Rapid procedure for large-scale isolation of plasmid DNA.
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Biomedical subjects
Publications and source records attributed to F S Kibenge.
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The nucleotide sequence of genome segment A cDNA of the STC strain of infectious bursal disease virus (IBDV) was determined and compared with sequences of the homologous genome segment of the 002-73 strain of IBDV and the Jasper strain of infectious pancreatic necrosis virus (IPNV). The STC-IBDV genome segment A was determined to be 3262 base pairs (bp), which is close to the estimated total length of 3300 bp for genome segment A in IBDV, although there is no proof that it is the real length of this genome segment. The STC-IBDV genome segment A contains two major overlapping open reading frames (ORFs). The large ORF of 3036 bp predicts a polyprotein of Mr 109,358, whereas the small ORF is 435 bp and predicts a protein of Mr 16,550 in STC-IBDV. STC-IBDV and 002-73-IBDV polyproteins are closely related (97.4% amino acid homology). Most of the amino acid mismatches are in VP2 sequences, mainly within the area of the conformation-dependent epitope. Comparison with the Jasper-IPNV polyprotein reveals levels of amino acid sequence homology of about 40% in VP2, 32% in VP3 and 21% in VP4. Within the VP2 molecule the conformation-dependent epitope area is again the least homologous, but the heterogeneity is more conspicuous than between the two IBDV strains, which is not surprising since IBDV and IPNV are serologically unrelated. The small ORF proteins have about 88% amino acid sequence homology between STC-IBDV and 002-73-IBDV, and 30% between each IBDV strain and Jasper-IPNV. There is no homology at all in the non-coding regions of IBDV and IPNV. These comparative sequence data will be useful for subgrouping the Birnaviridae family.
A molecular clone representing 445 base pairs at the 3' end of infectious bursal disease virus (IBDV) genome segment B was used in a dot blot hybridization assay to detect viral RNA from cell culture and from chicken bursa and spleen tissue specimens. The cloned nucleotide sequence represents approximately 14% of the virus-encoded polymerase (VP-1) gene. The lower detection limit of radiolabeled probes prepared from this clone was 0.1 ng of IBDV double-stranded RNA. The probe had broad specificity and was used to detect four serotype 1 IBDV strains and one serotype 2 IBDV strain. This probe, however, did not cross-react with nucleic acid extracted from nine unrelated poultry viruses. A rapid procedure for isolation of IBDV genomic RNA from bursa and spleen tissue specimens was developed and used with the dot blot hybridization assay to detect IBDV strains in tissue samples from experimentally infected and commercially reared chickens.
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The prevalence of reoviruses in commercial chickens with the runting/stunting syndrome, tenosynovitis, and normal chickens was investigated. Reoviruses were isolated from 3-week-old chickens affected with the runting/stunting syndrome and from older chickens with tenosynovitis; viruses were isolated from tissues with and without lesions. Reoviruses were also frequently isolated from rectal contents of normal 3-week-old chickens, and there was serological evidence of previous reovirus infection in all flocks of adult meat breeder chickens examined. The widespread occurrence of reoviruses in both normal and diseased chickens indicates that the isolation of reoviruses from tissues of chickens with lesions does not necessarily imply any aetiological relationship of reovirus with disease, even in the absence of other known causes. However, the occurrence of reovirus in normal chickens does not preclude an aetiological relationship with disease and further investigation of strain variation and possible virulence factors in avian reoviruses is required.
A comparison of the serological responses to a reovirus administered by the eye-drop, intramuscular, oral and subcutaneous routes was made in three-week-old chickens. Their patterns of virus shedding were also compared. All four routes resulted in an antibody response and no virus excretion in faeces more than two to three weeks after administration. The oral route represented the most favourable route for live reovirus vaccine administration.
Lysogeny was readily demonstrated among strains of Staphylococcus hyicus that were isolated from chickens. Susceptibility to phage lysis was affected by prophage immunity, but lipase activity and erythromycin resistance were not affected by the presence of temperate phage. In contrast to previously published results, lipase-negative strains of S. hyicus were relatively common and the use of selective media based on lipase activity would have been unsuitable for detection of the S. hyicus strains examined.
The phage typing and cultural characteristics of 574 strains of S. aureus of poultry origin in Australia were examined. With the avian phage set of Shimizu (1979) it was possible to type 74.2% of strains. A number of significant variations in the phage typing patterns of Australian strains compared to those reported from Japan and Europe were observed. A lower proportion of Australian strains were of avian phage group I and a higher proportion of group III. A high proportion of strains were of mixed lytic groups. No locally isolated phages were able to increase significantly the percentage of typeable strains, although four local phages appeared to be of greater value for phage typing poultry strains of S. aureus than some other phages of the avian phage set. The international (human) phage set was of limited value in typing Australian strains of poultry origin although four strains were identified which were indistinguishable from strains of human origin. Using cultural characteristics of the strains in conjunction with phage typing, the Australian strains of S. aureus were assigned to one of three major groups and nine subgroups. A list of typing phages considered to be valuable for use on Australian poultry strains of S. aureus is given.
Bacteriophage typing of Staphylococcus aureus from different outbreaks of tenosynovitis in broiler breeder replacement chickens showed that, although a mixture of phage types was present on affected farms, there was a predominant phage type isolated from lesions of affected chickens. The predominant phage type isolated from chickens in different outbreaks was variable. The source of S. aureus associated with tenosynovitis appeared to be a resident population present on the skin.
