W. A. Malmquist

East coast fever: Cultivationin vitro of bovine spleen cell lines infected and transformed byTheileria parva

SummaryThree spleen cell lines derived from 3 calves experimentally infected withTheileria parva have been established. After an apparent disappearance of parasitized cells from the cultures there was a resurgence of infected lymphoblasts in 5 to 6 weeks. The infected lymphoblasts can be readily subcultured without feeder layers in a medium consisting of Eagle’s Minimal Essential Medium and 20 per cent foetal calf serum. The plating of infected cells was greatly enhanced by implanting the cells on preformed monolayers. No evidence was obtained by the methods employed which might indicate the existence of an extracellular form of the parasite capable of reinfecting new cellsin vitro.RésuméTrois lignées de cellules de rate issues de trois veaux expérimentalement infectés parTheileria parva ont été établies. Après une apparente disparition des cellules parasitées dans les cultures, il y avait une résurgence de lymphoblastes infectés au bout de 5 à 6 semaines.Ceux-ci pouvaient être mis en subculture aisément sans couche de soutien dans un milieu constitué du M.E.M. de Eagle, enrichi par 20 p. 100 de sérum de foetus de veau. L’étalement des cellules infectées était grandement facilité par leur implantation sur des couches préformées. Par les méthodes employées, on obtient aucune preuve de l’existence d’une forme extracellulaire du parasite, capable de réinfecterin vitro des cellules neuves.SumarioSe han establecido 3 lineas celulares de bazo derivadas de tres terneros experimentalmente infectados conTheileria parva.Despuéś de una aparente desaparición de las células parasitadas de los cultivos, hubo una resurgencia de linfoblastos infectados en 5–6 semanas. Los linfoblastos infectados pueden ser subcultivados facilmente sin capas alimentadoras en un medio consistente de Eagles, medio mínimo esencial enriquecido con 20 por ciento de suero bovino fetal. El plaqueo de las células infectadas fue grandemente estimulado mediante la implantación de das células en monocapas previamente formadas. Con los métodos utilizados no se obtuvo evidencia para indicar la existencia de una forma extra celular de el parásito capaz de reinfectar neuvas célulasen vitro.

Production of equine infectious anemia antigen in a persistently infected cell line

Equine infectious anemia (EIA) virus replicated and produced antigen in cultures of horse leukocytes, equine embryonic spleen cells, and a line of equine dermal (ED) cells. A CPE was produced only in horse leukocyte cultures. Presence of the virus in spleen and dermal cell cultures was monitored frequently by inoculating fluids into leukocyte cultures where a CPE was produced or demonstrating specific EIA antigen by double immunodiffusion. Culture fluids from persistently infected equine dermal cells proved the most practicable source of antigen for quantity production purposes. A fraction precipitated from fluids with 8 per cent polyethylene glycol (MW 6000) was treated with diethyl ether to release the antigen. A semi-quantitative evaluation of antigen yield was performed using radial immunodiffusion.

Soluble antigen of bovine viral diarrhea virus

Complement-fixing (CF) soluble antigen (SA) was detectable intra-cellularly prior to the appearance of infectious NADL-MD bovine viral diarrhea (BVD) virus during synthesis in roller flask cultures of bovine embryonic kidney cells. The release of infective virus into the extracellular fluid was concomitant with the release of SA. The SA was separated from the infective virus by sedimentation in a sucrose density gradient and by DEAE-cellulose chromatography. The SA was heat labile at 56° C, but stable in buffers at pH 5, 7, and 9 at 37° C. The SA was irreversibly precipitated in buffers at pH of 3 or below. Trypsin and α-chymotrypsin completely inactivated the SA, whereas ribonuclease and deoxyribonuclease had no detectable effect on the CF activity. There was no apparent CF or neutralizing relationship between the SA and infectious NADL-MD BVD virus and arbovirus group B and lymphocytic choriomeningitis virus antisera.

Morphological variation of a syncytial virus from lymphosarcomatous and apparently normal cattle

Double-membrane enveloped, endoplasmic reticulum-associated (ERA) viral particles occurred in the cytoplasm of bovine embryonic splenic cell cultures previously infected with bovine syncytial virus (BSV) that had been isolated from lymphosarcomatous and apparently normal cattle. Previously, BSV was seen only in budding forms at the plasma membrane. It is proposed that the double membrane enveloped, ERA viral-like particle is an aberrant form of BSV because: (1) the ERA viral particle had a nucleocapsid morphologically similar to BSV; (2) it was associated with BSV immunofluorescence; (3) it occurred in the same cells and cell cultures as BSV; and (4) it was not demonstrated in cell cultures in the absence of BSV.

Antigenic relatedness of equine herpes virus types 1 and 3.

SummaryAntiserums prepared in specific pathogen free (SPF) ponies were used in direct and indirect immunofluorescence, immunodiffusion, complement fixation and serum neutralization procedures to study the interrelationships of the three types of equine herpes viruses (EHV-1, EHV-2, and EHV-3). Equine cell cultures infected with each type virus fluoresced when stained with homologous conjugated anti-serum. In reciprocal tests EHV-1 and EHV-3 cross-fluoresced, but EHV-2 did not cross-fluoresce. Non-infected cell cultures did not fluoresce when stained with the 3 conjugates. EHV-1 and EHV-3 cross-fluoresced in reciprocal indirect fluorescent antibody tests, but no cross-fluorescence was shown with EHV-2. Antigens representing each type of equine herpes virus reacted with their homologous antiserum in the immunodiffusion test. In reciprocal tests, a common line(s) of identity formed with EHV-1 and EHV-3; however, the precipitin line(s) was not common with EHV-2. Antigen prepared from noninfected embryonic mule skin (EMS) cell cultures did not react with any of the antiserums. Specific complement-fixing antibodies were present in antiserums when tested against their homologous antigens. In reciprocal complement fixation tests EHV-1 and EHV-3 cross-reacted, but no cross-reactivity was shown with EHV-2. Significant levels of neutralizing antibody were in an antiserum when tested against homologous virus, whereas cross-neutralization was not detectable in reciprocal tests. These studies indicate that each type of equine herpes virus contains specific antigenic components, and EHV-1 and EHV-3 share a common antigen(s) that is not shared with EHV-2.