Sources of error with in vitro digestibility assay of pasture feeds

A series of experiments was undertaken to determine population statistics for in vitro organic matter digestibility ( in vitro OMD) data and to examine the effects of basal diet, donor animal and precollection fasting interval on the activity and specificity of rumen fluid inoculum. The experiments utilized wether sheep, a diverse set of pasture grass and legume feeds prominent in the Australian subtropics and the Tilley and Terry in vitro digestibility procedure running under the operating pressure of a practicing feeds evaluation laboratory.
The standard errors of in vitro OMD estimates for within and between batch runs were ±0·88 × 10⁻² and 0·62 × 10⁻², respectively. These error terms were used to develop protocols to accept, reject or scale raw in vitro OMD data. Differences between donor animals in the activity of rumen fluid were highly significant. Extending the precollection fasting interval beyond 16 h was associated with a substantial decline in inoculum activity.
An in vitro-in vivo calibration relationship based on fifteen test feeds and using lucerne ( Medicago sativa ) as basal diet was described by the linear model y = 1·3 x-0·195±4·9 × 10⁻² r = 0·79 (y = in vivo OMD, x = in vitro OMD). Despite large effects of basal diet on both the absolute values and relative ranking of test feeds, neither the RSD nor r values were improved using alternative diets to Lucerne chaff.
The results highlight the need to formally standardize the analytical and biological components of the in vitro digestibility procedure to safeguard the integrity of data.     

Free-running rhythms of adrenocorticotropic hormone (ACTH), cortisol and melatonin in pigs.

In the domestic pig, a circadian rhythm of plasma cortisol occurs, with greatest concentrations in the morning and lowest concentrations in the afternoon. However, photic entrainment of the rhythms of ACTH and melatonin in pigs have not been defined clearly. This experiment was designed to evaluate free-running rhythms of ACTH, cortisol and melatonin in pigs housed in constant light (LL) and constant darkness (DD). Twelve crossbred barrows, maintained under ambient photoperiod, were catheterized and tethered individually in two environmentally controlled rooms, one with LL and the other with DD. For animals in LL, fluorescent lights provided 202 +/- 15 (mean +/- standard deviation) lux of light at 65 cm above the floors. Incandescent nightlights equipped with 7 watt red bulbs provided 7 +/- 2 lux and were illuminated continuously in both rooms. Pigs were given at least 14 d exposure to LL and DD, then samples of plasma and serum were obtained at hourly intervals for 48 hr. Plasma was assayed for ACTH, and serum for cortisol and melatonin. Periodograms were constructed to analyze the data. For this type of analysis, a statistic, Qp, is calculated, and circadian periodicity is suggested if maximum Qp (Qp max) occurs at or near 24 hr. The period of the free-running rhythms (tau) at Qp max for ACTH, cortisol and melatonin for pigs in LL (23.80 +/- .01, 23.78 +/- .01, and 23.21 +/- .02 hr, respectively) did not differ significantly from those for pigs in DD (23.39 +/- .01, 23.20 +/- .01, and 22.55 +/- .02 hr, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection and characterization of leptospiral antigens using a biotin/avidin double-antibody sandwich enzyme-linked immunosorbent assay and immunoblot.

A biotin/avidin double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of antigens of Leptospira interrogans serovars in experimentally inoculated bovine urine samples was evaluated. Immunoglobulin G (IgG) from rabbits immunized with L. interrogans serovar hardjo type hardjobovis sonicated, whole cell, and formalinized-heated antigen preparations were purified by a protein A-superose column coupled to fast protein liquid chromatography, and evaluated for species specificity in the ELISA. The ELISA using each specific IgG detected as few as 10(4) leptospires of the homologous serovar hardjo diluted in phosphate-buffered saline solution with Tween 20 (PBSS-Tween 20). On immunoblot analysis of proteinase-K-digested whole cell leptospiral preparations, each IgG revealed the presence of bands specific to serovar hardjo, suggesting the presence of serovar-specific epitopes on the lipopolysaccharide molecules. The minimum number of cells of heterologous serovars pomona, grippotyphosa, bratislava, icterohaemorrhagiae and copenhageni detected by each ELISA was greater, ranging from 10(6) to 10(7). The common antigenic determinants observed on immunoblot analysis were different for each specific IgG, except for a major cross-reacting, possibly flagellar, protein doublet at approximately 36-36.5 kDa. Leptospires were equally well detected by the ELISA in both bovine urine and PBSS-Tween 20.     

Mystery swine disease in The Netherlands: the isolation of Lelystad virus.

