First application of PCR-Luminex system for molecular diagnosis of fungicide resistance and species identification of fungal pathogens

Fungicide resistance in plant pathogens is often caused by a single point mutation in a gene encoding fungicide target proteins. Such is the case for resistance to MBI-D (inhibitors of scytalone dehydratase in melanin biosynthesis) fungicides in rice blast fungus (Magnaporthe oryzae), which is caused by a mutation in the scytalone dehydratase gene that results in a replacement of valine with methionine at codon 75 of the fungicide target protein. PCR-Luminex, a novel system developed for high-throughput analysis of single nucleotide polymorphisms (SNPs) was successfully introduced to diagnose MBI-D resistance using specific oligonucleotide probes coupled with fluorescent beads. The PCR-Luminex system was further tested for its potential in identifying species causing Fusarium head blight on wheat. Four major pathogens, Fusarium graminearum (=F. asiaticum), F. culmorum, F. avenaceum, and Microdochium nivale, known to cause the disease, were tested, and the species were identified using the PCR-Luminex method. So far, this report is the first on the application of the DNA-based PCR-Luminex system in the area of crop protection and/or agricultural sciences.     

Protective effect of anthocyanins from black soybean seed coats on UVB-induced apoptotic cell death in vitro and in vivo

UVB radiation proves to be one of the most relevant environmental risks because of its hazardous effects, such as premature skin aging and especially skin photocarcinogenesis. Anthocyanins, water-soluble pigments present in plants, are known to be powerful antioxidants that help protect plants from UV damage. In this study, we aimed at investigating the protective effect of anthocyanins from black soybean [Glycine max (L.) Merr] seed coats on UVB-induced apoptosis, and furthermore, we investigated the molecular mechanism responsible for regulation of apoptosis in vitro and in vivo. Pretreatment with anthocyanins reduced UVB-induced reactive oxygen species levels and inhibited UVB-induced apoptotic cell death through the prevention of caspase-3 pathway activation and reduction of proapoptotic Bax protein levels. UVB irradiation induced apoptotic cell death, which was inhibited by topical application of anthocyanins in hairless mice. It is concluded that anthocyanins from the seed coat of black soybeans may be useful compounds to modulate UVB-induced photoaging.     

Effect of Steeping Conditions on Wet-Milling Characteristics of Hard Red Winter Wheat

A batch-wise small-scale wet-processing laboratory for whole wheat kernel has been designed and constructed to produce wheat starch and gluten from wheat grains. Hard red winter wheat kernels were steeped in three steeping media: SO₂ solution, lactic acid, and hydrochloric acid. Acid concentrations of 0.1, 0.3, and 0.5%, were used for SO₂ solutions and hydrochloric acid, and 0.1, 0.6, and 3.0% for lactic acid. After 16, 20, and 24 hr of steeping, the wheat was wet-milled. Yields and protein contents of wet-milling fractions were compared. Both high concentration of steeping media and long steeping time increased the starch yield and decreased the protein contents of the starch. However, the steeping time and acid concentration could be reduced from 24 to 20 hr and from 0.5 to 0.3%, respectively, without any statistically significant difference in starch yields or protein contents of the starch. Consistency and color of the starch were affected by both steeping time and acid concentrations of steeping media.     

Experimental model of swine pneumonic pasteurellosis using crude Actinobacillus pleuropneumoniae cytotoxin and Pasteurella multocida given endobronchially.

This study was designed to develop and characterize a swine pneumonic pasteurellosis model by concurrent introduction of Pasteurella multocida type A and Actinobacillus pleuropneumoniae crude cytotoxin. After a series of preliminary experiments, a combination of 4 x 10(9) P. multocida and 4,000 toxic units of A. pleuropneumoniae crude cytotoxin was determined to produce optimal results. A total of 48 pigs were divided into four groups of 12 pigs each. The control group received buffered saline only. Four pigs from each group were randomly selected for necropsy 3, 7 and 14 days postinoculation (PI). Inoculation of pigs with P. multocida and A. pleuropneumoniae cytotoxin (group 1) resulted in moderate to severe pneumonia. Pasteurella multocida was isolated from pneumonic lesions, grossly normal lung, and bronchial lymph nodes of all group 1 pigs throughout the 14 day experimental period. Pathological changes typical of field cases of swine pneumonic pasteurellosis were produced. Pigs inoculated with P. multocida alone (group 2) had pneumonic lesions and P. multocida was reisolated from lungs at three days PI. Pasteurella multocida was not isolated from these pigs at 7 and 14 days PI, except for one pig in which an abscess developed in the thorax. Pulmonary lesions induced by A. pleuropneumoniae crude cytotoxin alone (group 3) were transient and resolved by seven days PI. Group 1 pigs had significantly greater lung lesion volumes than group 2 and 3 pigs at 3, 7 and 14 days PI. Statistical analysis indicated a significant interactive effect of P. multocida and A. pleuropneumoniae cytotoxin on the development of lung lesion volumes at 7 and 14 days PI (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of dietary energy source on in vitro substrate utilization and insulin sensitivity of muscle and adipose tissues of Angus and Wagyu steers

