Variations in automatically recorded rumination time as explained by variations in intake of dietary fractions and milk production, and between-cow variation.

Individual recording of rumination time (RT) is now possible in commercial dairy herds, through development of a microphone-based sensor, which is able to record RT by the sound of rumination activity. The objectives of this study were to examine the relationship between daily RT and intakes of different dietary fractions, the relationship between RT in minutes per kilogram of dry matter intake (DMI) and milk production, and to examine the variation in RT within and between mid-lactating dairy cows. Data from 3 production trials were used in which a total of 27 different diets were fed. The data contained 761, 290, and 203 daily recordings of RT, milk yield, milk components, DMI, and intake of dietary fractions recorded on 29, 26, and 24 Holstein and Swedish Red cows from trials 1, 2, and 3, respectively. The dietary fractions included forage neutral detergent fiber (NDF), concentrate NDF, crude protein, sugar, starch, and the remaining fraction represented by organic matter--(forage NDF+concentrate NDF+crude protein+sugar+starch). The relationship between the dietary fractions and RT was analyzed in 2 steps. In step 1, the dietary fractions, which were significantly related to RT, were selected and simultaneously checked for multicollinearity between the dietary components; in step 2, a multivariate model, including the effect of repeated measurements, the main effect of the selected dietary fractions from step 1, random effects of cow(trial) and trial, and information on breed, days in milk, and parity was used to analyze the relationship between RT and the selected dietary fractions. Relationships between RT in minutes per kilogram of DMI and milk yield and milk components were analyzed, using the same multivariate model as in step 2. Approximately 32% of the variation in daily RT could be explained by variations in intakes of the dietary fractions, whereas 48% of the total variation in RT was accounted for by individual variations between cows. Intakes of forage NDF and starch were positively related to daily RT, whereas intakes of sugar and the remaining fraction were negatively related to daily RT. Rumination time in minutes per kilogram of DMI was negatively related to milk yield and protein percentage, but positively related to milk fat percentage.

Effects of forage type, forage to concentrate ratio, and crushed linseed supplementation on milk fatty acid profile in lactating dairy cows.

The effects of an increasing proportion of crushed linseed (CL) in combination with varying forage type (grass or corn silage) and forage to concentrate ratio (F:C), and their interactions on milk fatty acid (FA) profile of high-producing dairy cows was studied using a 3-factor Box-Behnken design. Sixteen Holstein and 20 Swedish Red cows were blocked according to breed, parity, and milk yield, and randomly assigned to 4 groups. Groups were fed different treatment diets formulated from combinations of the 3 main factors each containing 3 levels. Forage type (fraction of total forage dry matter, DM) included 20, 50, and 80% grass silage, with the remainder being corn silage. The F:C (DM basis) were 35:65, 50:50, and 65:35, and CL was supplied at 1, 3, and 5% of diet DM. Starch and neutral detergent fiber content (DM basis) of the treatment diets ranged from 117 to 209 g/kg and 311 to 388 g/kg, respectively. Thirteen treatment diets were formulated according to the Box-Behnken design. During 4 experimental periods of 21 d each, all treatment diets were fed, including a repetition of the center point treatment (50% grass silage, 50:50F:C, 3% CL) during every period. Intake, production performance, and milk FA profile were measured, and response surface equations were derived for these variables. Shifting from 80% grass silage to 80% corn silage in the diet linearly increased dry matter intake (DMI), net energy for lactation (NE(L)) intake, cis-9,cis-12-C18:2 (C18:2n-6) intake, and milk yield, and linearly decreased cis-9,cis-12,cis-15-C18:3 (C18:3n-3) intake and milk fat content. Shifting from a high forage to a high concentrate diet linearly increased DMI, NE(L) intake, C18:2n-6 intake, and milk yield, and decreased milk fat content. Supplementation of CL linearly increased C18:3n-3 intake, but had no effect on DMI, NE(L) intake, milk yield, or milk fat content. Shifting from 80% grass silage to 80% corn silage linearly increased proportions of trans-10-C18:1 and C18:2n-6 in milk fat, whereas the proportions of trans-11,cis-15-C18:2 and C18:3n-3 linearly decreased. Significant interactions between CL supplementation and F:C were found for proportions of trans-10-C18:1, trans-15-C18:1, cis-15-C18:1, trans-11,cis-15-C18:2, and C18:3n-3 in milk fat, with the highest levels achieved when the diet contained 5% CL and a 35:65F:C ratio. The effect of supplementing CL on several milk FA proportions, including C18:2n-6 and C18:3n-3, depends significantly on the F:C ratio and forage type in the basal diet.

