Molecular epidemiology of Mycobacterium bovis isolates from free-ranging wildlife in South African game reserves.

Bovine tuberculosis is endemic in African buffalo and a number of other wildlife species in the Kruger National Park (KNP) and Hluhluwe-iMfolozi Park (HiP) in South Africa. It was thought that the infection had been introduced into the KNP ecosystem through direct contact between cattle and buffalo, a hypothesis which was confirmed in this study by IS6110 and PGRS restriction fragment length polymorphism (RFLP) typing. The molecular characterisation of 189 Mycobacterium bovis isolates from nine wildlife species in the HiP, including three smaller associated parks, and the Kruger National Park with adjacent areas showed that the respective epidemics were each caused by an infiltration of a single M. bovis genotype. The two M. bovis strains had different genetic profiles, as demonstrated by hybridisation with the IS6110 and PGRS RFLP probes, as well as with regard to evidence of evolutionary changes to the IS profile. While the M. bovis type in HiP was transmitted between buffaloes and to at least baboon, bushpig and lion without obvious genetic changes in the RFLP patterns, in the KNP a dominant strain was represented in 73% of the M. bovis isolates, whilst the remaining 27% were variants of this strain. No species-specific variants were observed, except for one IS6110 type which was found only in a group of five epidemiologically related greater kudu. This finding was attributed to species-specific behaviour patterns rather than an advanced host-pathogen interaction.

High Mycobacterium bovis genetic diversity in a low prevalence setting.

The genetic diversity among South African Mycobacterium bovis isolates from cattle was determined by genetic fingerprinting. The restriction fragment length polymorphism (RFLP) markers IS6110 and polymorphic GC-rich sequence (PGRS) as well as spoligotyping and determination of variable number of tandem repeats (VNTR) were used to characterize sub samples of 91 M. bovis field isolates. PGRS RFLP was the single most discriminatory method and combinations of typing methods, which included IS6110 and/or PGRS had the highest discriminatory power, able to reveal 29 distinct genotypes among 35 farms with no epidemiological link. Three of the farms were co-infected with two genetically unrelated strains. In contrast to reports from European and also other colonised countries on the African continent our findings are suggestive of a high genetic diversity of M. bovis in South Africa's cattle population, implying a variety of unrelated ancestor strains. Despite effective intervention through test-and-slaughter campaigns no indication of a 'founder effect' was apparent in the panel of isolates derived from all infected provinces.

Inhibition of tritiated thymidine incorporation in cultured cells by rat kidney extract.

KCl extract from rat kidney, rat liver, and Morris hepatomas inhibited [3H]thymidine incorporation into cultured cells. Tissues came from male inbred BUF rats. The most pronounced inhibition was achieved with the kidney extract. Protein synthesis was not inhibited during a 24-hour exposure of the cells to the inhibitor. Incorporation of [3H]deoxycytidine was inhibited, as was cell growth, when the kidney KCl extract was present for several days. [3H]thymidine incorporation was inhibited almost immediately after the addition of the extract. The inhibition was reversible. Regular [3H]thymidine incorporation was restored 24 hours after removal of the inhibitor, which was neither arginase nor a thymidine-degrading enzyme. The inhibitor was stable to heat (80 degrees C for 10 min) and resistant to trypsin, pronase, DNase, and RNase. Exposure of the extract to proteolytic enzymes, hyaluronidase, and neuraminidase resulted in a loss of inhibitory activity only after extensive dialysis of the treated extract. The inhibitor appeared to be a mucoprotein in which the carbohydrate moiety may be responsible for the inhibition. The KCl extract also inhibited RNA synthesis and DNA synthesis by the de novo pathway. The inhibition of phosphorylation of thymidine, however, appeared to be the primary action of the inhibitor.

Effect of a normal serum protein absent from hepatoma-bearing animals on cell cultures.

