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Potomac horse fever, caused by Ehrlichia risticii, is an important disease of equines. The major features of the disease are fever, leukopenia, and diarrhea. The organism has been detected from the blood mononuclear cells of infected horses, but its presence in the feces has not been known. A method for immunomagnetic separation of E. risticii from the feces of infected horses was developed, and the separated organisms were detected by PCR. Coating immunomagnetic beads (Dynabeads) with a 1:5 dilution of rabbit anti-E. risticii serum and incubating the Dynabeads with fecal samples for 25 min at room temperature gave optimum results. E. risticii was detected from the feces during the course of diarrhea from two experimentally infected horses. In horse 1, watery diarrhea occurred from days 11 to 16 postinfection (p.i.), after which the feces became soft on day 17 p.i. and then returned to normal. The organisms were first detected from the feces on day 11 p.i., peaked on day 13 p.i., and then gradually decreased until day 16 p.i., after which they became undetectable. In horse 2, first, on day 12 p.i., there was soft feces which continued and progressed to diarrhea on day 17 p.i. The feces became normal after day 18 p.i. The organisms in the feces of this horse were first detected on day 12 p.i. and peaked on day 14 p.i., after which they declined until day 16 p.i. and then became undetectable. In both horses, the number of organisms in the mononuclear cells peaked on days 10 and 11 p.i., respectively, 3 days prior to the respective peaks in the feces. E. risticii was not detected from the plasma samples obtained from these horses. There was a drastic reduction in PCR amplification of E. risticii DNA for fecal samples stored frozen at -20 degrees C in comparison with those stored at 4 degrees C. The presence of the organism in the feces only during the soft- or diarrheal-feces phase supports the previous hypothesis that the diarrhea is caused by the organisms replicating in cells lining the intestines. This rapid simple method of detection of the organisms from the feces will be helpful in diagnostic and epidemiologic studies of Potomac horse fever.

Ehrlichia risticii, the causative agent of Potomac horse fever, has recently been isolated from many vaccinated horses with typical clinical signs of the disease. The heterogeneity of the E. risticii isolates obtained from the vaccinated horses necessitates the identification of the molecular basis of strain variations to elucidate the vaccine failure and to aid in the development of an efficient vaccine against this disease. As an attempt, two major cross-reacting surface antigen genes of 50- and 85-kDa antigens, present separately in strains 25-D (isolated in 1984) and 90-12 (isolated in 1990 from a vaccinated horse), respectively, were cloned and sequenced. A comparative sequence analysis revealed differences and similarities between these two antigens with strain-specific sizes (SSA). The 2.5- and 1.6-kb genes coding for the 85- and 50-kDa proteins, respectively, contained many different tandem repeats. The identical repeat motifs were more frequent in the middle of both genes, but the numbers and positions of the repeats were altogether different in the genes. Many of these direct repeats of both genes had exact sequence homology and coded for the same amino acids. The homology of the 5*- and 3*-flanking regions of the two genes was greater than that of the regions in the central part of the genes. A comparative analysis of the deduced amino acid sequences of these two antigen genes indicated eight common domains, which were designated identical domains. Although the sequence homologies of these identical domains were the same, the positions of the domains in their respective strains were completely different. This finding might be one of the bases of antigenic variation between the strains. In addition, there were a few unique regions in both antigen genes where no sequence homology existed. These specific regions were designated unique domains. The 50-kDa protein had two such unique domains, and the 85-kDa protein had six such unique domains. The presence of such unique domains contributed to the large size variation of these SSA. The cross-reactivity of recombinant proteins confirmed the presence of conserved epitopes between these two antigens. The SSA have been determined to be apparent protective antigens of E. risticii. The obligate intracellular bacterium Ehrlichia risticii is the etiologic agent of Potomac horse fever (PHF). The disease is characterized by fever, leukopenia, depression, anorexia, and profuse diarrhea and affects horses of all ages, with a fatality rate as high as 20%. The mode of transmission of this disease remains unknown.

Ehrlichia risticii is the causative agent of Potomac horse fever (PHF), an acute infectious disease of horses. In the last few years, there have been several reports of PHF cases occurring even in vaccinated horses. We isolated a new strain of E. risticii (90-12 strain) from a vaccinated horse suffering from clinical PHF. The major pathogenic, immunologic, and molecular differences between the 90-12 strain and the 25-D stain, which was originally isolated during the outbreaks in 1984, were studied. The 90-12 strain was more pathogenic for mice and horses compared with the 25-D strain. In enzyme-linked immunosorbent assay and immunofluorescence assay with mouse and horse antisera of both the strains, two- to fourfold differences were observed between (immunoblot) with mouse and horse antisera and also with the recombinant clone-specific antibodies. Though several antigens were similar in both the strains, there were significant differences between them in the 110-, 85-, 70-, 51-, and 33-kDa antigens. The 85-kDa antigen was present only in the 90-12 strain but cross-reacted with a 50-kDa antigen of the 25-D strain. The 51-kDa antigens of both strains had different migration patterns, Southern blot hybridization of the genome from both the strains with DNA probes made from the 51-, 55-, and clones for both the strains, whereas the probe of the 85-kDa clone showed a completely different pattern. The 16S rRNA gene sequences from the two strains were identical. Neither strain replicated in gamma interferon-treated mouse peritoneal macrophages. In in vitro neutralization assay, sera from the 25-D strain-infected horse neutralized the homologous strain but did not neutralize the 90-12 strain, whereas sera from the 90-12 strain-infected horse neutralized both the strains. In mouse protection experiments, there was complete homologous protection. But in cross-protection, mice immunized with the 25-D strain were only partially protected against challenge with the 90-12 strain, whereas mice immunized with the 90-12 strain were completely protected against the 25-D strain challenge. These results clearly indicate that there are major differences between the 90-12 and 25-D strains which may have implications regarding the vaccine failure for PHF and the development of an efficient vaccine.