Long-term stability of liquid ionization chambers with regard to their qualification as local reference dosimeters for low dose-rate absorbed dose measurements in water.

The long-term sensitivity and calibration stability of liquid ionization chambers (LICs) has been studied at a local and a secondary standards dosimetry laboratory over a period of 3 years. The chambers were transported several times by mail between the two laboratories for measurements. The LICs used in this work are designed for absorbed dose measurements in the dose rate region of 0.1-100 mGy min(-1) and have a liquid layer thickness of 1 mm and a sensitive volume of 16.2 mm3. The liquids used as sensitive media in the chambers are mixtures of isooctane (C8H18) and tetramethylsilane (Si(CH3)4) in different proportions (about 2 to 1). Operating at a polarizing voltage of 300 V the leakage current of the chambers was stable and never exceeded 3% of the observable current at a dose rate of about 1 mGy min(-1). The volume sensitivity of the chambers was measured to be of the order of 10(-9) C Gy(-1) mm3. No systematic changes in the absorbed dose to water calibration was observed for any of the chambers during the test period (sigma < 0.2%). Variations in chamber dose response with small changes in the polarizing voltage as well as sensitivity changes with accumulated absorbed dose were also investigated. Measurements showed that the LIC response varies by 0.15% per 1% change in applied voltage around 300 V. No significant change could be observed in the LIC sensitivity after a single absorbed dose of 15 kGy. The results indicate that the LIC can be made to serve as a calibration transfer instrument and a reference detector for absorbed dose to water determinations providing good precision and long-term reproducibility.

Assessment of the relative dose distribution around an 192Ir line source using a liquid ionization chamber.

The relative absorbed dose distribution in water around an 192Ir line source of 50 mm length and 0.3 mm diam has been measured using a liquid ionization chamber (LIC). The sensitive volume of the chamber is a cylinder with 3.0 mm diam and a thickness of 1.0 mm. The sensitive medium in the LIC consists of a mixture of two dielectric liquids, tetramethylsilane and isooctane. The mixture has been optimized so that the LIC provides an almost energy-independent response for the radiation qualities present at different distances from the source in water. The measurements were carried out at distances of 2.5-50.0 mm along the source bisector and at distances of 0.0 to +/-40.0 mm along the source axis. The results were compared with measurements made with LiF chips in Solid Water and with calculated data based on an analytical solution to the Sievert integral as well as with Monte Carlo calculations of absolute dose rate in water. Considering the uncertainties involved, the dose distribution measured by the LIC is in reasonably good agreement with the theoretical data as well as the results held with LiF. In comparison with the Monte Carlo calculations the discrepancies range from 0.1% to 18% with the largest differences at points close to the source. This work demonstrates the ability of the LIC for mapping the dose distribution around a low-dose-rate brachytherapy source emitting photons of intermediate energies.

General collection efficiency in liquid iso-octane and tetramethylsilane used as sensitive media in a thimble ionization chamber.

