Persistence of Mycobacterium bovis Bacillus Calmette-Guérin in white-tailed deer (Odocoileus virginianus) after oral or parenteral vaccination.

Mycobacterium bovis is the cause of tuberculosis in cattle and a serious zoonotic pathogen, most commonly contracted through consumption of unpasteurized dairy products. To control this zoonosis, many countries have developed bovine tuberculosis eradication programmes. Although relatively successful, efforts are hindered in many regions by spillover from wildlife reservoirs of M. bovis to cattle. Such is the case in the United States where spillover of M. bovis from free-ranging white-tailed deer to cattle occurs. One approach to control such inter-species transmission is vaccination of wildlife. The live, attenuated human vaccine M. bovis Bacillus Calmette-Guérin (BCG) has been shown to reduce disease severity in white-tailed deer; however, vaccine persistence within tissues has also been noted. Consumption of venison containing BCG by hunters may present a public health concern as BCG exposure, although unlikely to cause disease, could cause false positive tuberculin skin test results. To examine BCG persistence further, 42 white-tailed deer were vaccinated orally or subcutaneously (SC) with BCG Danish. Three deer from each group were killed and examined at periods ranging from 2 weeks to 11 months after vaccination. BCG was recovered from orally vaccinated deer as late as 3 months after vaccination, while BCG persisted in SC vaccinated deer for as long as 9 months. At no time was BCG isolated from meat; however, prolonged persistence was seen in lymphoid organs. Although vaccine persistence was noted, especially in SC vaccinated deer, the distribution of culture-positive tissues makes human exposure through consumption unlikely.

Cancer initiating properties of fumonisin B1 in a short-term rat liver carcinogenesis assay.

The nature of cancer initiation by fumonisin B(1) (FB(1)) was investigated in rat liver by monitoring the effect of phenobarbital (PB) as cancer promoter and evaluating the involvement of spontaneously initiated cells. A PB promoting regimen (0.05% in the diet) stimulated the outgrowth of FB(1)-induced placental glutathione S-transferase (GSTP) positive initiated hepatocytes. Reversion of the FB(1)-induced GSTP(+) foci was noticed in the absence of a promoting regimen. Younger rats were shown to be more sensitive to the induction of GSTP(+) foci by FB(1). Cancer initiation by FB(1) was associated with a hepatotoxic effect, which was less pronounced in older rats presumably due to a reduced intake. A specific role of spontaneously initiated cells and their promotion by FB(1) into the development of eosinophilic clear cell foci could not be established under the present experimental conditions. The ability of different stimuli to selectively promote the outgrowth of FB(1) initiated cells further verifies the cancer initiating potency of this apparent non-genotoxic mycotoxin. The underlying mechanism(s) involved in the genesis of the initiated hepatocytes is not known at present.

Exposure of sheep scrapie brain homogenate to rumen-simulating conditions does not result in a reduction of PrP(Sc) levels.

AIMS: Experiments were designed to evaluate the potential of rumen-simulating conditions to reduce PrP(Sc) levels. METHODS AND RESULTS: Scrapie-positive brain material was incubated under rumen-simulating conditions. Time points were taken over a 24-h period and PrP(Sc) levels were analysed by Western blot. No loss of PrP(Sc) was observed over a 24-h time period. CONCLUSIONS: Our results indicate that a fully developed rumen fermentation does not provide significant protection against prion infection via the oral route. Developmental changes including senescence of immune system function or other developmental changes in the gastrointestinal tract are potential mechanisms by which relative bovine spongiform encephalopathy (BSE) susceptibility might vary with age. SIGNIFICANCE AND IMPACT OF THE STUDY: Epidemiology of the BSE outbreak in the United Kingdom indicates that younger animals were at higher risk of infection. The rumen undergoes pronounced developmental changes early in life, coinciding with the introduction of fibre into the diet. The timeframe of highest risk of infection overlaps the time in life prior to full rumen development. This work indicates that a fully developed rumen does not provide significant protection against prion infection via the oral route of infection. This result implicates other developmental changes that are responsible for the age-dependent susceptibility of cattle to BSE.