Angus (n = 8; 210 kg of BW) and 7/8 Wagyu (n = 8; 174 kg of BW) steers were used to evaluate the effects of dietary energy source on muscle and adipose tissue metabolism and insulin sensitivity. Steers were assigned to either a grain-based (corn) or hay-based (hay) diet and fed to similar final BW. At slaughter, LM and s.c. and i.m. adipose tissue samples were collected. Portions of the LM and adipose tissues were placed immediately in liquid N for later measurement of glycolytic intermediates. Fresh LM and s.c. and i.m. adipose tissues were incubated with [U-(14)C]glucose to assess glucose metabolism in vitro. All in vitro measures were in the presence of 0 or 500 ng/mL of insulin. Also, s.c. and i.m. adipose tissues were incubated with [1-(14)C]acetate to quantify lipid synthesis in vitro. Glucose-6-phosphate and fructose-6-phosphate concentrations were 12.6- and 2.4-fold greater in muscle than in s.c. and i.m. adipose tissues, respectively. Diet did not affect acetate incorporation into fatty acids (P = 0.86). Insulin did not increase conversion of glucose to CO(2), lactate, or total lipid in steers fed hay but caused an increase (per cell) of 97 to 110% in glucose conversion to CO(2), 46 to 54% in glucose conversion to lactate, and 65 to 160% in glucose conversion to total lipid content in adipose tissue from steers fed corn. On a per-cell basis, s.c. adipose tissue had 37% greater glucose oxidation than i.m. adipose (P = 0.04) and 290% greater acetate incorporation into fatty acids than i.m. adipose (P = 0.04). Insulin addition to s.c. adipose tissue from corn-fed steers failed to stimulate glucose incorporation into fatty acids, but exposing i.m. adipose tissue from corn-fed steers to insulin resulted in a 165% increase in glucose incorporation into fatty acids. These results suggest that feeding hay limited both glucose supply and tissue capacity to increase glucose utilization in response to insulin without altering acetate conversion to fatty acids. Because s.c. adipose tissue consistently utilized more acetate and oxidized more glucose than did i.m. adipose, these results suggest that hay-based diets may alter i.m. adipose tissue metabolism with less effect on s.c. adipose tissue.     

Exendin-4 improves resistance to Listeria monocytogenes infection in diabetic db/db mice

The incidence of diabetes mellitus is increasing among companion animals. This disease has similar characteristics in both humans and animals. Diabetes is frequently identified as an independent risk factor for infections associated with increased mortality. In the present study, homozygous diabetic (db/db) mice were infected with Listeria (L.) monocytogenes and then treated with the anti-diabetic drug exendin-4, a glucagon-like peptide 1 analogue. In aged db/db mice, decreased CD11b⁺ macrophage populations with higher lipid content and lower phagocytic activity were observed. Exendin-4 lowered high lipid levels and enhanced phagocytosis in macrophages from db/db mice infected with L. monocytogenes. Exendin-4 also ameliorated obesity and hyperglycemia, and improved ex vivo bacteria clearance by macrophages in the animals. Liver histology examined during L. monocytogenes infection indicated that abscess formation was much milder in exendin-4-treated db/db mice than in the control animals. Moreover, mechanistic studies demonstrated that expression of ATP binding cassette transporter 1, a sterol transporter, was higher in macrophages isolated from the exendin-4-treated db/db mice. Overall, our results suggest that exendin-4 decreases the risk of infection in diabetic animals by modifying the interaction between intracellular lipids and phagocytic macrophages.     

