Polymerase chain reaction (PCR) systems were developed for detection of 3 pathogenic bovine mycoplasmas, Mycoplasma alkalescens (Mak), M. bovigenitalium (Mbg) and M. bovirhinis (Mbr). The primers were selected from the sequences of the 16S rRNA from each of the mycoplasmas. Neither the Mbg-PCR system nor the Mbr-PCR system showed cross-amplification among 24 bovine, caprine-ovine and human mycoplasma species (including Acholeplasma and Ureaplasma species) tested. The Mak-PCR system showed cross-amplification with M. canadens ATCC29418T (T=type strain). The sensitivity of each PCR system to detect the proper mycoplasma was 103 colony forming units (CFU) when a pure culture was tested, and 2 × 10 3 CFU when the mycoplasma culture was spiked into a calf nasal swab sample. The target mycoplasmas in a clinical nasal swab sample that could be detected by the direct plate method could also be detected by these PCR systems.
A female 3-year-old Pug showing frequent epileptic fits during 8-months was shown on magnetic resonance imaging (MRI) to have dilated cerebral ventricles and inflammatory lesions in the whole cortex of the bilateral cerebral hemispheres as well as the lower left parietal and the middle right temporal lobes. Histopathology of the corresponding regions revealed meningoencepalitis characterized by wide distribution of degenerated neurons with glial satellitosis and neuronophagia and prominent perivascular cuffing with lymphoid cells.
The mammary tumor is one of the popular neoplastic diseases in female dogs. In the present study, the expression of canine c-kit proto-oncogene in mammary tumor specimens was investigated to evaluate its potential usefulness as a tumor marker. By comparing the homology among the nucleotide sequences reported for human, mouse, rat and feline c-kit c-DNA, a pair of primers was synthesized for the reverse transcriptase-polymerase chain reaction (RT-PCR) method. The RT-PCR product of canine spleen total RNA was shown to have 756 bp in size and to be highly homologous to the corresponding sequences reported for the mammalian species. The expression of c-kit transcript was detected in 11 mammary tumors of different histopathology including adenocarcinomas, benign and malignant mixed tumors. The level of the transcription in adenocarcinomas was significantly higher than those in malignant mixed tumors. Fifteen canine tumor specimens originated from various tissues were also tested for their c-kit transcript. In all of the mastocytoma samples examined, high expression of the mRNA was detected. Of other 12 tumors, only low level of RT-PCR products were detected in 5 samples, whereas no apparent amplification was observed in 7 tumors. These results indicate that the high expression of c-kit transcript is helpful for the diagnosis of canine mammary tumors.
A nuclear magnetic resonance (NMR) spectrometer equipped with a magnet producing a high and extremely uniform magnetic field (7.05 T) was combined with a strong field gradient coil (3.5 mT/cm) and applied to MR microimaging of the mouse brain to visualize its topographical structure. Since the proton-density-weighted condition (long repetition time (TR) and short echo time (TE); TR/TE=3,000 ms/10.4 ms) was found to be the most suitable for imaging the mouse brain, mid-sagittal and coronal sections in 1-mm- or 0.3-mm-thick slices were imaged according to the multislice spin echo sequence with 2 or 8 acquisitions, a 2 kHz pulse width and a 256 × 256 data matrix. As expected, the resolution of MR microimaging was comparable to that of the histological sections. The white matter especially, could be distinguished from the gray matter in some regions of the brain. Coronal sections of the brain also showed that the hippocampal CA1-CA3 regions were distinguishable from the other regions. The results suggested that the present MR microimaging technique might be a useful tool for the study of topological anatomy and submicroscopic research using brains of small laboratory animals.
Fluorodeoxyuridine, an inhibitor of thymidylate synthetase, is known to induce chromosomal fragile sites. The drug treatment may cause deprivation of intracellular thymidine nucleotide pool followed by a serious imbalance of deoxynucleotide pool. Though the stress is probably related to the induction of folate-sensitive fragile sites, the exact machanism is still to be investigated. The present study has been carried out to test the possibility that the fragile sites are originated, at least in part, from incorpolated uracil residues. The incorpolated uracil residue can be detected by a novel assay for abasic sites after treatment with uracil-DNA N-glycosylase (UDG). About 2.7 abasic sites per 104 nucleotides were detected in the DNA extracted from feline fibroblasts after the treatment with FUdR and caffeine. By digesting the DNA with UDG prior to the assay, significant increase in the number of abasic sites were observed. These results indicate that the large amount of uracil residues are present in the feline fibroblast DNA under the condition which induces chromosomal fragile sites.
