virus must play a more direct role in the transformation process rather than affecting the ability of the cell to absorb, retain, or metabolize the chemical.

Replacement of bovine mitochondrial DNA by a sequence variant within one generation.

Inheritance of mitochondrial DNA (mtDNA) in Holstein cattle was characterized by pedigree analysis of nucleotide sequence variation. mtDNA was purified from leukocytes of 174 individuals representing 35 independent maternal lineages, and analyzed for nucleotide sequence variation by characterization of restriction fragment length polymorphism and direct sequence determination. These data revealed 11 maternal lineages in which leukocytes from some individuals seemingly were homoplasmic for the reference mtDNA sequence at nucleotide 364, whereas those from other individuals were homoplasmic for a sequence variant at this position. Both alternative alleles were detected in all branches of these 11 lineages, suggesting that mutation at nucleotide 364 and fixation of the variant sequence occurred frequently in independent events. Thirteen instances were detected of mother-daughter pairs in which leukocytes of each of the two animals seemingly were homoplasmic for a different allele at nucleotide 364, demonstrating the bovine mitochondrial genome can be replaced completely by a nucleotide sequence variant within a single generation. The two alternative sequences seemingly arose de novo at similar frequency, ruling out replicative advantage or other selective bias as the explanation for rapid fixation of mutations at nucleotide 364. Another instance of intralineage sequence variation was detected at nucleotide 5602. This variation was detected in only one of the lineages examined, and evidently arose within three generations.

Myxoid chondrosarcoma with a translocation involving chromosomes 9 and 22.

Myxoid chondrosarcoma is an uncommon neoplasm thought to be derived from mesenchymal chondrocytic cells. Although cytogenetic abnormalities have been reported in sarcomas, too few cases have been studied to determine the frequency of nonrandom chromosomal changes in mesenchymal tumors. In this article, we describe a chondrosarcoma with a nonrandom reciprocal translocation t(9;22)(q22;q11). The cellular homologue to the retrovirus transforming gene of simian sarcoma virus is located on chromosome #22, and its possible significance in this case is discussed.

Cell transformation by chemical agents--a review and analysis of the literature. A report of the U.S. Environmental Protection Agency Gene-Tox Program.

The literature on cell transformation by chemical carcinogens has been critically reviewed. This subject is highly relevant to carcinogenesis in vivo, because the phenotypic changes that are collectively referred to as cell transformation usually involve the acquisition of tumorigenicity on inoculation into suitable rodent hosts. The systems chosen for review fall into 3 categories: cell strains (cells with a limited lifespan); cell lines (cells with an unlimited lifespan); and oncogenic viral-chemical interactions involving cells (Fischer rat embryo cells expressing an endogenous retrovirus, mouse embryo cells expressing the AKR leukemia virus, chemical enhancement of a simian adenovirus, SA7 transformation of Syrian hamster or rat embryo cells). Of the entire literature reviewed, 117 papers have been accepted for data abstraction by pre-defined criteria; these include 41 references to cell strains, 40 in cell lines, and 38 in viral-chemical interactions including cells. Because different systems have been reviewed, it would be meaningless to group all the compounds. The overall summary of the systems is as follows (many compounds have been tested in more than one system and, hence, are duplicated in these totals). (Chart: see text) In general, there is a reasonably good correlation between the results of the cell transformation systems and in vivo carcinogenesis. However, the many deficiencies of the EPA Merged Carcinogen List preclude definitive comparisons. Moreover, a number of 'false negatives' were obtained in systems that did not employ external metabolic activation. Further validation of all systems is required, but it seems very probable that several cell transformation systems will become valuable in assaying (with reasonable time and cost) the carcinogenic potential of environmental chemicals.

Bovine sire effects on daughters' in vitro blood neutrophil functions, lymphocyte blastogenesis, serum complement and conglutinin levels.