The general collection efficiency in the dielectric liquids iso-octane (CaH18; 2-2-4 trimethylpentane) and tetramethylsilane (Si(CH3)4), used as sensitive media in a thimble liquid ionization chamber (LIC) with a liquid layer thickness of 1 mm, has been studied. Measurements were made for continuous radiation at varying dose rates using 140 keV photons from the decay of 99mTc for chamber polarizing voltages of 50, 100 and 500 V. The maximum dose rate in each measurement session was about 150 mGy min(-1). The experimental results were compared with theoretical general collection efficiencies calculated by the equation for the general collection efficiency in gases. The results show that the general collection efficiency in a thimble LIC for continuous radiation can be calculated with the equation for the general collection efficiency in gas ionization chambers, using the same chamber geometry correction factors and analogous characteristic ion recombination parameters for the dielectric liquids.

Immunization with influenza A virus hemagglutinin and neuraminidase produced in recombinant baculovirus results in a balanced and broadened immune response superior to conventional vaccine.

Influenza A virus hemagglutinin (HA) and neuraminidase (NA) from A/Nanchang/933/95 were expressed by recombinant baculovirus-infected insect cell lines. HA and NA were chromatographically purified then combined in a single vaccine preparation. Immunization of mice with this preparation resulted in high titers of antibodies to both HA and NA equivalent for each antigen to titers in animals immunized with either antigen alone. Anti-NA antibody titers, measured by either enzyme linked immunoabsorbant assay or neuraminidase inhibition test were higher in the combined recombinant vaccine than in conventional monovalent inactivated vaccine. There was no difference in the anti-HA antibody titers between these two vaccine preparations. Homotypic and closely related heterotypic infections were suppressed and greater reduction in viral replication was observed following a distantly related heterotypic infectious challenge than was observed with conventional inactivated vaccine. The combined HA and NA vaccine takes advantage of the equivalent immunogenicity of dissociated HA and NA, to produce a broader and more balanced immune response to both antigens, without the HA-dominant antigenic competition that occurs with natural infection or immunization with conventional vaccine. Additionally, the recombinant baculovirus expression system offers a reliable rapid production system without the use of massive numbers of embryonated chicken eggs. These studies in a mouse model system suggest that production of a combined HA and NA vaccine from recombinant baculovirus offers an improved alternative to conventional inactivated influenza vaccine.

Supplementation of conventional influenza A vaccine with purified viral neuraminidase results in a balanced and broadened immune response.

Influenza virus neuraminidase was chromatographically extracted from A/Johannesburg/33/94 (H3N2) and used to supplement conventional monovalent H3JHN2JH inactivated influenza vaccine. Immunization of mice with this preparation resulted in high titers of antibodies to both hemagglutinin (HA) and neuraminidase (NA) equivalent for each antigen to titers in animals immunized with either antigen alone. Homotypic infection was suppressed and greater reduction in viral replication was observed following heterotypic infectious challenge than was observed following the non-supplemented vaccine. There was no evidence of suppression of the immune response to the HA despite the presence of high amounts of NA in the vaccine. Supplementation of conventional inactivated influenza vaccine with NA takes advantage of the equivalent immunogenicity of dissociated HA and NA, to produce a more balanced immune response to both surface antigens, without the antigenic competition tht occurs after immunization with conventional vaccine or infection. These studies in a mouse model system suggest that supplementation of current inactivated influenza vaccines offers the prospect of improved immunization of humans against influenza.

Immunization with dissociated neuraminidase, matrix, and nucleoproteins from influenza A virus eliminates cognate help and antigenic competition.

When hemagglutinin (HA) and neuraminidase (NA) are presented together on an intact influenza virus particle, the antigens are competitive, with HA dominant over NA in T- and B-cell priming. Immunization with mixtures of purified HA and NA eliminates antigenic competition between HA and NA, as well as between N1-N2 NA mixtures. Evidence that vaccine preparations contain influenza virus matrix (M1) and nucleoprotein (NP) prompted the investigation of possible competing effects of these proteins on the anti-HA and anti-NA immune response. However, in BALB/c mice immunized with mixtures of purified NA, M1, and NP no antigenic competition was demonstrated in either the primary or the secondary response. When mice were immunized with intact virus or by infection, a lesser antibody response to M1 and NP was observed. Furthermore, as measured by mean pulmonary virus titers after infection, no additional protective effects were conferred on mice immunized with M1 and NP either alone or in conjunction with other antigens. These studies of influenza virus antigen mixtures have implications for vaccination against influenza and other vaccines consisting of combinations of antigens.

