Recovery of infectious virus from full-length cowpox virus (CPXV) DNA cloned as a bacterial artificial chromosome (BAC)

ABSTRACT: Transmission from pet rats and cats to humans as well as severe infection in felids and other animal species have recently drawn increasing attention to cowpox virus (CPXV). We report the cloning of the entire genome of cowpox virus strain Brighton Red (BR) as a bacterial artificial chromosome (BAC) in Escherichia coli and the recovery of infectious virus from cloned DNA. Generation of a full-length CPXV DNA clone was achieved by first introducing a mini-F vector, which allows maintenance of large circular DNA in E. coli, into the thymidine kinase locus of CPXV by homologous recombination. Circular replication intermediates were then electroporated into E. coli DH10B cells. Upon successful establishment of the infectious BR clone, we modified the full-length clone such that recombination-mediated excision of bacterial sequences can occur upon transfection in eukaryotic cells. This self-excision of the bacterial replicon is made possible by a sequence duplication within mini-F sequences and allows recovery of recombinant virus progeny without remaining marker or vector sequences. The in vitro growth properties of viruses derived from both BAC clones were determined and found to be virtually indistinguishable from those of parental, wild-type BR. Finally, the complete genomic sequence of the infectious clone was determined and the cloned viral genome was shown to be identical to that of the parental virus. In summary, the generated infectious clone will greatly facilitate studies on individual genes and pathogenesis of CPXV. Moreover, the vector potential of CPXV can now be more systematically explored using this newly generated tool.     

Attempted in vitro maturation and fertilization of postmortem Sumatran rhinoceros (Dicerorhinus sumatrensis) oocytes

A study was conducted opportunistically to evaluate the potential of rescuing immature oocytes from the ovaries of the Sumatran rhinoceros postmortem. Recovered oocytes (n = 30) were placed in maturation culture for 36 hr and inseminated with frozen-thawed homologous spermatozoa. After culture, evaluation of nuclear maturation status revealed that a large number of oocytes were degenerated (n = 21), but nine oocytes were assessed at the germinal vesicle (n = 3), metaphase I (n = 3), and metaphase II (n = 3) stages. Frozen-thawed Sumatran rhinoceros spermatozoa were capable of binding to the zona pellucida of in vitro matured oocytes, but no fertilization or cleavage resulted. In conclusion, relatively large numbers of oocytes can be obtained by ovarian follicular aspiration postmortem in the Sumatran rhinoceros, and some of these oocytes are capable of achieving nuclear maturation in vitro. However, additional studies are required to improve maturation success and achieve fertilization in culture.     

The Effect of Different Soybean Tillage Systems on Infiltrability and Erosion Susceptibility of an Oxisol in Paraná, Brazil

Measurements of infiltrability were carried out during the soybean vegetation period in three different tillage systems on an Oxisol, with the aim of estimating relative erosion susceptibility. The tillage systems studied were conventional tillage (disc plow), minimum tillage (chisel plow) and no-tillage. Infiltration was determined as the difference between rainfall applied with a portable rainfall simulator and runoff collected from mini-plots.
During all growth stages of the soybeans, infiltrability under no-tillage was higher than under conventional tillage. Minimum tillage took an intermediate position. Lowest overall infiltrability and consequently, highest relative erosion susceptibility was observed in the early growth stage after planting, in a wet year, when the degree of soil cover was also lowest. Infiltrability was mainly affected by degree of surface seal development as a result of natural rainfall energy the soil surface had received prior to measurements, as well as degree of soil cover. Highly significant correlations were found between the calculated sum of erosivity indices one month before measurement and the total infiltration. Differences in bulk density and macroporosity had hardly any effect on infiltrability.
The better performance of no-tillage in controlling erosion observed in Brazil can thus be explained by the higher degree of soil cover in comparison to the other tillage systems studied.     

Clostridial peritonitis associated with a mast cell tumor in a dog

Recurrent peritonitis caused by Clostridium limosum was associated with a mast cell tumor of a cranial mesenteric lymph node in a dog. The diagnosis of mast cell tumor was obscured because of the peritonitis and the appearance and location of the mass, which resembled an abscess. Since clostridial infections frequently are associated with neoplasia in man, veterinarians should be aware of the possibility of a similar relationship in animals.     

