Partial isolation and characterization of a cysteine proteinase inhibitor from Lima bean (Phaseolus lunatus)

Lima beans (Phaseolus lunatus) have been shown to contain cysteine proteinase inhibitor (CPI) activity, but the CPI has not been isolated or characterized. Accordingly, our objective was to isolate and partially characterize a CPI from lima bean. The isolation scheme included water extraction of lima bean flour followed by a chromatography series using DEAE Sepharose, Phenyl Sepharose, hydroxyapatite, and reversed-phase high performance liquid chromatography. This scheme resulted in the partial purification of a approximately 20 000-dalton protein with high inhibitory activity against papain. This isolated lima bean CPI had an N-terminal sequence homologous with other members of the cystatin class of CPIs. The protein was relatively heat labile; suggesting it could be inactivated with normal cooking, which is favorable for its use in transforming plants to create insect resistance.     

The bovine immune response to Brucella abortus. III. Preparation of antisera against a Brucella component precipitated by sera of some infected cattle.

Two methods are described for the partial purification of a high molecular weight, heat-resistant component (CO1) of sonicates of smooth and rough Brucella abortus which is precipitated by sera of some infected cattle. Method 1, a combination of gel filtration chromatography and polyacrylamide gel electrophoresis, was used to prepare CO1 from sonicates of a smooth field strain of B. abortus. Method 2, a combination of gel filtration chromatography and heat treatment, was used to obtain CO1, from sonicates of rough B. abortus strain 45/20. Rabbit antisera produced against CO1 prepared by either method contained only CO1 precipitins but were negative in standard agglutination and complement fixation tests conducted with whole cell antigens. Evidence is presented that CO1 is identical to Brucella antigen A2, and it is proposed that in future the designation A2 be employed.     

A prediction equation for enteric methane emission from dairy cows for use in NorFor

Abstract

A data-set with 47 treatment means (N = 211) was compiled from research institutions in Denmark, Norway, and Sweden in order to develop a prediction equation for enteric methane (CH₄) emissions from dairy cows. The aim was to implement the equation in the Nordic feed evaluation system NorFor. The equation should therefore be based on input variables available in NorFor. The best equation to predict CH₄ (MJ/d) was based on dry matter intake (DMI, kg/d), and content of (g/kg DM) fatty acids (FA), crude protein (CP), and neutral detergent fiber (NDF). The equation was CH₄ = 1.36 (±0.10) × DMI – 0.125 (±0.039) × FA – 0.02 (±0.012) × CP + 0.017 (±0.005) × NDF (RMSE = 3.00 MJ CH₄/d; CV = 13.8%; R² = 0.77), where RMSE is the root mean square error and CV is the coefficient of variation. However, CP was on the borderline of being significant and did not quantitatively explain much variation in CH₄ emission. Based on the present research, we concluded, therefore, that the equation CH₄ = 1.23 (±0.08) × DMI – 0.145 (±0.039) × FA + 0.012 (±0.005) × NDF (RMSE = 3.10 MJ CH₄/d; CV = 14.3%; R² = 0.75) is most suited for being implemented in NorFor. However, the ability of the proposed equation to predict enteric methane emissions is uncertain until evaluated on an independent data-set.     

Designing and evaluating risk-based surveillance systems: potential unwarranted effects of applying adjusted risk estimates

Risk-based surveillance systems reveal occurrence of disease or infection in a sample of population units, which are selected on the basis of risk factors for the condition under study. The purpose of such systems for supporting practical animal disease policy formulations and management decisions are: A: to detect an emerging disease or infection, if it becomes introduced into a population; or B: to substantiate freedom from a condition in a population; or C: to detect cases and estimate the prevalence of an endemic condition in a population. In risk-based surveillance these aims should be met with prudent use of resources while maintaining acceptable system performance. High-risk category units are selected for testing by identification of the presence of specific high-risk factor(s), while disregarding other factors that might also influence the risk. On this basis we argue that the most applicable risk estimate for use in designing and evaluating a risk-based surveillance system would be a crude (unadjusted) relative risk, odds ratio or apparent prevalence. Risk estimates found in the published literature, however, are often the results of multivariable analyses implicitly adjusting the estimates for confounding from other risk factors. We describe some potential unintentional effects when using adjusted risk estimates in evaluating the efficacy and sensitivity of risk-based surveillance systems (SSe). In two examples, we quantify and compare the efficacy and SSe using adjusted and crude risk estimates. The examples use Danish surveillance data from previously published studies to evaluate systems aimed at risk-based detection of new cases of an endemic infection, i.e. Salmonella in dairy cattle herds (Example 1), and for substantiating the absence of a specific infection, i.e. Trichinella in the national slaughter pig population (Example 2), respectively.     

Experimental reproduction of postweaning multisystemic wasting syndrome (PMWS) in pigs in Sweden and Denmark with a Swedish isolate of porcine circovirus type 2