Neuraminidase-specific antibody responses to inactivated influenza virus vaccine in young and elderly adults.

Little information is available on the potential role of antibody to influenza virus neuraminidase (NA) in vaccine-induced immunity. In the present study, serologic responses to the N1Texas/91 and N2Beijing/92 NA components of trivalent inactivated influenza virus vaccine were measured by NA inhibition (NI) and enzyme-linked immunosorbent assay (ELISA), and the results for adults aged 18 to 45 (young) or > or = 65 (elderly) years were compared. The two age groups had comparable rates (32 to 50%) of NI response. In contrast, ELISA immunoglobulin G (IgG) antibody responses to N1 and N2 NAs occurred in 70 to 71 and 67 to 83%, respectively, of young subjects but in only 3 to 18 and 18 to 35%, respectively, of elderly subjects. prevaccination mean ELISA IgG and IgA NA antibody titers were generally lower for the young adults than they were for the elderly, whereas the corresponding NI titers were comparable. In young adults, plaque size-reducing NA antibody increases were positively associated with ELISA but not with NI antibody increases. There were no apparent age-related differences in the immunoglobulin isotype distribution of the anti-NA response, with IgG being the dominant class and IgG1 the dominant subclass of serum antibody. Anti-hemagglutinin antibody responses to H1Texas/91 and H3Beijing/92 were greater in magnitude and frequency than the corresponding NA-specific responses to N1Texas/91 and N2Beijing/92 when measured by hemagglutination inhibition and NI, respectively, but not when measured by ELISA. The discordance between NI and ELISA for measurement of NA-specific vaccine responses may reflect the relative insensitivity of NI in discriminating differences when initial antibody titers are low.

Purified influenza A virus N2 neuraminidase vaccine is immunogenic and non-toxic in humans.

The immunogenicity and toxicity of a purified influenza virus (N2) neuraminidase vaccine (NAV) were investigated in 88 human subjects aged 18-40, and compared to response to a conventional trivalent influenza vaccine, Fluogen (Parke-Davis). NAV doses ranged from 2.6 to 69.9 micrograms and were given intramuscularly. Serologic neuraminidase-inhibiting (NI) and neuraminidase-specific ELISA responses in this N2-primed population were roughly proportional to the dose administered. Maximal response was seen in 14-21 days and NI antibody titers persisted unabated for the 6-month post-vaccination follow-up period. All doses were well tolerated with respect to local and systemic reactions. NI tests performed with the putative (1975) priming N2 antigen demonstrated anamnestic response but did not reveal responses not already shown with the homologous (1992) antigen. Response to this purified, non-adjuvanted preparation encourages continuing investigation of the induction of infection-permissive immunity with influenza virus neuraminidase.

Immunogenicity of influenza A virus N2 neuraminidase produced in insect larvae by baculovirus recombinants.

Influenza A virus neuraminidase (NA) from A/Udorn/72 (H3N2) was expressed by recombinant baculovirus-infected insects. The recombinant NA was enzymatically active. Enzyme activity was neutralized by polyclonal antisera raised against virion-extracted NA. NA produced in whole insects by a baculovirus expression system is antigenically indistinguishable from virion NA by polyclonal antisera in functional assays (NI) and in ELISA, and is highly immunogenic without adjuvant. It is equivalent in immunogenicity to NA purified from influenza virus. Our results indicate that baculovirus-produced NA could be used as a source for large quantities of purified N2-NA for vaccine use.

Immunization with purified N1 and N2 influenza virus neuraminidases demonstrates cross-reactivity without antigenic competition.