Response of pups to modified-live canine parvovirus component in a combination vaccine

Twenty-eight pups from a general pet population were vaccinated for canine parvovirus (CPV) with a combination vaccine every 3 weeks until the pups were 11 to 16 weeks old. Canine parvovirus antibody titers were measured by serum neutralization before each vaccination and greater than or equal to 2 weeks after the final vaccination. Eighteen pups that initially were seronegative for CPV seroconverted after 1 to 3 doses of modified-live virus CPV vaccine administered when the pups were between 8 and 16 weeks old; 16 of 18 seroconverted after the 1st dose. Of 10 pups that were seropositive for CPV at initial examination, 7 did not develop protective titers after 3 doses of vaccine, with the last dose given when the pups were 14 to 16 weeks old. Maternally derived antibody was the primary cause of vaccination failure.     

Variation of neutrophil function with age in calves

Neutrophil function was evaluated on 2 occasions in 5 calves from each of the following age groups: 4 to 5 weeks, 9 to 11 weeks, 16 to 19 weeks, and 12 to 14 months. Of the neutrophil functions examined, the iodination reaction, which evaluates the activity of the myeloperoxidase-hydrogen peroxide-halide antibacterial system of the neutrophil, reflected the most marked differences among age groups. The iodination values for the 2 youngest age groups were approximately 52% of the value for the oldest cattle. This difference could not be attributed to the amount of myeloperoxidase in the neutrophil granules. Antibody-dependent cell-mediated cytotoxicity also tended to be lower in neutrophils from the 3 younger age groups. Nitroblue tetrazolium reduction, a measure of superoxide anion generation by neutrophils, was lower in the youngest age group only. The capability of neutrophils to ingest Staphylococcus aureus was higher in the 3 youngest groups of calves than in the oldest group. The observed differences in neutrophil function in young vs older calves may be partially responsible for the increased susceptibility of young calves to infectious disease.     

Light microscopic and electron microscopic changes in the livers of cats with extrahepatic bile duct obstruction

Light microscopic and transmission electron microscopic changes were studied in the livers of 6 cats at 25 to 54 days after their extrahepatic bile ducts were experimentally obstructed. Histologic findings included various degrees of bile duct dilatation, ductular proliferation, and peribiliary fibrosis. Concentric layers of dense, birefringent connective tissue surrounded midsized bile ducts. The degrees of bile duct proliferation, hepatocellular degeneration, and bile retention were similar in each cat, but the amount of periductular connective tissue increased with chronicity of bile duct obstruction. Ultrastructural changes included marked swelling of endoplasmic reticulum and mitochondrial inner compartments, marked dilatation and distention of the canaliculi by bile casts, and numerical reduction of canalicular microvilli. Microvilli were often swollen and blunt. Mitochondria were swollen and long, and the cristae lacked normal distribution and density. Cytoplasmic accumulations of granular, electron-dense material and concentrically laminated arrays of material indicative of bile substances were also observed.     

Einfluß von Monoethanolamin auf den Ertrag von Kulturpflanzen

Effect of monoethanolamine on yield of crops
I. Studies on the effect of monoethanolamine on the grain yield and the nitrogen household in cereal plants (pot experiments)
The effect of monoethanolamine (EA, applied as foliar spray, 10 mg per pot) on grain yield and yield components was investigated in pot experiments with spring barley, winter wheat, and winter rye. Under conditions of a moderate drought stress the applied EA increased the grain yield of spring barley from 5 % to 7 % (significance only at α= 0.05). A stimulating effect of EA on the grain yield of winter wheat and winter rye was also obtained.
The positive influence of EA on the grain yield of spring barley was reproducible under the conditions of a limited water supply in small-scale plot experiments (increase of yield about 9 %).
In case of spring barley and winter rye the increase of the grain yield by EA, applied at growth stage 31 (= DC 31) was connected with a greater number of ear-bearing tillers. The increase of the winter wheat yield resulted from more grains per ear of the tillers.
The enhanced formation of tiller grain mass and total tiller biomass by EA was in correlation with a higher nitrogen import into tillers (r = 0.8⁺). Since an export of N from the main shoots into the tillers was not observed a higher N uptake (≥ 6 %) was calculated from N balances. After a fertilization with ¹⁵N-labelled fertilizers the additional ¹⁵N uptake was 13 % to 20 %.
Possible stress reducing activities caused by EA are discussed.