In early 1991, the Dutch pig-industry was struck by the so-called mystery swine disease. Large-scale laboratory investigations were undertaken to search for the etiological agent. We focused on isolating viruses and mycoplasmas, and we tested paired sera of affected sows for antibodies against ten known pig viruses. The mycoplasmas M. hyosynoviae, M. hyopneumoniae, and Acholeplasma laidlawii, and the viruses encephalomyocarditis virus and porcine enterovirus types 2 and 7 were isolated from individual pigs. An unknown agent, however, was isolated from 16 of 20 piglets and from 41 of 63 sows. This agent was characterised as a virus and designated Lelystad virus. No relationship between this virus and other viruses has yet been established. Of 165 sows reportedly afflicted by the disease, 123 (75 per cent) seroconverted to Lelystad virus, whereas less than 10 per cent seroconverted to any of the other virus isolates or to the known viral pathogens. Antibodies directed against Lelystad virus were also found in pigs with mystery swine disease in England, Germany, and in the United States. We conclude that infection with Lelystad virus is the likely cause of mystery swine disease.     

Identification by isoelectric focusing of creatine kinase-MM isoforms in the plasma and striated muscle of pigs

Total creatine kinase (CK) and CK-MM isoforms were determined in plasma and longissimus dorsi muscle extracts from normal pigs. Based on their total CK activity, the pigs were divided into two groups. Pigs of group 1 (n=16) had a mean plasma total CK of 298±16 U/L and the distribution of the CK-MM isoforms was 65.7±2.5% CK-MM₃, 18.9±1.6% CK-MM₂ and 15.3±1.5% CK-MM. In group 2 (n=18; 826±75 U/L total CK) four isoforms were observed: 3.1±0.9% CK-MM, 67.9±3.0% CK-MM₃, 21.5±2.3% CK-MM₂ and 7.5±1.3% CK-MM₁. The differences between the two groups of pigs were significant (p<0.001) for CK-MM₁ and the presence of CK-MM. Four CK-MM isoforms were also detected in longissimus dorsi muscle homogenates: 45.6±8.1% CK-MM, 32.6±11.7% CK-MM₃, 16.6±2.3% CK-MM₂ and 5.1±2.8% CK-MM₁. The release of CK-MM isoforms from muscle into plasma seems to be unrelated to the concentration of these isoforms in striated muscle.     

Immunohistochemical detection of scrapie prion proteins in clinically normal sheep in Pennsylvania.

Following diagnosis of scrapie in a clinically suspect Suffolk sheep, 7 clinically normal flockmates were purchased by the Pennsylvania Department of Agriculture to determine their scrapie status using an immunohistochemical procedure. Two of the 7 euthanized healthy sheep had positive immunohistochemical staining of the prion protein of scrapie (PrP-Sc) in their brains, nictitating membranes, and tonsils. The PrP-Sc was localized in the areas of the brain where, histopathologically, there was neurodegeneration and astrocytosis. The PrP-Sc occurred within germinal centers of the affected nictitating membranes and tonsils and was located in the cytoplasm of the dendrite-like cells, lymphoid cells, and macrophages. These results confirm that immunohistochemical examination of the nictitating membrane can be used as a screen for the presence of scrapie infection in clinically normal sheep at a capable veterinary diagnostic laboratory. In sheep with a PrP-Sc-positive nictitating membrane, the diagnosis of scrapie should be confirmed by histopathology and immunohistochemical examination of the brain following necropsy. Following full validation, immunohistochemistry assays for detection of PrP-Sc in nictitating membrane lymphoid tissues can improve the effectiveness of the scrapie control and eradication program by allowing diagnosis of the disease in sheep before the appearance of clinical signs.     

Humoral immune responses of black-footed penguins (Spheniscus demersus) after DNA-mediated immunization with a beta-galactosidase reporter gene.

Humoral immune responses of black-footed penguins (Spheniscus demersus) to DNA-mediated immunization with a beta-galactosidase reporter gene expression plasmid were evaluated. Six male and 6 female adult penguins received either test plasmid, pCMV-beta, containing the beta-galactosidase gene or control plasmid, pCI, lacking a gene for expression. Three birds from each group were used previously in a diluent control group and given one injection of sterile saline. All samples were screened for anti-beta-galactosidase antibodies by indirect enzyme-linked immunosorbent assay with anti-chicken immunoglobulin G as secondary antibody. Antibodies to beta-galactosidase were detected in the sera of pCMV-beta-inoculated penguins, with a peak response on day 21. Antibody titers of the test plasmid group versus both control groups on days 21, 28, and 42 differed significantly. These results demonstrate that black-footed penguins can be safely transfected with the gene encoding beta-galactosidase and will mount a humoral response against the in vivo-expressed protein. Knowledge from this initial study can be applied to the development of DNA-mediated vaccines against specific infectious diseases of penguins.