Takumi Kanemaru

Pathogenicity and virulence of Rhodococcus equi in foals following intratracheal challenge

Twelve foals, between 27 and 83 days old, were infected with 2 strains of Rhodococcus equi by intratracheal administration. Ten of the 12 foals were inoculated with 10(4)-10(10) colony forming units (cfu) of ATCC 33701 strain. The other 2 foals were inoculated with 10(9) cfu of a plasmid-cured derivative of the ATCC 33701 strain (ATCC 33701P-). All of the 10 foals challenged with the ATCC 33701 strain showed clinical signs of pulmonary disease within 5-13 days, such as gross lesions associated with acute bronchopneumonia and microscopic lesions associated with granulomatous pneumonia. The two foals challenged with the ATCC 33701P- strain showed neither clinical signs of disease nor gross lesions. Apparently, when lacking plasmid, the virulent Rhodococcus equi lost its pathogenicity.

Detection of Babesia caballi infection by enzyme-linked immunosorbent assay using recombinant 48-kDa merozoite rhoptry protein

The 48-kDa Babesia caballi merozoite rhoptry protein was expressed using a pGEX4T expression vector in Escherichia coli as glutathione S-transferase fusion protein (GST-BC48), and the expressed GST-BC48 was used in an ELISA to detect specific antibodies in serum samples. No cross-reaction was observed with sera from horses experimentally infected with Babesia equi. GST-BC48 ELISA was a highly sensitive and specific test when compared with the CFT. A total of 209 horse sera obtained from Central Mongolia were examined with the GST-BC48 ELISA and 46.4% (97/209) were found to be seropositive for B. caballi, suggesting that the GST-BC48 ELISA can be successfully used for both quarantine and epidemiological studies.

Clinical and virological observations on swine experimentally infected with Getah virus

The pathogenicity of Getah virus for swine was examined. All 8 pigs (4 adults and 4 piglets) inoculated with Strains MIP-99 and MI-110 developed pyrexia ranging from 39.4 to 40.7 degrees C and anorexia. Mild depression and diarrhea were observed in 2 of the 4 piglets. These clinical signs were transient. Viremia occurred 1-2 days post-inoculation (p.i.) and the maximum titer was 10(3.0) TCID50 0.1 ml-1. The virus was recovered from a piglet autopsied on Day 3 p.i. from spleen and various lymph nodes. The maximum titer of virus (10(3.75) TCID50 0.1 g-1) was detected in the inguinal lymph node. Seroconversion was demonstrated in all the pigs on Day 6 p.i. These results suggest that Getah virus is mildly pathogenic for swine, which may play a role as an amplifying host in nature.

Morphological changes of the aorta and pulmonary artery in thoroughbred racehorses

The aorta and extrapulmonary artery were examined pathologically in 33 thoroughbred racehorses ranging in age from 1 to 5 years. Many of the great vessels of these horses exhibited degenerative or sclerotic changes in the media with neither lipidosis nor deposits of cholesterol. The severe lesions were predominantly observed at the bifurcation of the pulmonary artery. The severity of the lesions in both the aorta and pulmonary artery appeared to be associated with the racing career of the racehorse rather than with increasing age. Histopathogenetically, the medial changes in the great vessels were probably initiated by ischaemia and/or oedema in their walls due to pathological disturbances of the vasa vasorum and their vasomotor nerves. Whether hypervitaminosis D plays a role in the condition in racehorses needs to be determined by further study.

Protein characterization of Babesia equi piroplasms isolated from infected horse erythrocytes.

Proteins ofBabesia equi piroplasms were characterized. The piroplasms ofB. equi were purified by lysis of infected horse erythrocytes with N2 gas cavitation followed by separation in Percoll density-gradient centrifugation. The relative molecular weights (Mr) of major proteins separated by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 18, 28, 30, 41, 43, 54, 66.5, and 96 kDa. Immunoblot analysis using serum from an experimentally infected horse revealed six immunodominant proteins of 15, 18, 28, 30, 41, and 96 kDa. Two immunodominant proteins of 18 and 28 kDa were membrane-bound proteins as revealed by Triton X-114 phase partitioning.

Ultrastructure and immuno-cytochemistry of BHK-21 cells infected with a modified Bucyrus strain of equine arteritis virus

SummaryMorphogenesis of a modified Bucyrus strain of equine arteritis virus (EAV) in BHK-21 cells was studied. Bacillary tubules were first detected in the cytoplasm 8 h after infection, and mature virions 79 to 122 nm in diameter, 101 nm on average, were mostly observed in the cisternae of the rough endoplasmic reticulum (RER) at 12 h or later. They had isometrical cores and morphological subunits in the outer layer. Budding occurred from the RER and the outer nuclear membrane, but not from the cell surface. Structural linkage was detected between the tubule and the virus core. Aberrant strands were occasionally demonstrated within the nucleus 12 h after infection, and immunofluorescence and immunogold labeling revealed viral antigen also in the nucleus.