Blood neutrophil functions, lymphocyte blastogenic responses, serum complement, and serum conglutinin activity of 98 lactating Holstein cows from two genetic lines were evaluated. The genetic lines were produced in a selection experiment that created and perpetuated genetic differences in milk production for up to seven generations. No significant differences between the two genetic lines of cows were found for neutrophil function, lymphocyte blastogenic responses, serum complement levels, or serum conglutinin levels. Significant differences between sire progeny groups within lines were found for unstimulated and mitogen-stimulated lymphocyte blastogenesis (P less than 0.0001), and almost all neutrophil functions (antibody independent neutrophil cytotoxicity, antibody dependent neutrophil cytotoxicity, ingestion of bacteria, iodination, chemiluminescence, chemokinesis, and chemotaxis (P less than or equal to 0.05)). Sire progeny group differences (P less than or equal to 0.0001) within lines for serum complement and conglutinin activity were also found. Neutrophil chemiluminescence activity (positive relationship; P less than or equal to 0.001), concanavalin A-stimulated lymphocyte blastogenesis (positive relationship; P less than or equal to 0.004), and serum conglutinin activity levels (negative relationship; P less than or equal to 0.01) each had small but significant associations with the total milk somatic cell count. Cows seropositive for bovine leukosis virus had increased resting and mitogen-stimulated lymphocyte blastogenic activity and were associated with increased in vitro neutrophil random migration and production of superoxide anion. Estimates of genetic parameters of various immune cell functions, of serum complement and of conglutinin levels for daughters of 11 sires with 4-6 daughters in the data set were determined. In this report, genetic variation was demonstrated for nonspecific humoral and cellular immunity.

Study of immunological dysfunction in periparturient Holstein cattle selected for high and average milk production.

Data from twenty assays of traits associated with innate and adaptive immunity were evaluated from 137 periparturient Holstein cows. These cows had been selected through planned matings for four different levels of milk production (high and average pounds of milk, and high and average pounds of milk fat plus protein). For up to seven generations, the genetic lines were produced by mating females of each line to sires of corresponding merit. With the exceptions of neutrophil ingestion of Staphylococcus aureus and directed migration, all assays measuring neutrophil functions were depressed beginning 2 to 3 weeks before calving through 3 weeks after calving. Serum concentrations of immunoglobulin G1 decreased while those of immunoglobulin G2 increased around calving time. Serum complement and conglutinin concentrations decreased before calving and reached a minimum around calving time. Cows selected for high milk production (pounds of milk and pounds of milk fat plus proteins) had significantly higher (P < 0.10) numbers of circulating neutrophils and mononuclear cells, had higher (P < 0.10) neutrophil resting chemiluminescence and higher (P < 0.10) neutrophil directed migration than cows with average production potentials. There were significant (P < 0.001) sire progeny group differences for most traits associated with the immune system that we tested. These results can be considered encouraging, in that selection for high milk yield did not produce unfavorable correlated responses in the functional capacity of immune function traits, and that there is sufficient genetic variation in these immunological traits among sires of high genetic merit for milk production to potentially improve the immunocompetence of periparturient cows through planned mating experiments.

Association of class I bovine lymphocyte antigen complex alleles with in vitro blood neutrophil functions, lymphocyte blastogenesis, serum complement and conglutinin levels in dairy cattle.

Ninety-eight lactating Holstein cows from two genetic lines selected for high and average milk production were used in the study. Five peripheral blood samples were collected over a 60-day period from each cow for evaluation of neutrophil function, lymphocyte blastogenesis, leukocyte count, and serum complement and conglutinin levels. Blood samples were typed for antigens encoded by alleles at the bovine major histocompatibility complex (BoLA) A locus. Alleles w14(w8), w20A, and w19(w6) were the most frequent of 14 alleles present in this herd. Association of BoLA type with immune function results was examined by using gene substitution models including and ignoring sire effects. Alleles w15(w8) and w16 were associated with greater circulating mononuclear cell and total leukocyte numbers, while w27(w10), w11, and w20A were associated with lower numbers of these cell types. Alleles EU28D and w20A were positively and negatively associated with granulocyte percentage, respectively. Allele w16 was associated with greater antibody-independent neutrophil cytotoxicity, unstimulated lymphocyte proliferation, serum conglutinin activity, and with lower antibody-dependent neutrophil cytotoxicity. Allele w19(w6) was associated with decreased conglutinin activity and decreased neutrophil iodination. Increased antibody-dependent neutrophil cytotoxicity was observed for animals bearing allele w14(w8), and decreased neutrophil iodination, serum conglutinin, and nonstimulated lymphocyte blastogenesis were observed in individuals carrying w20A or EU28D. Significance of both sire and BoLA complex effects suggests that both major histocompatibility complex genes and background genes of the sire significantly affect immune function. This research suggests BoLA-A locus genes may be major genes or markers for closely linked major genes involved in regulation of nonspecific immune function.

Pregnancy-associated glycoprotein and decreased polymorphonuclear leukocyte function in early post-partum dairy cows.

