Chiharu Suto

Ehrlichia muris sp. nov., identified on the basis of 16S rRNA base sequences and serological, morphological, and biological characteristics.

The 16S rRNA gene of a new infectious agent, strain AS145T (T = type strain), which was isolated from a wild mouse in Japan, was amplified by using the PCR. The amplimers were directly sequenced by dideoxynucleotide methods with Taq DNA polymerase. Sequence comparisons with other members of the tribe Ehrlichieae and related species revealed that the infectious agent isolated from the mouse is a new species of the genus Ehrlichia that is most closely related to Ehrlichia chaffeensis (level of sequence similarity, 97.9%), an agent of human ehrlichiosis in the United States. This result was consistent with the results of an immunoblot analysis performed with immune sera against different ehrlichiosis agents. On the basis of these findings and other morphological, biological, and serological characteristics of the organism, we propose that ehrlichiae with these properties belong to a new species, Ehrlichia muris.

Impaired Antigen Specific Responses and Enhanced Polyclonal Stimulation in Mice Infected with Ehrlichia muris

The immune status of BALB/c mice infected by intraperitoneal inoculation with Ehrlichia muris was examined. The level of E. muris infection in both peritoneal cavity and spleen was greatest at day 10 postinoculation (PI). Thereafter, the infection level was dramatically reduced while the organism persisted for up to 400 days PI. The greatest intraperitoneal infiltration of leukocytes, splenomegaly, and leukocytosis were observed on days 10, 15, and 20 PI, respectively. Infected mice developed marked hypergammaglobulinemia of IgG and IgM that peaked at day 20 PI; however, IgA plummeted at day 15 PI. Of IgG, G2a and G3 increased while G1 and G2b remained constant. Despite hypergammaglobulinemia, both IgG and IgM antibody titers against E. muris were very low throughout the 30‐day study. Antibody development and plaque‐forming cells against sheep red blood cells (SRBC) were abolished when the antigen was inoculated on day 10 PI. IgM antibody development against SRBC was more severely inhibited than IgG antibody development. However, when mice were immunized with SRBC prior to E. muris infection, antibody development against SRBC was not reduced. Delayed type hypersensitivity reaction to dinitrofluorobenzene was also maximally inhibited when the antigen was administered on day 10 PI. The IFN‐γ level in the blood was maximal at day 10 PI. These results indicate that although the vigorous polyclonal activation and protective IFN‐γ responses occurred by day 10 PI—which cleared most of the ehrlichial infection—antigen‐specific immune stimulation was impaired primarily at the level of antigen‐priming at peak parasitemia.

Immunoglobulins Specific to Mosquito Salivary Gland Proteins in the Sera of Persons with Common or Hypersensitive Reactions to Mosquito Bites

Using the immunoblot technique, we analyzed the quality and quantity of IgG, IgG4, and IgE specific to mosquito salivary gland (hereafter abbreviate as SG) components of Aedes albopictus in the sera of volunteers with common reactions and of 3 patients with severe reactions.

A new virus isolated from the cockroach, Periplaneta fuliginosa (Serville).

Despite the remarkable progress in insect virology, we have not known of any virus isolated from cockroaches. In this paper, isolation of a parvovirid virus from the smoky-brown cockroach, Periplaneta fuliginosa (Serville), and some properties of the isolate will be reported. In 1975-76, while we were examining the spontaneous hindgut ulcer of the cockroach, we noticed unusually high mortalities of nymphs and adults among colonies of P. fuliginosa that had been collected in Nagoya district and reared in our laboratory. Before death, hindlegs of the sick cockroaches were paralysed and their movement became incoordinated. At autopsy, the abdomen appeared. swollen with enlarged fat body colored milk-white, which differedfrom the brownish white ones observed in healthy cockroaches. More than half of these dead cockroaches developed the hindgut ulcer (7). Light microscopy of the dead cockroaches exhibited no parasitic causality. However, electron microscopy of negatively stained smears prepared from macerated tissue of the dead cockroaches disclosed two kinds of virus-like particles with diameters of approximately 10 nm and 20 nm, respectively. Therefore, isolation of viral agents from the sick cockroaches was attempted. The sick cockroaches were homogenized in PBS (0.15 M NaCl, M/100 phosphate buffer, pH 7.2) containing streptomycin (100 ƒÊg/ml) and penicillin (500 u/ml), and centrifuged twice at 10,000 •~ g at 4 C for 20 min. The supernatant (Sup-l) was inoculated into the hemocoel of twelve healthy adults. About 20 days after the inoculation most of them died showing the same symptoms as described above. The Sup-l was passed through a 450 nm Millipore filter, and the filtrate was subjected to serial passage through healthy cockroaches. After the fifth passage it was determined that the filtrate was as potent as the Sup-l in the infectivity to the cockroach. The agents contained in the filtrate could pass through membrane filters of 220, 100, and 50 nm porosity. Electron microscopy of thin sections prepared from the infected cockroaches revealed that many virus-like particles with a diameter of about 20 nm were accumulated in the nuclei and sometimes arranged in crystalline array in the cytoplasm of the fat body-, muscle-, and pericardial cells (Fig. 1). Judging from the size and shape of these particles as well as from their arrangement in the infected cells,

Hemocytes of Pomacea canaliculata: I. Reversible aggregation induced by Ca2+

Using a platelet aggregometer, factors involved in hemocyte aggregation of a prosobranch snail Pomacea canaliculata were investigated. Aggregation-inducing tests revealed that extracellular Ca2+ is required to provoke the cellular response which is reversible. The aggregation-dispersion response can be induced by adding Ca2+ and EDTA alternately. Aggregation was inhibited by a calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) in a dose-dependent manner, and by a protein kinase-C inhibitor 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine dihydrochloride (H-7). Thus, calmodulin and protein kinase-C may contribute to the calcium-mediated aggregation of hemocytes, which resembles a phenomenon that occurs in vertebrate leucocytes. Unlike the aggregation of mammalian leucocytes, no stimulus other than Ca2+ is required to induce the response of Pomacea hemocytes. Thus, this system may serve as a simple model for analysing cellular aggregation responses.