Complement-dependent serum neutralization with virulent and avirulent Bucyrus strains of equine arteritis virus.

Virulent and avirulent strains of Bucyrus equine arteritis virus (EAV) were used to raise antiserum in horses. Serum neutralization (SN) tests were performed with and without the addition of guinea pig complement. The inclusion of ten percent guinea pig serum in the virus suspension was sufficient for optimal enhancement of SN titres at any immune stages after immunization. Immune serum prepared against avirulent virus reacted only with homologous virus and there was no complement enhancement. Immune sera raised against live or inactivated virulent virus neutralized both virulent and avirulent virus. The reaction with virulent virus demonstrated complement enhancement. There was also moderate potentiation in the presence of complement when serum raised against inactivated virulent virus reacted with avirulent virus.

Transplacental infection in mice inoculated with Getah virus

Transplacental transmission was demonstrated in pregnant mice subcutaneously inoculated with Getah virus. Viremia was shown in the infected dams, and high-titered virus was detected in the placenta and later in the fetus, suggesting virus invasion of the fetus through hematogenous infection of the placenta. High-titered virus was shown in the fetal brain and muscle and in the brain of the young dying soon after birth. Intrauterine infection resulted in a reduction of the litter size, number of young born alive and survival rate to 1 week of age. These results were further corroborated by necropsy performed several days after virus inoculation. The stage of gestation at the time of virus inoculation greatly influenced these results. Dams inoculated at 12 days of gestation delivered all dead babies, whereas virus inoculation at 5 days of gestation had no effect on the number of young born alive. The dams inoculated at 8 days of gestation had reduced litter sizes and those inoculated at 16 days of gestation produced slightly fewer live babies. Gestational stage at the time of virus inoculation also influenced viral growth in fetuses and placentas. The infection rate was low in dams inoculated at 5 days of gestation, high in dams inoculated at 8 or 16 days of gestation and 100% in dams inoculated at 12 days of gestation. High-titered virus was shown in placentas and fetuses of the dams inoculated at 8, 12 or 16 days of gestation. These results suggest that Getah virus may readily cross the placental barrier through hematogenous infection of the placenta in mice.

Venereal infection of mares by equine arteritis virus and use of killed vaccine against the infection

Venereal infection with equine arteritis virus (EAV) was established in each of seven mares by inoculation via the cervix with 20 ml of viral suspension (> or = 8 x 10(6) plaque-forming units; PFU), following treatment with prostaglandin and oestradiol. A dose of < or = 8 x 10(5) PFU produced infection in only five of eight mares. Serum neutralizing antibody developed in mares manifesting clinical signs of equine viral arteritis (EVA), and a weak antibody was detectable in one apparently healthy mare inoculated with 8 x 10(5) PFU. Virus isolation was demonstrated not only in the buffy coat but also in nasal swabs of infected mares. EAV was isolated frequently from the body tissues of the mares (killed 10 to 34 days post-inoculation) up to day 12, but rarely from the reproductive tissues later than day 12. The virus persisted longest in the splenic and deep inguinal lymph nodes, followed by the spleen and internal iliac lymph nodes. Four mares immunized with a killed vaccine for EVA showed no clinical disease after venereal challenge with EAV; the virus was recovered from the buffy coat of three mares and from the nasal swab of one of them, but not from the remaining animal.

Molecular characterization of a 30 KdaBabesia equi merozoite surface protein

Equine babesiosis caused by Babesia equi and B. caballi is present in most Asian countries including China. At present, Japan is free from these parasites, but risks of their invasion are unavoidable in the international trade of horses. Intraerythrocytic B. equi merozoites express an immunodominant 30 kDa surface protein (EMA-1, Kapprmeyer et al., 1993), which is genetically related to the immunodominant piroplasm surface proteins expressed by Theileria species, including Theileria sergenti/buffeli/orientalis, T. mutans and T. parva. We have amplified the coding region of p30 from genomic DNAs of B. equi stocks and isolates from USA, Russia and South Africa by PCR and the PCR products were subjected to RFLP analysis. We could differentiate at least four allelic forms of the p30 gene by digestion with Xhol and Stul. Further cloning and sequencing analysis revealed that p30 alleles of four B. equi stocks (2 sequences from Florida stocks, USA, and one each of Russian and South African stocks) showed about 80,010 homology at the nucleotide level. Current diagnosis is done by CF test in Japan, but development of alternative methods is required to improve the sensitivity and specificity of the serological diagnosis. In addition, production of CF antigens which are prepared from infected horse blood is expensive and laborious. We are currently developing a recombinant antigenbased ELISA. The bacterial recombinant products of p30 was reactive with sera from B. equi-infected horses by ELISA. Epitope analysis using overlapping synthetic peptides revealed the sequence recognized by horse antibodies to be localized in the central region of p30. Further evaluation of the use of the recombinant p30 as ELISA diagnostic antigen is necessary because the regions bearing the epitope of 4 aUelic forms of p30 contain amino acid variation.