Phagocytosis and oxidative burst activity of polymorphonuclear neutrophil leukocytes (PMN) isolated from blood and pregnancy-associated glycoprotein (bPAG) concentrations in plasma were evaluated in two longitudinal studies in dairy cows from 3 weeks before until 5 weeks after calving, carried out in the United States and in Europe. Ingestion of Staphylococcus aureus by blood PMN increased during the first week after calving and normalised 3 weeks post-partum. Phagocytosis of Escherichia coli did not change in the early post-partum period. In both studies, a significant decrease in oxidative burst activity of PMN was observed between 1 and 3 weeks after calving. In all cows, a very significant increase in plasma bPAG concentration was found between 1 week before and 2 weeks after calving. The peak of bPAG concentration in plasma immediately preceded the alterations of blood PMN functions. These results suggest that bPAG may be associated with inhibition of PMN function of dairy cows during the early post-partum period.

Molecular analysis of cytoplasmic genetic variation in Holstein cows.

Mitochondrial DNA from Holstein maternal lineages implicated to express cytoplasmic genetic effects on lactation traits was subcloned and screened for molecular polymorphisms. Sixteen of 35 lineages sampled differed from the most common mitochondrial DNA form by at least one restriction endonuclease cleavage site in the 4.3 kilobase segment examined. Variation existed in the region that regulates DNA replication and transcription as well as in transfer and ribosomal ribonucleic acid coding regions of the DNA. The index of nucleotide diversity calculated from polymorphism frequencies indicated that the minimum extent of variation between two random lineages was 1.16 x 10(-4) nucleotide differences per base pair in the segment examined. Presence of a HpaII marker near nucleotide 360 was associated with lower (P less than .001) milk fat percentages. Molecular markers indicated that pedigrees may not be sufficient to separate true cytoplasmic lineages for quantitative genetic analyses. These findings provide a molecular genetic basis for further study of cytoplasmic effects on phenotypic variation in Holstein cattle.

Comparison of natural transmission of bovine leukemia virus in Holstein cows of two genetic lines selected for high and average milk production.

One hundred and fifty lactating Holstein cows from 2 genetic lines selected for high and average milk production were used in the study. Sera from 6 annual herd tests were analyzed by agar-gel immunodiffusion test for antibodies to bovine leukemia virus. Odds of being seropositive were analyzed by use of stepwise and backward logistic regression procedures. Analysis within birth year revealed that estimated in odds increased by 0.19/year of age among cows of the high genetic line and by 0.43 among cows of the average genetic line. This was accompanied by a more important cohort effect among high producers than among average producers.

Genetic control of disease resistance and immunoresponsiveness.

A great deal of evidence points to substantial genetic control over at least some of the immune responses, although genetic parameters for clinical disease have been less favorable. The past two decades have illustrated that single genes with a large impact on food animal health do exist and can be used to improve the health of domestic populations. The current focus on molecular genetics within food animal species will likely unveil numerous other examples of single genes with large effects, although the use of animals possessing favorable genotypes for disease resistance may represent a compromise in selection for increased production of raw product. Moreover, it is also clear that genetic control over the immune system is not limited to a few genes but is more likely influenced by many genes, each with small effects. The use of this information in animal improvement programs is not straightforward because of factors complicating the identification of superior individuals within the population. The scarcity of information dealing with phenotypic and genetic relationships between measures of disease resistance and aspects of immune response complicates the situation even further. Despite these potential hurdles, the potential for permanent improvement of disease resistance within food animal species in the future is tantalizing and merits intensified future study.

Direct and correlated responses to multitrait, divergent selection for immunocompetence.

Leghorn lines had been selected for an immunocompetence index based on four traits: antibody production to Mycoplasma gallisepticum (MG) and Pasteurella multocida (PM) vaccines, reticuloendothelial clearance of colloidal carbon (CCA), and cell-mediated, wing web response to phytohemagglutinin (PHA). The purpose of this study was to produce replicated lines of chickens with divergent levels of multitrait immunocompetence by index selection. The objectives of analyses of Generations 5 to 7 of this study was to characterize these lines with respect to immune-response traits, correlations among these traits, and correlated responses in other important production traits. Differences (P < .05) existed between the lines selected for high or low immune response and between the two replicates in mean breeding values and in individual immune-response traits. Averages of heritability estimates, weighted by number of offspring and pooled across three generations (two cycles of selection), estimated by using sire variance components and parent-offspring correlations were, respectively, .16 and .09 for the index, .31 and .08 for MG, .21 and -.02 for PM, .06 and .05 for CCA, and .08 and .12 for PHA. Realized heritabilities (response divided by effective selection differential) pooled across the two selection cycles, were .19 and .11 for the index, .06 and -.01 for MG, .44 and .32 for PM, 1.52 and -1.21 for CCA, and .48 and .15 for PHA, for Replicates 1 and 2, respectively. Phenotypic correlations among traits were generally small, and several estimates were negative. Estimates of genetic correlation varied widely. Juvenile and adult body weights, age of first egg, 32-wk egg weight, and rate of egg production were analyzed to evaluate effects of selection on these traits of direct economic importance. Very few differences were noted.