Comparison of rapid methods for the extraction of bacterial DNA from colonic and caecal lumen contents of the pig.

AIMS: The increasing uses of DNA methodologies to study the micro flora of the pig gastrointestinal tract requires an efficient recovery of bacterial DNA from the intestinal sample. Thus, the objective of this study was to determine which DNA extraction methods are most effective for luminal samples from pigs. Several routinely used nucleic acid extraction procedures were compared based upon quantity and purity of extracted DNA. METHODS AND RESULTS: DNA was extracted from pig colonic and caecal lumen samples using 19 methods for bacterial DNA extraction. The quantity of total DNA recovered by each extraction method was determined and compared. Two methods using extraction with polyvinylpolypyrrolidone (PVPP) or phenol and two methods involving bead mill homogenization were found to provide the greatest quantity of extracted DNA for both colonic and caecal lumen. Extracted DNA from these four methods was further analysed for purity based upon the presence of PCR inhibitors, which was ascertained by determining the efficiency of amplification of a segment of the 16S rDNA. PCR amplification could be readily achieved with DNA extracted by each of these four methods, but efficiency of amplification tended to be higher with DNA from two of the methods (one extracted with PVPP and one with bead mill homogenization). CONCLUSIONS: Four extraction methods proved to be significantly superior in quantity of DNA extracted from luminal samples. Of these four, no strong inhibitors of PCR amplification were detected in any of the extracted DNA. However, the efficiency of amplification tended to be lower in DNA samples from two of the methods, suggesting the presence of low levels of PCR inhibitors. SIGNIFICANCE AND IMPACT OF THE STUDY: Results of this study provide a basis for choosing which DNA extraction procedures are most effective for use with samples of pig lumen.

Interaction of fumonisin B(1) and aflatoxin B(1) in a short-term carcinogenesis model in rat liver.

The co-existence of the fumonisin and aflatoxin mycotoxins in corn merited studies to investigate their possible synergistic toxicological and carcinogenic effects. When utilising a short-term carcinogenesis model in rat liver, both the compounds exhibited slow cancer initiating potency as monitored by the induction of foci and nodules stained positively for the placental form of gluthatione-S-transferase (GSTP(+)). However, when rats were treated in a sequential manner with AFB(1) and FB(1) the number and size of GSTP(+) lesions significantly increased as compared to the separate treatments. Histopathological analyses indicated that the individual treatments showed far less toxic effects, including occasional hepatocytes with dysplastic nuclei, oval cell proliferation and, in the case of FB(1), a few apoptotic bodies in the central vein regions. The sequential treatment regimen induced numerous foci and dysplastic hepatocyte nodules, and with oval cells extending from the periportal regions into the centrilobular regions. This would imply that, in addition to the cancer promoting activity of FB(1) of AFB(1)-initiated hepatocytes, the AFB(1) pre-treatment enhanced the FB(1) initiating potency, presumably by rendering the liver more susceptible to the toxic effects of FB(1). The co-occurrence of AFB(1) and FB(1) in corn consumed as a staple diet could pose an increased risk and should be included in establishing risk assessment parameters in humans.

Toxicological effects in rats chronically fed low dietary levels of fumonisin B(1).

