The application of tissue culture methods to the study of the vesicular diseases of animals
Sellers, R. F.
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The application of tissue cultures in the study of vesicular diseases of animals has been illustrated by reference to pig kidney tissue cultures and the virus of foot-and-mouth disease.Investigation ^ras carried cut on the most suitable method of producing monolayer tissue cultures from pig kidneys by the trypsinisution technique and it was found possible to produce monolayers in suitable quality and quantity. The monolayers were used for titration of the virus of foot-and-mouth disease and study was made of the factors affecting the formation of plaques on the monolayers. It was found that the number, size and shape of the plaques formed were affected by the state of the cells, the nature of the overlay, the conditions of culture incubation arid the strain of virus used. By adherence to a standard techhique and by comparison of titrea obtained from assay of a standard virus on different sets of cultures it was possible to obtain accurate and useful results.Strains of the virus of foot-and-mouth disease, which had been passaged through mice and chick embryos did not multiply or cause eytopathogenic effect in calf and ox kidney monolayers to the same extent as strains passagsci in cattle or in kidney cultures and It was possible to correlate their behaviour with non-infactivity tests in cattle. All strains of the virus tested grew in pig kidney mono¬ layers and caused cytopathogenic effect but gave rise to plaques of varying size and shape. The plaques arising from the different strains were compared and it was found that the relative number of the different plaque sizes varied with the origin of the virus and that the plaque population changed on peerage in different cultures or animals.The adsorption and multiplication of the virus was followed in pig kidney cell euepensions and monolayers, It was found difficult to infect all the cells after 30 minutes absorption with a high input of virus. The latent period was found to be 2.5 - 3 hours for cell suspensions and monolayers and at the end of that time there was a rapid virus increase. The titra of the culture reached a peak of 10(6.7)-10(7.5) pfu/ml at 6 - 8 hours after which it remained constant for a period up to 18 hours and then fell gradually,Virus grown in pig kidney monolayers was inactivated by adsorption on aluminium hydroxide and treatment with 0,03$ formalin. The vaccine thus produced protected guinea pigs agniast challenge with liomologous culture virus arid stimulated the production of neutralising antibody. Investigatione were made into the optimum time of virus harvest. It was found that vaccine made from virus harvested 24 - 42 hours after infection of the monolaye -B, when the cytopathogmic effect was complete, gave the best protection to guinea pigs on challenge- The siae of challenge dose which distinguished between different levels of protection,was found to be 104 guinea pig IX^q, and & tentative scheme for a potency teat was put forward baaed on the dilution of vaccine protecting 5$* the guinea pigs from generalisation after challenge with 104ITjq of virus.Pig kidney monolayer tissue cultures were also used &3 a means of differential diagnosis of vesicular diseases and for the assay of neutralising antibody.These findings ware discussed in relation to diagnosis, identification and classification of outbreaks of foot-and-mouth disease in the field and to the selection of virus strains for preparation of attenuated and inactivated vaccines. The method of virus titration by the plaque technique was discussed with regard to vaccine production, fundamental virus research, plaque analysis of strains and genetic studies. The importance of virus growth studies and production of high tltre virus stocks was emphasised in relation to virus-cell interactions and in relation to provision of suitable material for biophysical, biochemical and biological investigations on the virus.