Diploid and triploid specimens of Japanese and Korean Fasciola sp. showed abnormality in their spermatogenesis. Live germ cells obtained from the testes were observed under a differential interference contrast microscope. In the stages from spermatogonium to spermatid, the cells combined together at the central cytoplasmic bridge during a series of divisions. One spermatogonium becomes a cell group of 8 primary spermatocytes through 3 mitoses. Until the primary spermatocyte stage, cells are divided in a uniform manner. In most of the diploid specimens, the primary spermatocytes are irregularly divided into non-uniform secondary spermatocytes, however, some specimens perform a regular division. In the majority of triploid flukes, the primary spermatocytes are divided in a regular pattern, but some of the specimens perform an irregular division. The non-uniform spermatids do not perform a spermiogenesis. In the diploid specimens, no spermatozoa were found that were produced by spermiogenesis. Whereas in the triploid specimens, some spermatids distributed uniformly on the surface went through a spermiogenesis. We observed some moving spermatozoa in one triploid specimen. The spermatozoa possibly retain their normal reproductive function.
Exploring the antigenic and genetic diversities of Babesia ovata, we obtained several field isolates from grazing cattle in the Okushiri island, Japan. Parasite isolation was greatly facilitated by using bovine red blood cell-substituted SCID mice (Bo-RBC-SCID mice), into which the blood samples of the cattle were inoculated. Isolates from different individuals within a herd of cattle were compared in immunoblot analysis with an anti-B. ovata serum and also in Southern blot analysis with a probe for the small subunit ribosomal RNA gene. In both analyses, the isolates exhibited banding patterns that were significantly different from each other. We were also able to obtain a series of parasite isolates from a single cow in different seasons of a nine months period, including winter when active vector ticks were not in the field environment. Different seasonal isolates showed different banding patterns in both immunoblot and Southern blot analyses. By contrast, these analyses detected little difference among the parasites that had been passed various times in Bo-RBC-SCID mice, where no specific immune responses should be generated. These results indicate that individual animals within a herd of cattle were infected with antigenically and genetically diversified populations of B. ovata, and that the parasites could persistently infect a single animal with dynamic change in their predominant subpopulations.
The histological and ultrastructural characteristics of an adenocarcinoma of the lung are described in an about 16-year-old female Steller sea lion with a 1.5 month history of cough and anorexia. The animal had multiple neoplastic nodules in the lungs and diaphragmatic pleura. The bronchial and mediastinal lymph nodes were replaced by neoplastic tissue, and there were several metastatic lesions in the liver and spleen. The lung tumor was characterized by accumulations of encapsulated lesions with central necrosis, and the neoplastic cells showing a papillary growth pattern produced small amounts of mucin. Ultrastructurally, some cells contained basal bodies, and cilia were rarely seen. This neoplasm was considered to be of ciliated bronchial or bronchiolar epithelium origin.
To develop a rapid and specific method to detect and/or identify enterohemorrhagic Escherichia coli O157:H7, two mouse monoclonal antibodies (MAbs) were prepared. Specificities of these two MAbs (1D9 and 3E8) were determined by flow cytometry method (FCM). MAbs 3E8 and 1D9 were found to react with E. coli O157:H7, Citrobacter freundii and Salmonella group N (O:30), but not with Escherichia hermannii. With a mixture containing strains of E. coli O157:H7 and E. coli O6:H1, two different peaks appeared in FCM with MAbs, whereas a single peak appeared with polyclonal rabbit antiserum. From these findings, FCM with MAb is suggested to be a rapid, specific, and useful method to detect and identify strain(s) of E. coli O157:H7 in food ingredients.
The Listeria monocytogenes-carrying rates were 100% for listeriosis patients and 1.3% for healthy humans. The L. monocytogenes contamination rates for retail sliced beef (34.2%) and pork (36.4%) were significantly higher (p<0.05) than those for cattle (2.0%) and pigs (0.8%) and for cattle (4.9%) and swine (7.4%) carcasses. The percentages of serotypes 1/2a, 1/2b and 4b which are most dominant in human patients were high in isolates from fresh (90.0%) and processed (100%) fish and shellfish and imported natural cheese (96.7%).
