Nobuaki Shimada

Fate of maize intrinsic and recombinant genes in calves fed genetically modified maize Bt11.

The presence of maize intrinsic and recombinant cry1Ab genes in the gastrointestinal (GI) contents, peripheral blood mononuclear cells (PBMC), and visceral organs of calves fed genetically modified Bt11 maize was examined by PCR in a subchronic 90-day performance study. Samples were collected from six Japanese Black/Holstein calves fed Bt11 maize and from six calves fed non-Bt maize. Fragments of maize zein (Ze1), invertase, chloroplast, and cry1Ab were detected inconsistently in the rumen fluid and rectal contents 5 and 18 h after feeding. The chloroplast DNA fragments of ribulose-1,5-bisphosphate carboxylase/oxygenase and tRNA were detected inconsistently in the PBMC, the visceral organs, and the longissimus muscle, while the cry1Ab gene was never detected in PBMC or in the visceral organs. These results suggest that feed-derived maize DNA was mostly degraded in the GI tract but that fragmented DNA was detectable in the GI contents as a possible source of transfer to calf tissues. These results also suggest that the recombinant cry1Ab genes were not transferred to the PBMC and tissues of calves fed Bt11 maize.

BACILLUS THURINGIENSIS INSECTICIDAL CRY1AB TOXIN DOES NOT AFFECT THE MEMBRANE INTEGRITY OF THE MAMMALIAN INTESTINAL EPITHELIAL CELLS: AN IN VITRO STUDY

Abstract The mammalian intestinal epithelium has been found, based on in vivo experiments, to be resistant to insecticidal Cry toxins, which are derived from Bacillus thuringiensis and fatally damage insect midgut cells. Thus, the toxins are commonly used as a genetic resource in insect-resistant transgenic plants for feed. However, Cry toxins bind to the cellular brush border membrane vesicle (BBMV) of mammalian intestinal cells. In this study, we investigated the affinity of Cry1Ab toxin, a lepidopteran-specific Cry1-type toxin, to the cellular BBMV of two mammalian intestinal cells as well as the effect of the toxin on the membrane potential of three mammalian intestinal cells compared to its effects on the silkworm midgut cell. We found that Cry1Ab toxin did bind to the bovine and porcine BBMV, but far more weakly than it did to the silkworm midgut BBMV. Furthermore, although the silkworm midgut cells developed severe membrane potential changes within 1 h following the toxin treatment at a final concentration of 2 μg/ml, no such membraneous changes were observed on the bovine, porcine, and human intestinal cells. The present in vitro results suggest that, although Cry1Ab toxin may bind weakly or nonspecifically to certain BBMV components in the mammalian intestinal cell, it does not damage the cells membrane integrity, thus exerting no subsequent adverse effects on the cell.

Toxicological evaluation and bioaccumulation potential of lolitrem B, endophyte mycotoxin in Japanese black steers

Lolitrem B, a causative toxin for ryegrass staggers, is produced by Neotyphodium lolii infecting perennial ryegrass (Lolium perenne). Japanese black cattle have been suspected to be more sensitive to lolitrem B than to other strains, and there has been a concern about the public health hazard of eating beef contaminated with lolitrem B. We carried out a feeding experiment to examine the sensitivity of Japanese black cattle to lolitrem B and the residual level of lolitrem B in several animal tissues. Japanese black steers were fed a 0, 500, 750, 1000, 1500 or 2000 µg kg−1 diet of lolitrem B provided by endophyte-infected perennial ryegrass straw for 12 weeks. All six animals in the 2000 µg kg−1 diet group exhibited ryegrass staggers symptoms. Furthermore, two out of three animals in the 1500 µg kg−1 diet group, three out of six animals in the 1000 µg kg−1 diet group and one out of three animals in the 750 µg kg−1 diet group presented clinical signs of ryegrass staggers. These results suggest that a daily intake of 18 µg kg−1 body weight of lolitrem B can produce ryegrass staggers in Japanese black steers. Perirenal fat tissues of the steers from those groups having one or more animals exhibiting ryegrass staggers symptoms contained approximately 150 ng g−1 of lolitrem B, while only small amounts of lolitrem B were detected in muscle, liver and kidney. Because the residual amount of lolitrem B in tissues of Japanese black cattle is small, the exposure to lolitrem B in consumers of the beef is likely to be low.