Twenty-six samples known to contain infectious bursal disease virus (IBDV) were examined by virus-isolation attempts on ovine kidney (OK) cell line, Vero cell line, and chicken embryo fibroblast (CEF) cultures. Virus was isolated from two of 26 samples, three of 26 samples, and three of 25 samples on OK, Vero, and CEF cultures, respectively. However, in contrast to IBDV replication in Vero and CEF cultures, isolated virus was unable to induce serially sustained cytopathic effects (CPE) during successive passages in the OK cell line, unless cell lysates were treated with chloroform between every other passage. The cytopathogenicity of the untreated virus passaged in OK cells was revived and maintained upon passage in Vero cells. An initial single passage of laboratory or field material in OK cells followed by further passages in Vero cells resulted in virus isolation from six of 26 samples, which was better virus recovery than when either cell line was used alone or when CEF cultures were used. Twenty of the 26 test samples were originally positive when examined by nucleic acid hybridization with radiolabeled IBDV cDNA, indicating that some of the samples that were negative upon virus isolation using OK and Vero cells may have contained inactivated virus.
A cDNA library was prepared from the double-stranded RNA genome of the infectious bursal disease virus (IBDV) strain ST-C. The cDNA molecules were annealed into the plasmid pUC9 and used to transform Escherichia coli strain JM107. A cDNA clone that contained IBDV-specific nucleotide sequences was selected and designated STC-1. Radiolabeled probes were prepared from STC-1 and hybridized to genome segment A of ST-C in a northern blot hybridization assay. The STC-1 cDNA was 448 base pairs in length, and its nucleotide sequence indicated that it is located near the VP-2/VP-4 junction in IBDV genome segment A. Biotin-labeled probes were prepared from STC-1 and used in a dot-blot hybridization assay to detect IBDV. Under relatively low stringency conditions of hybridization, the biotinylated probes detected four subtypes of IBDV serotype 1 and a serotype 2 IBDV isolate.
The lesions and etiologic agents associated with 13 outbreaks of respiratory disease in commercial chickens were investigated. Adenoviruses were isolated from tracheal and lung tissues of affected chickens in all 13 outbreaks. Escherichia coli was isolated from the lung of an occasional bird. The tracheal specimens were consistently negative for Bordetella avium, but E. coli and occasionally Staphylococcus aureus were isolated. There was also serological evidence in one outbreak, and pathological evidence in another, of a concurrent infectious bursal disease virus (IBDV) infection of chickens affected with the disease. Gross and microscopic alterations in the tracheas and lungs of affected chickens were similar in all outbreaks and consisted of catarrhal tracheitis and occasionally multifocal pneumonia with mononuclear cell infiltrates. Hepatitis and splenitis with heterophil infiltrates occasionally were seen in birds with coliform septicemia. The tracheal and lung lesions in the present investigation were considered primarily of adenovirus etiology, complicated by secondary bacterial infection.
The growth of five strains of infectious bursal disease virus--three strains of serotype I (SAL, D-78, 2512), one of serotype II (OH), and one variant strain (Variant-A)--were compared in Vero and chicken embryo fibroblast (CEF) cell cultures in order to characterize the replication of different strains of IBDV in Vero cells. For all five virus strains, the latent period in Vero cells ranged from 12 to 18 hr, which was longer than the 4-to-6-hr latent period observed in CEF cultures for strains SAL, D-78, and OH. Virus strains SAL, D-78, and OH, which were examined in both Vero and CEF cultures, also had a more extensive maturation phase and higher yields of virus in Vero than in CEF cultures. Total titers of these viruses of 5.35 to 6.10 log10 TCID50/ml in CEFs occurred 24 to 30 hr postinoculation (PI), although the cytopathic effect (CPE) was not seen until 72 hr PI. By comparison, their total infectious virus titers of 6.85 to 8.35 log10 TCID50/ml in Vero cells occurred from 48 hr PI, coinciding with the appearance of CPE. The growth curve of Variant-A in Vero cells differed from the other viruses by showing steadily rising extracellular and cell-associated virus titers throughout the 72-hr observation period. Only very low titers of Variant-A were obtained in CEF cultures, and thus no growth curve in CEFs was performed.
In 1983, twenty-two outbreaks of viral arthritis/tenosynovitis were diagnosed in a 6-month period on 18 fryer farms of one commercial operation located in western Washington. The main source of the reovirus infection was traced to a breeder flock that supplied progeny chicks to all of the affected farms.
Four avian reoviruses were orally inoculated into 1-day-old chickens to determine pathogenicity, virus persistence in the intestinal tract, and effects on body weight gains. Avian reoviruses Reo-25 and W3-492 belonged to two separate serotypes, and viruses TC 897 and W3-410 were antigenically related to W3-492. Isolate W3-492, which was highly pathogenic, was very rarely recovered from cloacal swabs collected 2 weeks postinoculation, but inoculated chickens gained significantly less weight (P less than or equal to 0.001) than uninoculated controls during the 5-week test study. Isolate Reo-25 persisted the longest in the intestinal tract, and isolates TC 897 and W3-410, of intermediate persistence, had no significant effect on body weights. There was no apparent correlation between serotype and pathotype of avian reoviruses.