DNA:DNA hybridization studies of the homothallic species ofNeurospora showed that the repeated DNA sequences provided no means of distinction among them. Hybridization with nonrepeated DNA sequences, however, showed that theN. terricola species was quite unlike the others. These studies suggest that heterothallism might have evolved from homothallism inNeurospora.

Studies were conducted with chicken tracheas infected in vivo and in vitro with infectious bronchitis virus. For vivo studies, chickens were infected intratracheally with the virus. At intervals, they were sacrificed, tracheal rings were cut in 1-1.5 mm widths, washed, examined in the light microscope for ciliary movement and processed for scanning electron microscopy (SEM). For in vitro studies, tracheal rings of similar dimension were planted in tissue culture media and were infected with the virus. At intervals the tracheal rings were examined in the light microscope for ciliary movement and processed for SEM.

DUTTA, S. K., SHEIKH, I. & CHOPPALA, J. 1976. Morphological variation in the genus Neurospora: ascospore and DNA studies. Trans. Amer. Micros. Soc., 95: 462-469. The genus Neurospora shows remarkable morphological variations based on mating type compatibility and cellular variations caused by mutations. Scanning electron microscopical and cytological studies of ascospore development were carried out on three homothallic (8-ascospore ascus) species, N. africana, N. dodgei, and N. lineolata, and one pseudohomothallic (4-ascospore ascus) species, N. tetrasperma. Although N. tetrasperma ascospores were the largest in size quantitatively, the highest yield of pure ascospores was obtained from N. africana culture, and the shape of ascospores of all species studied were elliptical, though those of N. tetrasperma were more elongated. N. africana ascospores matured within 15-20 days, whereas ascospores of other species matured after 21 days when grown on agar medium. Pictures were taken by SEM to study patterns of wall sculpturing of ascospores of different species. DNAs isolated from all of these species showed biomodal thermal denaturation curves typical for DNAs of heterothallic species N. crassa; however, minor fractions of ascospore DNA were significantly smaller, quantitatively, in comparison with those of mycelial and conidial DNAs.

Ribosomal RNA genes (rDNAs) of Neurospora crassa contain DNA sequences which code for 17S, 5.8S, and 26S rRNAs, in addition to internal and external spacers. As has been reported for many eukaryotes, the DNA sequences which code for 17S, 5.8S, and 26S rRNAs in Neurospora species are probably conserved while the internal and external spacer regions are probably variable sequences. Extensive electron microscopic studies of 45S precursor rRNA of several cold and warm blooded animals confirm that spacer regions vary extensively from species to species. It was desirable to know whether such differences in rDNA sequences exist between Neurospora species. Any such difference should be detectable using standard procedures for DNA homology studies rDNA sequences were isolated from N. crassa mycelial cells using the procedure described previously. The purified rDNA was /sup 3/H-labeled (by nick translation) and reassociated with total DNA isolated from the heterothallic species N. crassa and from three homothalliospecies: N. dodgei, N. lineolata, and N. africana. In addition, /sup 32/P-labeled total DNA of N. crassa was reannealed with unlabeled bulk DNA from N. crassa, N. dodgei, and N. lineolata.

Be₄B was hot-pressed to theoretical density at 1030 deg C and 3000 psi. It contained 5% impurities, mainly BeO and Be, which accounted for the high density (1.96 vs 1.94 g/cm/sup a/ theoretical). The various phases in its microstructure were analyzed. The average grain diameter was 17 mu m, with a distribution from 5 to 31 mu m. Microhardness was measured to be 1350 kg/mm/sup 2/; the average modulus of rupture and the elastic modulus were 25,608 plus or minus 3,000 and 34.5 x 10/sup 6/ psi, respectively. (DLC)

1. Price, R. J., and K. C. Seager. Evaluation of immunity to fowl pox. 2. Immunization of young chicks with fowl-pox vaccine. Poultry Sci. (35)2, 379-384, 1956. 2. Seager, K. C., and R. J. Price. Evaluation of immunity to fowl pox. 1. Immunization of young chicks with pigeon and fowl-pox vaccines. Poultry Sci. (35)2, 372-379, 1956. 3. Winterfield, R. W., and E. H. Seadale. Newcastle disease immunization studies. 2. The immune response of chickens vaccinated with B-1 Newcastle disease virus administered through the drinking water. Poultry Sci. (36)1, 54-64, 1957.