Genome variability of foot-and-mouth disease virus during the short period of the 2010 epidemic in Japan.

Foot-and-mouth disease virus (FMDV) is highly contagious and has a high mutation rate, leading to extensive genetic variation. To investigate how FMDV genetically evolves over a short period of an epidemic after initial introduction into an FMD-free area, whole L-fragment sequences of 104 FMDVs isolated from the 2010 epidemic in Japan, which continued for less than three months were determined and phylogenetically and comparatively analyzed. Phylogenetic analysis of whole L-fragment sequences showed that these isolates were classified into a single group, indicating that FMDV was introduced into Japan in the epidemic via a single introduction. Nucleotide sequences of 104 virus isolates showed more than 99.56% pairwise identity rates without any genetic deletion or insertion, although no sequences were completely identical with each other. These results indicate that genetic substitutions of FMDV occurred gradually and constantly during the epidemic and generation of an extensive mutant virus could have been prevented by rapid eradication strategy. From comparative analysis of variability of each FMDV protein coding region, VP4 and 2C regions showed the highest average identity rates and invariant rates, and were confirmed as highly conserved. In contrast, the protein coding regions VP2 and VP1 were confirmed to be highly variable regions with the lowest average identity rates and invariant rates, respectively. Our data demonstrate the importance of rapid eradication strategy in an FMD epidemic and provide valuable information on the genome variability of FMDV during the short period of an epidemic.

Experimental infections using the foot-and-mouth disease virus O/JPN/2010 in animals administered a vaccine preserved for emergency use in Japan

The effectiveness of a vaccine preserved for emergency use in Japan was analyzed under experimental conditions using cows and pigs in order to retrospectively evaluate the effectiveness of the emergency vaccination performed in the 2010 epidemic in Japan. Cows and pigs were administered a vaccine preserved for emergency use in Japan at 3 or 30 days before virus infection (dbv) and were subsequently infected with the foot-and-mouth disease virus (FMDV) O/JPN/2010, which was isolated in the 2010 epidemic in Japan. All animals vaccinated at 30 dbv and one of three pigs vaccinated at 3 dbv showed no vesicular lesions during the experimental period. The virus titers and viral RNA loads obtained from clinical samples were lower in the vaccinated cows than in the non-vaccinated cows. The viral excretion periods were shorter in the vaccinated cows than in the non-vaccinated cows. In contrast, in the vaccinated pigs, the virus titers and viral RNA loads obtained from the samples, except for those obtained from sera, were not decreased significantly, and the viral excretion periods were not sufficiently shortened. These results suggest that the vaccine can protect against clinical signs of infection by the FMDV O/JPN/2010 in animals; however, it should be noted that in vaccinated and infected animals, especially pigs, clinical samples, such as saliva and nasal swabs, may contain excreted viruses, even if no clinical signs were exhibited.

Reverse transcription-PCR using a Primer Set Targeting the 3D Region Detects Foot-and-Mouth Disease Virus with High Sensitivity

Because foot-and-mouth disease (FMD) has the potential to spread extensively, methods used for its diagnosis must be rapid and accurate. Therefore, reverse transcription-PCR (RT-PCR) plays an important diagnostic role. Here we designed the primer set FM8/9 to amplify 644 bases of the conserved 3D region of all seven serotypes of FMD virus (FMDV). We compared the performance of RT-PCR assays using FM8/9 with that using the primer set 1F/R targeting the 5’-UTR described in the manual of the World Organization for Animal Health. The detection limits of the RT-PCR assays were determined for 14 strains representing all serotypes. Compared with the sensitivities of the RT-PCR assay using 1F/R, those using FM8/9 were 101-to 104-fold higher for eight strains. To assess the validity of the methods for analyzing clinical samples, sera and saliva samples from pigs and cows infected with FMDV were collected daily and analyzed using the two PCR assays. The FM8/9 assay detected FMDV from all infected pigs and cows for longer times compared with the 1F/R assay, therefore revealing higher sensitivity for the clinical samples. Our results suggest that the FM8/9 RT-PCR assay is highly sensitive and is therefore suitable for the diagnosis of FMD.