The toxicity of low dietary levels of fumonisin B(1) (FB(1)), i.e. 1, 10 and 25 mg FB(1)/kg diet, were monitored in rats over a period of 24 months. No effects on the body weight gain and feed intake profiles were noticed, while the relative liver weight was significantly (P<0.05) reduced in the FB(1)-treated rats. Mild toxic effects, including single cell necrosis (apoptosis), proliferation of bile duct epithelial cells (DEC), and early signs of fibrosis, bile duct hyperplasia and in one case, adenofibrosis, were noticed in the liver of the rats fed the highest (25 mg/FB(1)/kg diet) dietary level. A significant (P<0.05) increase in the level of oxidative damage was also noticed in the liver of the rats of high dosage dietary group. The toxic effects were less severe in the 10 mg FB(1)/kg dietary group, whilst only a few ground glass foci were observed in the 1 mg FB(1)/kg dietary group. Hepatocyte nodules, staining positively for glutathione-S-transferase (placental form, PGST), were observed macroscopically in the 25 mg FB(1)/kg treated group and to a lesser extent in the 10 mg FB(1)/kg treated rats. The most prominent toxic lesions by FB(1) (10 and 25 mg FB(1)/kg dietary groups) in the kidneys were restricted to the tubular epithelium manifesting as granular cast, necrosis, apoptosis, calcification and the presence of regenerative foci in the proximal convoluted tubules. The existence of a cytotoxic/proliferative threshold with respect to cancer induction by FB(1) in rat liver became apparent, with a dietary level of <10-mg FB(1)/kg diet as a no effect threshold for the induction of hepatocyte nodules.

The cancer-promoting potential of fumonisin B1 in rat liver using diethylnitrosamine as a cancer initiator.

The cancer-promoting potential of fumonisin B1 (FB1) was investigated by feeding different dietary levels (10, 50, 100, 250, 500 mg FB1/kg) to diethynitrosamine (DEN)-initiated rats for 21 days. Dietary levels containing 50 mg FB1/kg and higher, markedly increased the number and size of the placental form of glutathione-S-transferase-positive (GSTP+) foci in the liver of the rats. The cancer-promoting activity of FB1 was associated with an inhibitory effect on partial hepatectomy (PH)-induced regenerative hepatocyte proliferation, as the incorporation of 3H-labelled thymidine was significantly (P < 0.05) reduced by those FB1-containing diets that exhibited cancer promotion. In vitro studies on the mitogenic activity of epidermal growth factor (EGF) in primary rat hepatocytes further supported the in vivo data in that FB1, similar to other cancer promoters such as phenobarbital and 2-acetylaminofluorene (2-AAF), alters growth stimulatory responses in primary hepatocytes. No significant (P > 0.05) changes in the sphinganine/sphingosine (Sa/So) ratio were observed in the liver of the rats fed the lowest FB1-containing diet (50 mg FB1/kg diet) that effected cancer promotion. The present study indicated that FB1 exhibited cancer-promoting activity in the absence of adverse hepatotoxic effects and at dietary levels that failed to effect cancer initiation.

Hepatotoxicity and -carcinogenicity of the fumonisins in rats. A review regarding mechanistic implications for establishing risk in humans.

Cancer induction by the non-genotoxic mycotoxin, fumonisin B1, has been investigated by studying the mechanisms involved during cancer initiation and promotion in rat liver. Cancer initiation is effected through a toxic-proliferative response while the inhibitory effect on hepatocyte cell proliferation appears to be a key aspect determining cancer promotion. Dose-response effects of the fumonisins on the induction of early neoplastic lesions in both long- and short-term animal experiments have been established. The biphasic response of FB1 on hepatocyte proliferation will be discussed in relation to the known mechanisms of cancer induction by the genotoxic hepatocarcinogens. Recent investigations regarding the effect of the fumonisins on lipid biosynthesis and its inhibitory effect on hepatocyte growth stimulatory responses in vitro will be highlighted. Integration of our current knowledge regarding the carcinogenic potential of the fumonisins in setting a realistic and applicable risk assessment model for this non-genotoxic carcinogen will finally be addressed.

Fed before diethylnitrosamine, Fusarium moniliforme and F proliferatum mycotoxins alter the persistence of placental glutathione S-transferase-positive hepatocytes in rats.