Based on the absence of serologic cross-reactivity, the neuraminidases (NAs) of influenza A viruses are divided into antigenically discrete subtypes, analogous to the hemagglutinin (HA) major antigens with which they share the virion surface. An innovative approach to influenza vaccination takes advantage of the infection-permissive nature of immunization with NA as the minor surface antigen. However, evidence that HA dominates immune response when HA and NA are presented together in the intact virion prompted investigation of possible competing effects during immunization of NA subtype mixtures ultimately required for human vaccination. Immunization of BALB/c mice with purified N1- and N2-subtype NAs demonstrated no antigenic competition in primary or secondary response. However, when homotypic or heterotypic infection followed immunization, cross-reactive antibodies between N1 and N2 were found and "reverse antigen competition" occurred with initial NA priming suppressing response to HA following infection with virus containing homologous NA. These studies of antigen mixtures have implications for the use of combined and chimeric vaccines for diseases other than influenza.

Dissociation of influenza virus hemagglutinin and neuraminidase eliminates their intravirionic antigenic competition.

When presented together on the intact influenza virus particle, the external hemagglutinin (HA) and neuraminidase (NA) antigens are competitive, with HA dominant over NA in both T- and B-cell priming (B. E. Johansson, T. M. Moran, and E. D. Kilbourne, Proc. Natl. Acad. Sci. USA 84:6869-6873, 1987). Dissociation and purification of HA and NA from virus and their injection separately or in combination into BALB/c mice eliminates their antigenic competition as measured by antibody response, confirming that it is their structural association that leads to what we have termed intravirionic antigenic competition. We discuss this phenomenon with respect to previously described intermolecular antigenic competition and with regard to its probable mechanism. Our findings are relevant to contemporary interest in viral vaccine vectors and multicomponent vaccines.

Influenza A virus haemagglutinin polymorphism: pleiotropic antigenic variants of A/Shanghai/11/87 (H3N2) virus selected as high yield reassortants.

Genetic reassortment of the A/Shanghai/11/87 (H3N2) variant of influenza A virus with A/PR8/34 (H1N1) virus [the standard donor of high yield (hy) genes for influenza vaccine viruses] resulted in the isolation of two reassortants with differing H3 haemagglutinin (HA) phenotypes, X-99 and X-99a. The two HA phenotypes were derived from individual subpopulations of the H3N2 wild-type virus during the reassortment event. The HA mutants and their respectively derived reassortants (identical in RNA genotype) differed in antigenicity, replication characteristics, yield in chick embryos and haemagglutinin gene sequence. Despite antigenic differences in reactions to polyclonal rabbit antisera of 60%, both X-99 and X-99a, the hy reassortants, were equally immunogenic and protective in BALB/c mice to challenge by parental wild-type virus. Differences in HA phenotype were related to a Ser to Ile change at amino acid position 186. These findings emphasize the polymorphism of influenza virus strains as well as the need for caution in selection of vaccine strains from among antigenically distinct viral subpopulations.

Infection-permissive immunization with influenza virus neuraminidase prevents weight loss in infected mice.

In studies of infection of young Balb/c mice with a mouse virulent strain of X-31 (H3N2) influenza A virus we have shown a profound virus dose-related effect of infection on body weight. Most of this effect is prevented by prior administration of either inactivated whole virus vaccine, which prevents infection, or purified influenza virus neuraminidase, which is infection-permissive, but reduces pulmonary virus replication by 1.5 to 3 orders of magnitude. These studies support the concept of infection-permissive immunization and suggest that levels of virus replication previously shown to be antigenic can be sustained without significant systemic effects.

Influenza vaccine strain selection: equivalence of two antigenically distinct haemagglutinin variants of 1989 H3N2 influenza A virus in protection of mice.