The effect of a serum factor purified from normal rat serum on growth and incorporation of 3-H-thymidine and 3-H-leucine was investigated with the use of 3T3 and L cells. The factor was not present in serum of hepatoma-bearing animals at a time when the weight of the hepatomas was equal to or greater than liver weight. The factor disappeared gradually from the serum of hepatoma-bearing animals, and the disappearance was proportional to the increase in the size of the hepatomas. Diminished amounts of the factor could be detected as early as 10 days after hepatoma transplant, and the factor was absent at 30 days after hepatoma transplant from the serum of rats bearing hepatoma 7777. Medium supplemented with 5% normal rat serum supported incorporation of lavel into 3T3 and L cells and growth of these cells, as did medium supplemented with 10% calf serum. Serum from hepatoma-bearing animals was only approximately equal to 50% as efficient as normal rat serum in supporting growth and label incorporation. The addition of purified factor to the serum from hepatoma-bearing animals restored the ability of the serum to support growth and label incorporation to between 70 and 90% of that found with normal rat serum as a medium supplement. The factor also enhanced incorporation of 3-H-thymidine following release of 3T3 cells from contact inhibition.

Correlation of circulating levels of a serum protein with triiodothyronine levels and hepatoma growth.

In this communication, we provide evidence that proliferation of transplantable Morris hepatoma 7777 might to some extent be regulated by triiodothyronine and/or a specific serum protein, the levels of which are correlated with levels of triiodothyronine. The protein has an estimated molecular weight of 80,000 and migrates as one band on polyacrylamide gel electrophoresis. In normal rats, this protein accounts for approximately 1% of the total serum protein. Both the circulating levels of the serum protein and proliferating of transplanted hepatoma cells were decreased in thyroidectomized rats. Elevated levels of the serum protein and increased cell proliferation were observed when animals had 70% of their lives removed prior to transplant, were given injections of triiodothyronine, or had a 10-day-old first transplant surgically removed. Some evidence is also provided suggesting that the synthesis of the serum protein is stimulated by thyroid hormone.

Effect of bleomycin on [3H]Thymidine 5'-Triphosphate incorporation into host liver and hepatoma nuclei.

The effect of bleomycin on [3H]thymidine 5'-triphosphate ([3H]TTP) incorporation into isolated sucrose nuclei from host liver and Morris hepatomas has been compared. Bleomycin stimulates [3H]TTP incorporation 13-fold in host liver and hepatoma 16 nuclei, 8-fold in hepatoma 7800 nuclei, and 3-fold in hepatoma 7777 nuclei. Differences in the nuclear membranes are not responsible for the different response of the nuclei. Nuclei, denuded of their membranes by Triton X-100 treatment, give similar results to sucrose nuclei. Analysis of DNA extracted from liver or hepatoma nuclei incubated with bleomycin indicates that bleomycin produces scissions in the nuclear DNA and that some repair synthesis takes place. Incubation of nuclei with 111indium-labeled bleomycin shows an equal binding capacity of liver and hepatoma nuclei for bleomycin. Bleomycin also stimulates incorporation of [3H]TTP in a system using chromatin or calf thymus DNA as primer. Host liver or hepatoma chromatin incubated with a DNA polymerase extracted from normal rat liver nuclei is stimulated approximately to the same extent by bleomycin. When DNA polymerase extracts from host liver and hepatoma nuclei are assayed with calf thymus DNA as primer, bleomycin has a greater stimulatory effect on [3H]TTP incorporation with host liver DNA polymerase than with hepatoma DNA polymerase in the system. We suggest that a defect in the repair system in hepatoma nuclei is responsible for the relatively lower response to bleomycin.

DNA synthesis in membrane-denuded nuclei and nuclear fractions from host liver and Morris hepatomas.

Incorporation of [3H]TTP into membrane-denuded nuclei and fractions of these nuclei from host liver and Morris hepatomas has been compared. Treatment of sucrose nuclei with Triton X-100 removed 95% of the phospholipids and 15 to 20% of the protein. These membrane-denuded nuclei remained physically stable. The Triton X-100-extracted nuclei incorporated label into their DNA in nuclear-incorporating system similar to sucrose nuclei with their membranes intact. Triton X-100-treated nuclei from hepatoma 7777 incorporated six times more label and those from hepatoma 7800 incorporated three times more label than Triton X-100-treated host liver nuclei. Nuclei from the three sources incorporated more label when exogenous DNA was added to the incubation system, but the difference in incorporation between the hepatoma nuclei and the host liver nuclei disappeared. When Triton X-100-treated nuclei, prepared from a tumor-bearing animal given injections of [3H]thymidine for 10 min were fractionated on sucrose gradients after disruption by high Mg2+ concentration, the fractions from hepatoma 7777 nuclei contained six times as much label as the host liver nuclear fractions. Nuclear fractions prepared from unabeled hepatomas or host livers had DNA polymerase activity. The activity, however, is the same in fractions prepared from hepatoma 7777 or host liver nuclei. It is suggested that the nuclear membrane does not play an important role in nuclear DNA synthesis. It is further suggested that the increased incorporation found with hepatoma nuclei is dependent on a physical or chemical arrangement of components within the nucleus and not solely on different enzyme levels.