Groups of 5-w-old F344/N female rats were fed a semipurified diet for 13 w with or without 20 mg fumonisin B1/kg provided from an aqueous extract of Fusarium moniliforme-corn culture. After 1 w, a single dose of 30 mg diethylnitrosamine (DEN)/kg was given orally. Twelve weeks later, the presence of placental glutathione S-transferase-positive (PGST-[+]) hepatocytes were immunohistochemically quantified. Rats given DEN and the FB1-containing diet for 1 or 13 w developed 4-fold more PGST-[+] hepatocytes than rats given DEN alone. In a second study, male and female F344/N rats were fed 20 mg purified FB1/kg diet or F proliferatum-corn culture material containing 20 mg FB1/kg diet for 1 w before DEN treatment. One week after DEN treatment, male rats fed the F proliferatum-corn culture material had significantly fewer PGST-[+] hepatocytes than those fed DEN with or without purified FB1. At 9 w after DEN treatment, PGST-[+] cells in female rats given DEN and fed F proliferatum-corn culture material were more persistent than in rats given DEN alone. Males given DEN and fed FB1 or F proliferatum culture material had significantly fewer PGST-[+] hepatocytes than males given DEN alone. These results suggest that F moniliforme and F proliferatum components are cocarcinogens in females. In males, however, FB1 and unidentified F proliferatum components reduced the persistence of DEN-initiated preneoplastic hepatocytes.

Fumonisin B1 is fetotoxic in rats.

Groups of 5-6 pregnant F344/N rats were dosed (po) from d 8 to 12 of gestation with 30 or 60 mg purified fumonisin B1 (FB1)/kg body weight, or with a fat-soluble extract of Fusarium proliferatum/corn culture derived from an amount of corn culture that would provide approximately 60 mg FB1/kg. Control rats were dosed with water or corn oil. Food intake was monitored daily during dosing. Fetal bone development was examined after staining with alizarin red, whereas internal organ development was examined in hematoxylin and eosin-stained tissue sections. Although group differences in maternal body weight were not statistically significant, weight was 6% less in dams dosed with 60 mg FB1/kg compared with the control group (p < 0.12). Relative litter weight was significantly suppressed by 60 mg FB1/kg. Ossification of the sternebrae and vertebral bodies was significantly impaired by FB1 treatment. Litters from mothers treated with a fat-soluble extract of F proliferatum/corn culture did not have suppression of weight or impairment of bone development. Fumonisin B1 is fetotoxic to rats by suppressing growth and fetal bone development.

Fusarium proliferatum-fermented corn stimulates development of placental glutathione S-transferase-positive altered hepatic foci in female rats.

Groups of 8 6-w-old female Sprague-Dawley rats were initiated with 30 mg diethylnitrosamine (DEN)/kg. Control and initiated groups were fed a semipurified diet or diets supplemented with Fusarium proliferatum-contaminated corn to contain 20 or 50 mg fumonisin B1 (FB1)/kg. Histochemical staining for gamma-glutamyltransferase (GGT) and immunochemical staining for placental glutathione S-transferase (PGST), markers of altered hepatic foci (AHF), were performed on serial frozen hepatic sections. Gamma-glutamyltransferase -(+) AHF were not found in any group. Dosing with DEN significantly increased the number of PGST-(+) hepatocytes compared to the uninitiated groups. Groups fed F proliferatum-containing diets also had a significantly increased number of PGST-(+) AHF compared with those fed no F proliferatum. The volume percentage of liver occupied by PGST-(+) foci was significantly greater in the groups treated with DEN or F proliferatum. The number of PGST-(+) AHF/liver in the groups given DEN was also significantly greater than in the uninitiated groups. Fusarium proliferatum exposure also significantly increased the number of PGST-(+) AHF/liver. Feeding F proliferatum containing 20 mg FB1/kg promoted the development of DEN-initiated AHF in rats. Placental glutathione S-transferase was a more useful marker than GGT in detecting AHF produced by small amounts of F proliferatum mycotoxins fed after initiating dosing with DEN.