An experimental model using 3-day-old snatch-farrowed colostrum-deprived piglets co-infected with porcine circovirus type 2 (PCV2) and porcine parvovirus (PPV) is at present one of the best methods to study factors affecting development of postweaning multisystemic wasting syndrome (PMWS). A Swedish isolate of PCV2 (S-PCV2) retrieved in 1993 from a healthy pig has been used in this model to reproduce PMWS in pigs from Northern Ireland. This virus has been present in the Swedish pig population for at least a decade without causing any known PMWS disease problems, despite its potential pathogenicity. The reasons for this are unknown, but could be related to genetics, absence of triggers for PCV2 upregulation (infectious agent and/or management forms) within Swedish pig husbandry. In order to confirm the pathogenicity of S-PCV2, Swedish and Danish pigs were experimentally infected with this isolate according to the established model. Swedish pigs were also infected with a reference isolate of PCV2 (PCV2-1010) to compare the severity of disease caused by the two isolates in Swedish pigs. Both Danish and Swedish pigs developed PMWS after the experimental infection with S-PCV2. Antibodies to PCV2 developed later and reached lower levels in serum from pigs infected with S-PCV2 than in pigs inoculated with PCV2-1010. In general, pigs infected with S-PCV2 showed more severe clinical signs of disease than pigs infected with PCV2-1010, but pigs from all PCV2-inoculated groups displayed gross and histological lesions consistent with PMWS. All pigs inoculated with PPV, alone or in combination with PCV2, displayed interleukin-10 responses in serum while only pigs infected with PPV in combination with PCV2 showed interferon-alpha in serum on repeated occasions. Thus, the pathogenicity of S-PCV2 was confirmed and a role for cytokines in the etiology of PMWS was indicated.     

Exercising stall-housed gestating gilts: effects on lameness, the musculo-skeletal system, production, and behavior

Lameness in breeding-age gilts and sows is a major cause of culling, resulting in increased economic losses and welfare concerns. This study determined if exercise during gestation would affect the musculo-skeletal system, production variables, and behavior. Gilts were blocked by BW and assigned to 1 of 3 treatment groups: control (n = 10; no exercise), low exercise (n = 14; 122 m/d for 5 d/wk), and high exercise (n = 14; 122 m/d for 2 d/wk and 427 m/d for 3 d/wk). All gilts were stall-housed during gestation, and gilts were exercised between d 35 and 110 of gestation. Lameness score, BCS, BW, and blood were taken at multiple points before gestation, and during gestation and lactation. Blood serum was analyzed for carboxy-terminal telopeptide of type I collagen. Sow lying behavior was recorded for 3 d after farrowing. Farrowing data included litter weight and size at birth and weaning, and preweaning mortality. After weaning, 38 sows were slaughtered and muscles and the bones of the left fore- and hind-limbs were harvested. Bone density and quality were determined by computed tomography (CT) scans, dual energy x-ray scans, and bone-breaking force tests. The control group took longer to lie down than both exercise groups, and the low exercise group took longer to lie down than the high exercise group (P < 0.05). The number of pigs weaned was greater in the high exercise group than the control group (P < 0.05). Piglet preweaning mortality was greatest in the control group compared with both exercise groups (P < 0.05). The low exercise treatments exhibited a greater bone density (CT) in the humerus, radius, and tibia compared with that of the control group (P < 0.05). The bone density (CT) of the humerus in the low exercise group was greater than that of the high exercise group (P = 0.03). Breaking force in the humerus and femur was greater (P < 0.05) in the low exercise group than the control group. Breaking force in the tibia of the high exercise group was greater than the control group (P = 0.01). The tibia of both the low and high exercise groups had a greater breaking force (P < 0.05) than the control group. Although there was no benefit of exercise on lameness, differences in bone density and quality, lying behavior, and piglet survivability may provide useful insight into alternative housing for sows.     

Prion protein in sheep urine.

The misfolded form of cellular prion protein (PrP(C)) is the main component of the infectious agent of transmissible spongiform encephalopathies and the validated biomarker for these diseases. The expression of PrP(C) is highest in the central nervous system and has been found in peripheral tissues. Soluble PrP(C) has been detected in cerebrospinal fluid, urine, serum, milk, and seminal plasma. In this study, attempts were made to characterize prion protein in urine samples from normal and scrapie-infected sheep. Urine samples from scrapie-infected sheep and age-matched healthy sheep were collected and analyzed by Western blot following concentration. A protease K-sensitive protein band with a molecular weight of approximately 27-30 kDa was visualized after immunoblotting with anti-PrP monoclonal antibodies to a C-terminal part of PrP(C), but not after immunoblotting with monoclonal antibodies to an N-terminal epitope of PrP(C) or with secondary antibodies only. The amount of PrP(C) in the urine of 49 animals (control group: n = 16; naturally scrapie-infected group: n = 33) was estimated by comparison with known amounts of ovine recombinant PrP in the immunoblot. Background concentration of PrP(C) in urine was found to be 0-0.16 ng/ml (adjusted to the initial nonconcentrated volume of the urine samples). Seven out of 33 naturally scrapie-infected animals had an elevated level (0.3-4.7 ng/ml) of PrP(C) in urine. The origin of PrP(C) in urine and the reason for the increased level of PrP(C) in scrapie-infected sheep urine has yet to be explored.     

二穗短柄草幼胚再生体系及农杆菌介导转化的初步研究

以二穗短柄草3个品系BDR018,BD21,BD21-3的幼胚为外植体,研究了幼胚大小对胚性愈伤组织诱导和再生的影响。采用携带gus和bar基因双元表达载体(pDM805)的根癌农杆菌菌株AGL1对BDR018品系的幼胚愈伤组织进行了遗传转化。本实验探讨了影响幼胚愈伤组织诱导再生及遗传转化的几个因素。结果发现,幼胚大小介于0.5～1.0mm之间的愈伤组织诱导率最高。随愈伤组织年龄的增加,BDR018,BD21,BD21-3愈伤组织的再生率下降,白化率上升。愈伤组织年龄在5～8周范围内转化效率较高,平均转化效率为38.5%。真空处理5min和0.01%的SilwetL-77处理均可提高转化效率。对转化植株进行GUS基因化学组织检测和PCR鉴定,初步证明外源基因已整合再生植株基因组中。