Differences in major histocompatibility complex frequencies after multitrait, divergent selection for immunocompetence.

White Leghorn chickens from lines selected for four immune-response traits (IR lines) were serotyped for B system alloantigens characterizing the haplotypes and genotypes to examine the effect of divergent selection for multitrait immunocompetence on MHC haplotype and genotype frequencies. The selected lines were derived from the Ottawa Strain 7. The selection index included four immunocompetence traits: antibody production against Mycoplasma gallisepticum (MG) and Pasteurella multocida, inflammatory response to phytohemagglutinin, and reticuloendothelial carbon clearance. The four lines include two replicates of high and low multitrait-immunocompetence lines. After four cycles of selection, significant differences (P < .05) in several B system haplotype frequencies were observed, both among IR lines and between the IR lines and the Ottawa Strain 7. The B2 haplotype frequency was greater in all IR lines than in the Ottawa Strain 7. The B21 frequency was less in both high lines than in the Ottawa Strain 7. In comparisons among lines, frequencies of B21 were greater in both replicates of the low lines and the B12 and B19 frequencies were significantly greater (P < .05) in the high lines. A gene substitution model showed effects (P < .10) of specific haplotypes on MG and on the index. The B2 haplotype had a positive effect associated with MG. Haplotype B21 was positively associated with the multitrait index. Haplotype B13 had a negative effect on both MG and the index. Significant differences (P < .01) in genotype frequencies were also noted among the IR lines. Associations between specific MHC haplotypes or genotypes and immune-response traits may offer insight into MHC-mediated mechanisms of disease resistance.

Validation and long term performance characteristics of a quantitative enzyme linked immunosorbent assay (ELISA) for human anti-PA IgG.

Accurate, reliable and standardized quantification of anti-protective antigen (PA) IgG antibody levels is essential for comparative analyses of anti-toxin immune responses in anthrax cases, recipients of PA-based anthrax vaccines and for evaluation of anti-PA based immunotherapies. We have previously reported the early performance characteristics and application of a quantitative anti-PA IgG enzyme linked immunosorbent assay. The principal application of this assay was in a Phase 4 human clinical trial of anthrax vaccine adsorbed (AVA, BioThrax), the central component of the CDC Anthrax Vaccine Research Program (AVRP) and in humans following bioterrorism associated Bacillus anthracis infection (Quinn et al., 2002; Quinn et al., 2004; Marano et al., 2008). The objective of the AVRP was to determine the feasibility of reducing the number of priming series and booster doses of the licensed Anthrax Vaccine Adsorbed (AVA) (BioThrax®; Emergent BioSolutions, Lansing, MI) and changing the route of administration from subcutaneous (SC) to intramuscular (IM) (Marano et al., 2008). In this paper we report the validation and long term performance characteristics of the assay during its six year application in the AVRP (2002-2008). The critical features are 1) extensive validation of the assay using two standard reference sera; 2) long term stability and 3) consistency of the data for quantitative analysis of human long term anti-PA IgG responses. The reportable value (RV) of the assay was expressed as anti-PA IgG concentration (µg/ml). Accuracy of the assay was high with a percent error (%ER) range of 1.6-11.4%. Overall intra-operator and intermediate precision were high with Coefficients of Variation (%CVs) of 2.5-15.4% and 6.3-13.2%, respectively. The assay demonstrated excellent dilutional linearity for human sera using log(10) transformed data with the slope=0.95 to 0.99, intercept=0.02 to 0.06 and r(2)=0.980-0.987. The assay was robust, tolerating changes in serum incubation temperatures from 35 to 39°C, serum incubation times from 55 to 65min and changes in key reagents. The long-term assay stability over 6years using consecutive reference sera AVR414 and AVR801 demonstrated sustained high accuracy and precision for the assay, confirming its suitability for long term studies of PA protein-based anthrax vaccines.