Flow cytometric method (FCM) with fluorescent-labeled anti-CPE antibody was applied to develop a rapid, specific, and convenient method to detect enterotoxin (CPE) exposed on the surface of spores of Clostridium perfringens. The results obtained indicate that FCM can specifically detect CPE exposed on C. perfringens spores for a short time. Thus, FCM is found to be a rapid, specific, and convenient assay method for detection of CPE exposed on C. perfringens spores, suggesting that it will be hopefully useful to diagnose food poisoning caused by C. perfringens.
Three dogs were evaluated in our study using magnetic resonance imaging (MRI) to reveal the anatomical deformity and the degree of the lesion of cauda equina. In all dogs, MRI revealed soft tissue, such as cauda equina, epidural fat, and intervertebral disc, at the lumbosacral region clearly without contrast medium. Our results suggest that MRI has some advantages in evaluating cauda equina syndrome in dogs.
To investigate the influence of maternal antibody to porcine reproductive and respiratory syndrome (PRRS) virus infection, the following examination was done using conventional and SPF pigs. Ten 17-day-old conventional pigs with maternal antibody against PRRS virus and 6 44-day-old SPF pigs seronegative were inoculated intranasally with 105.0 TCID50 of PRRS virus. Two conventional and 4 SPF pigs were served as non-inoculated control. In conventional pigs, coughing and febrile response were observed after inoculation, and mean rate of weight gain reduced. One of the inoculated conventional pigs died on post-inoculation-day (PID) 28 and Haemophilus parasuis was isolated from the lung. Although febrile response was also observed in the inoculated SPF pigs, reduction in weight gain rate was not recognized. Virus was isolated from all the sera of inoculated conventional and SPF pigs except one conventional pig between PID 7 and 49, and between PID 7 and 28, respectively. Onset of viremia in the several conventional pigs delayed. Virus was isolated from the tissues of the 5 conventional pigs on PID 65 and from the tissues of the dead pig. On the other hand, virus was not isolated from the tissues of non-inoculated conventional pigs, and inoculated and non-inoculated SPF pigs. At the virus inoculation, antibodies by the indirect fluorescent antibody (IFA) assay against PRRS virus were detected in the sera of conventional pigs with antibody titers of 1:20. Antibody titers gradually decreased after inoculation and rose from PID 21 or 28 and were between 1:160 and 1:640 on PID 63. Virus neutralization (VN) antibody titers were 1:2 or 1:4 at the inoculation and gradually decreased. Apparent rise in VN antibody titer was not observed after the inoculation. In the sera of control pigs, both antibody titers gradually decreased and did not rise. In the sera of the SPF pigs, antibodies by the IFA assay were first detected on PID 7 or 14. The titers of antibodies rose and reached their maximum with 1:320 to 1:2,560 on PID 21 to 35. VN antibodies were first detected on PID 42 to 56 and thereafter, the titers ranged between 1:1 to 1:4. Control SPF pigs were free of antibody throughout the examination. Antigenic variability was not recognized between the inoculated and recovered viruses by the VN test. The prolonged duration of viremia and virus isolation from the tissues on PID 65 in conventional pigs with low maternal antibody might support the present of antibody-dependent enhancement activity of PRRS virus infection.
To get basic information to control persistent virus infection among domestic animals by cytokines, the antiviral activity of four natural human cytokines against bovine viral diarrhea virus (BVDV) was evaluated. Normal bovine peripheral blood mononuclear leukocytes (PBML) and fetal bovine muscular cells (FBMC) were treated with varying doses of human interferon (IFN)-α, IFN-γ, tumor necrosis factor (TNF)-α and TNF-β. The antiviral activity in treated cells was measured by the titration of virus infectivity in comparison with non-treated controls. IFN-α significantly suppressed virus growth in both PBML and FBMC. The growth of two cytopathogenic and two noncytopathogenic strains was suppressed in the presence of more than 103 u/ml of IFN-α. Addition of either TNF-α or TNF-β to IFN-α did not potentiate the suppressive effect. IFN-α also suppressed the replication of BVDV in PBML from cattle persistently infected with BVDV.
Equine infectious anemia virus (EIAV) core proteins (Gag and p26) obtained from a baculovirus expression system were used in agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA) antigens to test seventy-six horse sera. Those sera showed false-positive reaction in AGID test using Nisseiken antigen. However, none of them showed false-positive reaction with both of the expressed antigens. The 76 horse sera were also tested by ELISA. The sera gave a high background in ELISA using Nisseiken antigen. Gag and p26 reacted strongly against positive sera from horses immunized with Nisseiken antigen.