Immunoperoxidase plaque staining for the detection of pseudorabies virus.

A quantitative indirect immunoperoxidase plaque staining method was developed for the detection of pseudorabies virus infection in a pig kidney cell line (PK-15). The method is rapid and specific and foci of infection, represented by stained plaques, are easily counted by the unaided eye. Possible modification of this technique in a plaque reduction assay for the detection of antipseudorabies virus antibody is also discussed.     

Development and evaluation of an indirect enzyme immunoassay for detection of porcine antibodies to pseudorabies virus.

An indirect enzyme immunoassay is described for detection of porcine serum antibody to pseudorabies virus. The analytical sensitivity of the enzyme immunoassay was found to be approximately 4.5 log 4 X 10 (5120 times) greater than the serum neutralization test, based on parallel end point titrations. The diagnostic sensitivity of the enzyme immunoassay was comparable or superior to that of the serum neutralization, based on the earliest detectable antibody after infection of swine with pseudorabies virus by intranasal or intrauterine routes or by contact with infected pigs. The enzyme immunoassay, at a screening dilution of 1:20, gave 100% agreement with ELISA results provided with a U.S. Department of Agriculture-Animal and Plant Health Inspection Service proficiency panel of 40 sera. One serum having demonstrable antibody by the enzyme immunoassay was seronegative by the serum neutralization test.     

Combining ability and gene action for maturity and agronomic traits in different heterotic groups of maize inbred lines and their diallel crosses

Combining ability information is necessary for selection of suitable advanced lines for hybridization and identification of promising hybrids for development of improved varieties. A number of 14 maize (Zea mays L.) inbred lines and 91 related crosses were evaluated over two years, 2008 and 2009, in a temperate-zone of Iran. The objectives of the study were to identify the best general and specific combiners, heterosis and type of gene actions responsible for agronomic traits. Except for grain yield and growing degree day to milky, significant general (GCA) and specific (SCA) combining ability were observed for all traits. The Baker ratio for plant height (0.15), ear height (0.26), growing degree day to milky stage (0.04), and grain yield (0.002) showed the predominance of non-additive gene effects in the expression of these traits. The heterosis observed for grain yield, grain number, pollination period, ear and plant height was considerably higher than that observed for other traits. The correlations (r) of F₁ means and SCA effects were positive and significantly higher than that of r (F₁, mid-parents) and r (F₁, heterosis) for all the traits except cob percent, growing degree days to silking, and physiological maturity. MO17, K3547/5, and K3615/2 had negative GCA effects for growing degree day to milky stage and maturity. Among hybrids, MO17 × K3653/2, B73 × K3651/2, and K3545/6 × K3493/1 with positive SCAs for pollination period and grain yield had also negative SCA effects for degree day to silking and milky stages. Therefore, the use of these inbred lines and hybrids increases the response to selection for increasing grain yield and early maturity in maize.     

Production of an Antibody Fragment (scFv) Targeting PcrV Protein of Pseudomonas aeruginosa in Fed-Batch Cultivation Mode

Background: Pseudomonas aeruginosa is one of the opportunistic pathogens causing frequent hospital-acquired life-threatening infections in mechanically ventilated patients. The most significant virulence factor of P. aeruginosa is T3SS. PcrV is an important structural protein of the T3SS. Methods:In the current investigation, a recombinant scFv mAb against the PcrV protein was expressed in EnBase® (fed-batch) cultivation mode. The pETiteTM N-His SUMO Kan vector, including anti-PcrV scFv gene, was transformed into Escherichia coli (BL21) cells. The expression and solubility of anti-PcrV scFv protein were investigated at two different temperatures (25 °C and 30 °C) and at different induction times (4, 6, 8, 12, and 24 hours). Results:Increased efficiency was achieved by EnBase® compared to LB broth; owing to the slow release of glucose, the maximum level of solubility and total protein expression was observed in EnBase® cultivation system at 30 °C and 24 h post induction. Furthermore, IC₅₀ for anti-PcrV scFv protein was determined to be approximately 7 μg/mL. Conclusion:Anti-PcrV scFv produced in this study showed promising in vitro results, protecting RBC from lysis by P. aeruginosa (exoU+). Key Words: Fed Batch, recombinant protein, Pseudomonas aeruginosa, scFv     

Development and evaluation of an enzyme-linked immunosorbent assay for detection of bovine antibodies to epizootic hemorrhagic disease of deer viruses.

An indirect enzyme-linked immunosorbent assay (I.ELISA) is described for detection of bovine serum antibody to epizootic hemorrhagic diseases of deer virus (EHDV). Serum samples, at a dilution of 1:200, were incubated with group-specific EHDV antigens, pre-adsorbed to microtiter plates. Bound antibodies were detected by a murine monoclonal antibody to bovine immunoglobulin (Ig)G1 (heavy-chain specific) conjugated with horseradish peroxidase. The performance of the I.ELISA in detecting antibodies to EHDV in sequential serum samples from calves experimentally infected with serotypes 1,2,3 and 4 was evaluated. The I.ELISA detected EHDV antibodies from 14 days postinfection when seroconversion by the standard agar gel immunodiffusion (AGID) test was also evident. The group-specific antibodies to EHDV increased exponentially during the first two to four weeks postinfection and remained relatively stable for about 12 months in some calves. Unlike observations with the AGID test, no reaction was seen in the I.ELISA between blue-tongue virus (BTV) antigen and sera from calves given a single dose of EHDV. The performance of the I.ELISA and AGID were compared using 3,135 AGID negative bovine field sera from herds in Ontario, Alberta and British Columbia and 130 AGID positive samples collected from cattle in 1987 and 1988 during and after outbreaks of EHD in the Okanagan Valley, British Columbia. The specificity and sensitivity of the assay relative to the AGID test were 99.3% and 91.5% respectively, with an overall agreement of 99.0% between the tests.(ABSTRACT TRUNCATED AT 250 WORDS)     

Experimental peste des petits ruminants (goat plague) in goats and sheep.

In order to study the pathomorphology and immunohistochemistry of peste des petits ruminants, four goats and two sheep were inoculated intranasally with the Malig-Yemen strain of peste des petits ruminants virus. The animals developed fever, nasal discharge, oral erosions, cough and diarrhea. One goat and one sheep died and one moribund goat was killed. Three animals survived the infection. At necropsy, erosive stomatitis, pneumonia and gastroenteritis were found. Histopathologically the pneumonocytes and epithelial cells of the ileum had eosinophilic cytoplasmic and nuclear inclusions. By an indirect immunoperoxidase method, the nuclei and cytoplasm of the ileal epithelial cells of one goat contained positively (brown) stained antigen, which corresponded to viral nucleocapsids by electron microscopy. Virus appeared to be released through the microvilli of the epithelial cells. We also confirmed the formation of giant cells due to peste des petits ruminants virus.