A continuous bovine kidney cell line for routine assays of foot-and-mouth disease virus.

A continuous bovine kidney cell line, LF-BK, arose from primary bovine calf kidney cells that survived infection with a temperature-sensitive mutant of foot-and-mouth disease virus. No virus was recovered after the first passage. Cells of Passage 48 were inoculated into two steers which remained healthy and did not develop neutralizing antibodies to the virus. The karyotype of cells of the 53rd and 87th passages was similar and revealed that the cells were markedly transformed. The modal number of diploid chromosomes was 52 at both passage levels. LF-BK cells and primary bovine kidney cells were equally susceptible in plaque assays to each of the 7 types of foot-and-mouth disease virus. The cell line and primary bovine kidney cells were less susceptible than primary bovine thyroid cells to several subtypes of the virus in suspensions of tongue epithelium. The LF-BK continuous cell line is recommended for routine plaque assays or plaque neutralization tests as a substitute for primary bovine kidney cells.

Genome analysis of African swine fever virus isolated in Italy in 1983.

An African Swine Fever virus (ASFV) isolated in an 1983 outbreak of the disease in Piemonte, Italy, was related by restriction endonuclease analysis of the viral genome to ASFV strains isolated in the Dominican Republic (1978), Haiti (1981) and Cameroon (1982).

Immune response of mice and sheep to bluetongue virus inactivated by gamma irradiation.

Gamma irradiation is being tested as a means of inactivating bluetongue virus (BTV) for use in vaccines. Exposure of BTV 17 to various levels of irradiation revealed that a dose of approximately 0.6 megarad was required to reduce the virus titer by one log10, or 90%. To test the immunogenicity of irradiated BTV, mouse brain passaged virus and concentrated cell culture passaged virus were inactivated by 6 megarads of gamma irradiation, and vaccines were prepared by emulsifying the virus preparations in equal volumes of a modified incomplete Freund's adjuvant. These vaccines stimulated the production of neutralizing antibodies in mice and sheep, a cell mediated immune response in mice, and a protective immune response in sheep. The results suggest that gamma irradiation would be an effective means of inactivating BTV for the preparation of vaccines.

Ultrastructure of Haemophilus equigenitalis, causative agent of contagious equine metritis.

Haemophilus equigenitalis, a proposed new species of Haemophilus and the causative agent of contagious equine metritis, a venereal disease of the horse, had ultrastructural characteristics of gram-negative bacteria. The organism additionally had a small, threadlike capsule that was removed by heating in phosphate-buffered saline solution. Heating also detached the outer membrane from the cytoplasmic membrane. The capsule could only be demonstrated when bacterial were stained with ruthenium red during the preparation of ultrathin sections. The gross morphology of newly isolated organisms (rodlike or coccal) depended upon the medium on which they were grown.

Isolation and characterization of Escherichia coli phase variants and mutants deficient in type 1 pilus production.

Type 1 pili of Escherichia coli are the prototype of the somatic class of pili found on many strains of bacteria. As a first step in the genetic analysis of type 1 piliation, an extensive series of nonpiliated derivatives of E. coli K-12 strain AW405, was characterized to produce attached or free pili when examined in the antiserum or appeared to produce attached or free pili when examined in the electron microscope. The derivatives fell into two classes; phase variants and mutants. Phase variants that formed colonies of two distinctive types, one associated with a predominantly piliated (P+), and the other associated with a nonpiliated (P-) phase, were obtained. Each phase could give rise to the other at a relatively high rate, which was greater in the P- to P+ direction during culture in unshaken liquid medium. In addition, 77 Pil- mutants were selected on the basis of a subtle difference in colonial morphology. The mutants reverted, if at all, at a much lower rate than that of the P- to P+ change. The stability of Pil- derivatives grown in unshaken liquid medium was used as a criterion for distinguishing between phase variants and mutants, Phase variation also effected colonial morphology and chemotactic swarming. These properties did not directly depend upon piliation since Pil- mutants were only slightly altered in colonial form and unaltered in chemotactic swarming. Piliation of Pil+ bacteria was quantitatively affected by growth conditions.

Genetic complementation analysis of Escherichia coli type 1 somatic pilus mutants.

A genetic complementation analysis of 75 stable nonpiliated mutants of a type 1 piliated strain of Escherichia coli K-12, AW405, was performed. Strains containing pairs of pil mutations were constructed by the infectious transfer of an F101 plasmid containing one pil mutation into E. coli K-12 AW 405 containing another pil mutation. The presence or absence of type 1 pili on the merodiploid strains was determined by agglutination with type 1 pilus antiserum. All 75 mutants fell into one of four complementation groups. The pattern of complementation defined three cistrons involved in pilus formation, pilA, pilB, and pilC. The fourth complementation group was composed of a large number of mutants defective in both pilA and pilB functions.

Selection of a stable clone of the MVPK-1 fetal porcine kidney cell for assays of foot-and-month disease virus.

The MVPK-1 cell line, derived from fetal porcine kidney cells, supports the replication of foot-and-mouth disease (FMD) virus. The cell line was adapted to grow in medium containing 5% bovine serum. The susceptibility of the adapted cells decreased as they aged at 37 degrees C. Various clones were isolated from the adapted cells and their growth characteristics and sustained susceptibility to FMD virus were compared. Clone 7 maintained uniform susceptibility to FMD virus over a 3-day period at 37 degrees C and proved superior to other clones in the characteristics studied. The clone has maintained satisfactory susceptibility to FMD virus through 40 subcultures. Clone 7 can replace primary bovine kidney cells for routine viral assays, but cannot detect as much FMD virus in animal specimens as primary bovine kidney, bovine thyroid, or swine kidney cells.

Susceptibility of a new fetal pig kidney cell line (MVPK-1) to foot-and-mouth disease virus.

The Mengeling-Vaughn Porcine Kidney (MVPK-1) cell line, derived in October, 1970, from fetal pig kidneys, is susceptible to all 7 types of foot-and-mouth disease (FMD) virus. A plaque assay was developed for FMD virus that depended on washing MVPK-1 cells in serum-free medium before infection and excluding serum from 0.6% gum tragacanth overlay during plaque formation. The numbers of plaques that formed on MVPK-1 cells by a representative strain of each FMD type were comparable with the numbers of those on primary bovine calf kidney (BK) cells. Virus passaged in BK cell cultures did not have to be adapted to the cell line to obtain these results. The cell line lost susceptibility rapidly at 37 C after confluency was reached but retained susceptibility if maintained at room temperature. The cell line has the potential of replacing BK cells for many diverse purposes.