Dairy-cattle health in Gyeongnam, Korea.

An animal-health monitoring system in the Gyeongnam area was started in 1997 to develop statistically valid data for use in estimating disease frequencies in dairy cattle, and the associated costs. The objectives of this study were to: (1) describe what was done to implement and maintain the system in Gyeongnam; (2) present selected disease frequencies; (3) discuss the epidemiological consideration of what was done and implications for results obtained.Veterinary medical officers (VMOs), comprising professors and graduate students from Gyeongsang National University, faculty of Gyeongnam Livestock Promotion Institute and clinic veterinarians, served as data collectors. After training on current disease and management problems of dairy cattle, interview techniques, sampling methods and data-collection instruments, the VMOs participated in selection of the sample herds and data gathering. Forty (n=40) of 167 dairy herds were selected randomly using a computer-generated list of random numbers and the VMOs visited farms once in a month for 12 months to collect data about management, disease, inventory, production, preventive treatment, financial and other relevant data. Strict data-quality control devices were used. Specific feed-back was developed for the producers and data collectors.The six disorders found most frequently in cows (from the highest to the lowest) were breeding problems, clinical mastitis, birth problems, gastrointestinal problems, metabolic problems and lameness. In young stock, respiratory, multiple system, breeding and gastrointestinal problems were predominant, whereas in calves, gastrointestinal, respiratory and integumental problems predominated.     

Immunoproteomic analyses of outer membrane antigens of Actinobacillus pleuropneumoniae grown under iron-restricted conditions

Actinobacillus pleuropneumoniae, a bacterial pathogen of swine and agent of porcine pneumonia, causes a highly infectious disease of economic importance in the pig industry. Commercial vaccines for A. pleuropneumoniae include whole-cell bacterins and second generation subunit vaccines but they only confer partial protective immunity. Our search for new vaccine candidates identified antigens that are expressed during conditions that mimic infection; the outer membrane (OM) proteome of A. pleuropneumoniae serotype 5b was examined under iron restriction. Quantitative profiling by 2D-DiGE technology revealed that iron restriction induced expression of previously described transferrin binding proteins (TbpA, TbpB) plus four lipoproteins including spermidine/putrescine binding periplasmic protein 1 precursor (PotD2). Immunoproteomic analyses with antisera from na?ve animals and from infected pigs were able to differentiate antigens within the OM proteome that were specifically recognized during A. pleuropneumoniae infection. Immunoblots of iron-restricted profiles detected PotD2, heme-binding protein A (HbpA), and capsule polysaccharide export protein (CpxD) as well as surface antigens TbpA, TbpB, and OmlA. These data identify OM proteins that demonstrate immunogenicity and upregulation under conditions mimicking infection, providing emphasis on lipoproteins as an important class of antigens to exploit for vaccine development for A. pleuropneumoniae.     

Receptor-dependent and independent degradation of insulin by isolated swine adipocytes at 37 C

Insulin binding and degradation were measured at 37 C in isolated swine adipocytes. In preliminary experiments, binding decreased rapidly with increasing incubation time. This was associated with a marked increase in insulin degradation. Insulin binding was suppressed by some lots of bovine serum albumin (BSA), which suggests that some commercial preparations of BSA are contaminated with insulin-like molecules. In adipocyte suspensions, greater than 90% of the insulin degraded was due to a nonreceptor mediated process (i.e., insulin degrading activity present in the media). Despite the presence of insulin degrading activity in the media the cells were metabolically (as judged by lipogenic capacity and lactic dehydrogenase activity) and morphologically (greater than 98% excluded trypan blue) intact indicating that the cells were not leaking during the incubation. In subsequent experiments it was found that the specific step associated with transfer of cells during adipocyte isolation resulted in the release of insulin degrading activity. Implementation of a 30-min preincubation and washing sequence after adipocyte isolation removed the media insulin degrading activity, resulting in a marked reduction (approximately 70%) of insulin degradation by adipocyte suspensions. As a result of this modification, binding of tracer quantities of insulin attained steady-state binding conditions and maintained this for 2 h. These results demonstrate that techniques can be used to minimize nonreceptor mediated insulin degradation in adipocyte suspensions. As a result in vitro studies can be conducted that measure insulin binding and biological action in swine adipocytes at physiological temperatures.