Comparative evaluation of two ELISA kits for detecting antibodies to a nonstructural protein of foot-and-mouth disease virus using serum samples collected from naturally and experimentally infected cows

When foot-and-mouth disease (FMD) occurs and a “vaccination-to-live” policy is adopted in a country, the country must perform serological surveillance of a nonstructural protein (NSP) of FMD virus. The NCPanaftosa kit is the only kit for detecting antibodies to NSPs that is officially recognized as the reference regent by the World Organization for Animal Health; however, it is only used in South American countries. In this study, the specificity and sensitivity of the NCPanaftosa kit were compared with those of the PrioCHECK kit sold by an international company. Results in this study suggest that the PrioCHECK kit performs similarly to the NCPanaftosa kit in detecting antibodies to the NSP in the cattle population.

Pathogenesis of the attenuated foot-and-mouth disease virus O/JPN/2000 in experimentally infected pigs

We examined the pathogenesis of the attenuated foot-and-mouth disease virus (FMDV) O/JPN/2000 in pigs. The virus used in this study was passaged three times in primary bovine kidney (BK) cells and once in baby hamster kidney-21 (BHK-21) cells after isolation. A plaque assay demonstrated that this virus exhibited the small plaque (SP) phenotype. There was no clinical or histological evidence of vesicular lesions in pigs intraorally inoculated with 106 50% tissue culture infectious dose (TCID50)/ml of the SP virus (SPV) of FMDV O/JPN/2000. Although fever was detected from 2 or 3 days post inoculation (dpi), there was no other prominent clinical sign up to 6 dpi. Virus shedding from saliva and nasal swab samples was not observed in any pigs inoculated with the SPV of FMDV O/JPN/2000. In the foot, mild lamellar degeneration of prickle cells in the upper layer of the stratum spinosum was histologically observed without development into vesicular or necrotic lesions. Immunohistochemical virus antigen- and terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL)-positive reactions observed in the foot at 1 dpi seemed to disappear after 3 and 6 dpi. Our findings suggest that the SPV of FMDV O/JPN/2000 had low pathogenicity against pigs by intraoral inoculation.

Early pathogenesis of the foot-and-mouth disease virus O/JPN/2010 in experimentally infected pigs

We examined the histological distribution of the lesions and the viral antigen associated with the virus and virus RNA in multisystemic organs in the early stages of foot-and-mouth disease virus (FMDV) O/JPN/2010 infection in pigs. Characteristic lesions commonly observed in pigs with FMD arise following inoculation with 106 tissue culture infectious dose (TCID)50/ml of FMDV O/JPN/2010 in pigs at 3 days post inoculation (dpi) by a natural infectious route. However, none of the six pigs inoculated with 103 TCID50/ml of FMDV O/JPN/2010 showed any evidence of infection up to 6 dpi. Immunohistochemical detection for the FMDV antigen and terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) showed that FMDV predominantly infected prickle cells in the stratum spinosum in the tongue, coronet and bulb of the heel, and caused these infected cells to undergo cell death by apoptosis. However, there was no evidence that FMDV O/JPN/2010 infected epithelial/epidermal basal cells in the basal layer. Epithelial lesions with viral antigen in the tongue were distributed in the dorsal surface but not in the papillae, corpus linguae or inferior surface of the tongue. Non-suppurative myocarditis and epithelial lesions in the esophagus with FMDV antigen were observed in all three pigs examined at 3 dpi.