Response of cultured hepatocytes to a hepatomitogen after initiation by conditioned medium or other factors.

Injection of a substantially purified hepatomitogen into recipient rats that had 40% of their liver removed resulted in a significant stimulation of hepatic DNA synthesis as determined by the labeling index and the mitotic index. Normal or sham-operated rats did not respond to the injection of the mitogen. The extraction and partial purification of this hepatomitogen have previously been reported (A. Francavilla et al., Cancer Res., 47:5600-5605, 1987). Addition of the factor to an epithelial-like liver-derived cell line in culture (clone 9) or to a hepatoma cell line (HTC-SR) resulted in a dose-dependent stimulation of DNA synthesis. Hepatocytes in primary culture, on the other hand, were not stimulated by the addition of the factor. However, when the mitogen was added to hepatocytes in primary culture, together with conditioned medium, obtained from the responsive cell lines, a significant stimulation of DNA synthesis could be demonstrated in hepatocytes in culture. The stimulation was dose dependent with respect to the mitogen, was abolished by 10 mM hydroxyurea, and was independent of epidermal growth factor. The conditioned medium could be replaced by a protein factor extracted from the two cell lines as previously reported (P. Ove et al., J. Cell. Physiol., 131: 165-174, 1987). It appears that a cofactor is provided by the conditioned medium or by the cell extract, enabling the hepatomitogen to act on hepatocytes in primary culture.

Epidermal growth factor and proliferation in rat hepatocytes in primary culture isolated at different times after partial hepatectomy.

Primary hepatocyte cultures have been prepared from normal adult rat liver and from rat liver at 4, 8, 12, 24, and 48 h following partial hepatectomy (removal of 70% of the liver). Cells were maintained in minimal essential medium alone or supplemented with hormones. Comparing DNA synthesis in normal adult rat hepatocytes with DNA synthesis in hepatocytes isolated from regenerating livers, we found with minimal essential medium alone little DNA synthesis in normal adult rat hepatocytes and in hepatocytes isolated 4, 8, or 12 h after 70% hepatectomy. In hepatocytes isolated 24 h after partial hepatectomy, however, the incorporation of [3H]thymidine was 3 times the rate of normal hepatocytes. The addition of insulin to minimal essential medium had minimal effect on DNA synthesis in all hepatocytes. Addition of epidermal growth factor alone or in combination with insulin resulted in a dramatic increase in DNA synthesis in hepatocytes from regenerating rat liver. Increased incorporation was detectable as early as 4 h after partial hepatectomy and reached a maximum at 24 h after the operation. Results obtained with [3H]thymidine incorporation were confirmed by autoradiography and by direct DNA determinations in hepatocyte cultures. Epidermal growth factor binding to the hepatocytes was determined and agreed with previously reported binding studies. Binding of epidermal growth factor in hepatocytes isolated at 4 h after partial hepatectomy was the same as in normal hepatocytes but was undetectable in hepatocytes isolated from rats at 12 and 24 h after partial hepatectomy.

Some biochemical characteristics of rat liver and Morris hepatoma nuclei and nuclear membranes.

Nuclei prepared from host liver and from Morris hepatomas 7777 and 7800 have been compared with respect to some of their biochemical characteristics. Several criteria were used to ensure that liver and hepatoma nuclei were of equal purity. These criteria include equal specific activity ratios (homogenate nuclei) for several marker enzymes. Phospholipids, proteins, and sialic acid content were compared in liver and hepatoma sucrose nuclei and in membrane and chromatin fractions obtained from liver or hepatoma nuclei. As determined by sodium dodecyl sulfate polyacrylamide electrophoresis, the only qualitative difference in protein that could detected was in 2 of the 4 nuclear fractions. There was an extra band in each of the 2 hepatoma fractions. Sialic acid was increased in hepatoma nuclei. In addition, a fraction containing most of the inner nuclear membrane from liver nuclei had no sialic acid, whereas the equivalent hepatoma fraction did have sialic acid. Total phospholipids were increased in hepatoma nuclei. This increased phospholipid concentration in hepatoma nuclei as compared to liver nuclei was apparent with sucrose nuclei, citric acid nuclei, membrane-denuded nuclei, chromatin, and nuclear fractions. Determination of the percentages of individual phospholipids making up the total phospholipids extracted revealed that the only significant change in the phospholipid composition of hepatoma nuclei was an increase in sphingomyelin. A large amount of this sphingomyelin was found to be associated with chromatin. The possible significance of chromatin-associated phospholipids is discussed.