Precise antigenic analysis with haemagglutinin-inhibition (HI) tests of 1989 H3N2 influenza A viruses with polyclonal ferret, rabbit and mouse antisera has shown, first, significant differences among 1989 wild-type isolates, second, antigenic differences between two high-yield vaccine candidate reassortant viruses, third, significant antigenic differences of one reassortant (X-105) from the wild-type virus (A/Guangdong A/39) from which it was derived, and fourth, dependence of antigenic characterization of viruses upon the host species used in immunization. Nevertheless, the two reassortant viruses (only 43% similar by HI test) were equally protective in preventing homovariant or heterovariant infection in either previously unimmunized or infection-primed mice. These results not only confirm the known antigenic heterogeneity of influenza A viruses, but raise questions about the adequacy of current methods of antigenic characterization of influenza viruses and the basis for decisions on vaccine strain selection.

Comparison of intranasal and aerosol infection of mice in assessment of immunity to influenza virus infection.

A comparison was made of intranasal and aerosol routes of infection with X-31 influenza A virus in Balb/c mice. Mice were first infected with 100 MID50 by either route then challenged 42 days later with the same virus given by the same or alternative route. Three days following each infection, pulmonary virus was measured by inoculation of chick embryos. Mice initially infected under ether anesthesia by intranasal inoculation experienced higher initial mortality but proved most resistant to subsequent challenge by either method. In contrast, mice first infected by aerosol were least resistant to intranasal challenge, as indicated by increased rate of infection and pulmonary virus titers, but, like mice previously infected intranasally, were not reinfected by the aerosol route. Thus, intranasal infection appears to be more effective both in inducing and challenging immunity from infection. These results should be considered in the design of experiments utilizing influenza virus infection of mice as a model system.

Programmed antigenic stimulation: kinetics of the immune response to challenge infections of mice primed with influenza inactivated whole virus or neuraminidase vaccine.

Mice were immunized with either inactivated whole virus influenza A (H3N2) virus (WV) vaccine or with purified N2 neuraminidase (NA) vaccine then challenged with mouse-adapted homologous infective virus at intervals of 1-141 days later in order to ascertain the optimal vaccine-infection interval for induction of resistance to subsequent infection. Measured by serological or infection suppressing response, this interval was 15 days for both vaccines. Maximal reduction in pulmonary virus replication during initial (postvaccination) infection was achieved with WV vaccine, but in second infection by NA vaccine. This study provides further support for the concept of infection-permissive immunization with NA vaccines and suggests the promise of programmed antigenic stimulation by coupling of non-replicating and replicating antigens in the induction of solid immunity.

Comparative long-term effects in a mouse model system of influenza whole virus and purified neuraminidase vaccines followed by sequential infections.

A comparison of inactivated whole influenza virus vaccine and purified influenza neuraminidase (NA) in BALB/c mice repeatedly challenged by homologous or heterologous H3N2 variant infections demonstrated an initial superiority of whole virus vaccine but the ultimate superiority of NA vaccine in immunization after one or two boosting infections. Parenteral administration of either vaccine followed by infection was much more effective than infection alone in the induction of either homologous or heterologous immunity. On the basis of this model simulation of human experience and other earlier studies, it seems that purified NA vaccine may offer an important new strategy as an initial step in immunization against influenza in humans.

Independent and disparate evolution in nature of influenza A virus hemagglutinin and neuraminidase glycoproteins.

The hemagglutinin (HA) and neuraminidase (NA) external glycoprotein antigens of H1N1 and H3N2 subtypes of epidemiologically important influenza A viruses prevalent during recent decades were subjected to intensive antigenic analysis by four different methods. Prior to serological analysis with polyclonal rabbit antisera, HA and NA antigens of four viruses of each subtype were segregated by genetic reassortment to forestall nonspecific steric hindrance during antigen-antibody combination. This analysis has demonstrated that with respect to antigenic phenotype, HA and NA proteins have evolved at different rates. With H1N1 viruses, an arrest of significant evolution of the NA discordant with the continuing antigenic drift of HA was found in the 1980-1983 period. It is probable that the different and independent rates of evolution of HA and NA reflect the greater selective pressure of HA antibodies, which forces the more rapid emergence of HA escape mutants. The slower antigenic change found for NA further supports the potential for NA-specific infection-permissive immunization as a useful stratagem against influenza.