Hiroyuki Hirumi

Continuous cultivation of Trypanosoma brucei blood stream forms in a medium containing a low concentration of serum protein without feeder cell layers.

Blood stream forms (BSF) of Trypanosoma brucei brucei GUT at 3.1 were propagated in vitro in the absence of feeder layer cells at 37 C, using a modified Iscoves medium (HMI-18). The medium was supplemented with 0.05 mM bathocuproine sulfonate, 1.5 mM L-cysteine, 1 mM hypoxanthine, 0.2 mM 2-mercaptoethanol, 1 mM sodium pyruvate. 0.16 mM thymidine, and 20% (v/v) Serum Plus (SP) (Hazleton Biologics, Lenexa, Kansas). The latter contained a low level of serum proteins (13 micrograms/ml). Each primary culture was initiated by placing 3.5-4 x 10(6) BSFs isolated from infected mice in a flask containing 5 ml of the medium (HMI-9) supplemented with 10% fetal bovine serum (FBS) and 10% SP. The cultures were maintained by replacing the medium every 24 hr for 5-7 days. During this period, many BSFs died. However, from day 4 onward, long slender BSFs increased in number. On days 5-7, trypanosome suspensions were pooled and cell debris was removed by means of diethylaminoethyl cellulose (DE52) column chromatography. Blood stream forms then were collected by centrifugation, resuspended in fresh medium at 7-9 x 10(5)/ml, and transferred to new flasks. Subcultures were maintained by readjusting the BSF density to 7-9 x 10(5)/ml every 24 hr. Concentrations of FBS were reduced gradually at 5-7-day intervals by alternating the amounts of FBS and SP in HMI-9 with 5% FBS and 15% SP, with 2% FBS and 18% SP, and finally with 20% SP (HMI-18). By this method, 2-3 x 10(6) VSFs/ml were obtained consistently every 24 hr. for more than 80 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Mycoplasma diseases of plants and insects.

Publisher Summary This chapter explains the Mycoplasma diseases of plants and insects. It discusses the nomenclature and classification of the order Mycoplasmatales. The nonviral nature of the numerous graft-transmissible, arthropod-borne disease agents of plants is established. Electron microscopy reveals the characteristic morphology of the presumptive micro-organisms observed in diseased phloem cells and in various vectors, but absent from healthy phloem or from non-infected vectors. Plants can recover from yellows-type diseases when treated with sulfa drugs that definitely do not affect mycoplasmas, and they sometimes recover when treated solely with fertilizers. The chapter discusses on only specific criteria, accepted by microbiologists, should be used in the determination of the actual etiology of the presumptive mycoplasma diseases. These criteria require the cultivation of the isolated agents on solid media with the production of mycoplasma colonies, the reinoculation of susceptible healthy plants with the cultured mycoplasmas, and the subsequent reproduction of the original disease.

Insect Tissue Culture: Use of Blastokinetic Stage of Leafhopper Embryo

To find the proper material for cultivation in vitro of cells of leafhopper vectors of plant viruses, embryonic tissues of the six-spotted aster leaf-hopper (Macrosteles fascifrons) were tested during early developmental stages, during blastokinetic movement, and in late developmental stages. Growing cells were only obtained from the stage of blastokinetic movement. This stage can be determined visually in leafhopper eggs.

Ultrastructure of the feeding apparatus of the nematode Trichodorus christiei

The ultrastructure of the feeding apparatus, as well as the stoma and the pharynx, of Trichodorus christiei Allen, a vector of tobacco rattle virus, was studied by electron microscopy. The stomatal opening is triradiate. The stomatal lumen, which extends to the pharynx, is lined by the outer body wall cuticle. The feeding apparatus consists of two parts, an outer spear and a fine inner spear. The anterior half of the outer part, appearing in the stomatal and the anterior pharyngeal lumens, consists of solid cuticle in its apical region. The posterior half fuses to the dorsal cuticle of the posterior pharynx. Ih the middle region of the outer spear, the dorsal side of the spear has an oblique opening. Through this opening, the anterior half of the inner spear is inserted into the anterior outer spear. The pharynx is mostly composed of muscle cells containing both thin and thick myofilaments, approximately 80 A and 220 A in diameter, respectively. The pharyngeal muscles attach to the dorsal surface of the posterior half of the outer spear, thus enabling its protrusion. The food can pass continuously from the stomatal opening to the esophagus through the stomatal and the pharyngeal lumens.

Electron microscopic evidence for wound-tumor virus accumulation in various organs of an inefficient leafhopper vector, Agalliopsis novella

Abstract Various organs were excised from the leafhopper Agalliopsis novella infected with wound-tumor virus (WTV) and were studied by electron microscopy in an attempt to determine the ability of the inefficient vector to support virus multiplication. Small accumulations of WTV virions were detected in the midgut epithelium, muscles, blood cells, fat bodies, tracheoblasts, and the ventral ganglia. Many small virus microcrystals were found in the ventral ganglia. The findings provide the first direct evidence for the presence of a plant-pathogenic virus inside of the nervous system of an insect. Thus, the virus has been demonstrated to cause an infection in its arthropod host. Lack of sizable accumulations of WTV not only in the salivary gland but also in other organs of A. novella might account for the inefficiency of this species as a vector of WTV.

Metacyclic form-specific variable surface glycoprotein-encoding genes of Trypanosoma (Nannomonas) congolense.

A complementary DNA expression library in phage lambda gt11 was synthesized using mRNA from in vitro-produced metacyclic forms of a clone of Trypanosoma (Nannomonas) congolense. The unamplified library was screened with antiserum from a goat immune to infection with metacyclic (m)-forms of T. congolense ILRAD Nannomonas antigen repertoire 2(ILNaR2). Of the 100 antiserum-reactive phage clones identified, 22 were analyzed further: 21 of the clones contained overlapping portions of a single transcript, while one other contained a different transcript. Northern blot analyses indicated that the sequences contained in the clones were transcribed only by m-forms of ILNaR2. Immunological and sequence analyses indicated that the two different cloned sequences encode m-form-specific variable surface glycoproteins.

Phytoferritin accumulations in leaves of diseased coconut palms

SummaryA study by electron microscopy of coconut palm (Cocos nucifera L.) leaves from trees infected by the Cape St. Paul wilt (Kaincopé) disease of West Africa was carried out. Samples were obtained during the dry season (Dec.–Jan.) and fixed immediately upon removal from the trees in buffered glutaraldehyde. Further processing for electron microscopy was carried out within a week. No virus particles, mycoplasma-like organisms (MLO), fungi, or bacteria were detected in thin sections. Crystalline or paracrystalline accumulations of electron-opaque granules, approximately 5.5–6 nm in diameter, were observed in disintegrated chloroplasts of mesophyll cells. Based upon their morphological characteristics, formation of the slightly curved, “fingerprint” arrays or linear rows running parallel, and the visualization of electron-opaque cores in unstained preparations, the granules were identified as phytoferritin particles.