A comparison of DNA repair synthesis in primary hepatocytes from young and old rats.

DNA repair synthesis has been compared in primary hepatocyte cultures obtained from 3-month-old and 16-20-month-old rats. Several morphological and metabolic characteristics were determined to assure cultures of comparable quality. DNA damage was induced by the addition of bleomycin or the exposure of the culture to UV irradiation. DNA repair (unscheduled DNA synthesis) was determined by measuring [3H]thymidine incorporation. After UV irradiation, there was almost twice as much [3H]thymidine incorporation in cells obtained from young rats as in those obtained from old rats. Equal amounts of bleomycin resulted in substantially greater damage to DNA in cells from old rats than from young rats. For equal amounts of DNA damage there was again diminished [3H]thymidine incorporation in cells obtained from old rats. Finally equal amounts of bleomycin resulted in equal damage to DNA when the bleomycin was added to isolated rat liver nuclei from young or old rats. Bleomycin treated nuclei from young rats incorporated substantially more [3H]thymidine triphosphate (TTP) than bleomycin treated nuclei from old rats. The results indicate that hepatocytes from old rats are much more susceptible to bleomycin than hepatocytes from young rats and that the capacity for DNA repair synthesis is impaired in hepatocytes from old rats.

Inhibition of bleomycin-induced [3H] thymidine 5'-triphosphate incorporation into liver and hepatoma nuclei by N-ethyl maleimide and daunomycin.

The addition of bleomycin to a nuclear incorporating system results in an increased incorporation of 3H-thymidine 5'-triphosphate (3H-TTP) into the DNA of liver and hepatoma nuclei. Bleomycin added to the nuclear incorporating system also produces scissions of DNA as determined by sucrose density gradient centrifugation of the extracted DNA. The action of bleomycin is dependent on the presence of sulfhydryl agents in the incubation mixture. Two compounds, N-ethyl maleimide and daunomycin, inhibit the bleomycin-induced incorporation of 3H-TTP preferentially. N-Ethyl maleimide inhibits bleomycin-induced activity in liver and hepatoma 7777 nuclei equally. Lower levels of daunomycin inhibit the bleomycin-induced activity in the hepatoma 7777 nuclei than are required to inhibit the activity in liver nuclei. The two compounds inhibit the bleomycin effect by different mechanisms. The addition of N-ethyl maleimide to bleomycin in the incubation system prevents bleomycin from causing breaks in the DNA. The addition of daunomycin, despite inhibition of bleomycin-induced 3H-TTP incorporation, does not affect the bleomycin-produced breaks in the DNA. N-Ethyl maleimide acts by binding to the DNA and by competing with a sulfhydryl agent for bleomycin-sensitive sites on the DNA. Daunomycin apparently inhibits a repair enzyme that is responsible for the increased incorporation following bleomycin treatment.

The occurrence of Theileria and Cowdria parasites in African buffalo (Syncerus caffer) and their associated Amblyomma hebraeum ticks.

The polymerase chain reaction and oligonucleotide probing were used to detect Theileria and Cowdria species in DNA extracted from blood and ticks recovered from 24 African buffalo during a gamecapture operation in the Kruger National Park, South Africa. Species-specific probing indicated that all but one of the buffalo were carrying at least one Theileria species. Indirect fluorescent antibody (IFA) serology indicated that all animals had been exposed to Theileria parva infection but only 33% were positive for T. parva by probing. Twelve (50%) of the animals but only six of the 214 adult Amblyomma hebraeum ticks examined (2.8%) were probe-positive for Cowdria. Only one Cowdria 16S genotype was detected in the animals and ticks.