Morphometric method for evaluating the neurovirulence of live poliomyelitis vaccine*

Bulletin of the World Health Organization, 57 (5): 815-817 (1979) Morphometric method for evaluating the neurovirulence of live poliomyelitis vaccine * A. V. TYUFANOV1 & S. B. STEFANOV2 A method for measuring morphological changes caused by residual neurovirulence in live poliomyelitis vaccine is described. The entire area of 16 standard parts of the central nervous system is measured directly under the microscope using a morphometric eyepiece grid. The method can be used to supplement the conventional system of scoring morphological lesions. In our opinion, there is a need for a standardized method of evaluating the residual neurovirulence of both commercial poliomyelitis vaccine lots and candidate vaccine strains, since the currently used methods for the assessment of central nervous system (CNS) lesions using a semiquantitative no- tation (scores or crosses) are not sufficiently objec- tive. It would thus be advisable to supplement them with strictly quantitative morphometry. The present paper is aimed at demonstrating the possibilities of classical morphometry in the de- scription of pathological findings. This study has been carried out on two vaccine lots that had been thoroughly tested previously. MATERIALS AND METHODS The type 3 vaccine lots No. 102 and 106 were prepared from attenuated poliovirus strains with infectious titres of 10718 and 10725 PFU/ml. Rhesus monkeys (Macaca mulatta) were inoculated intra- spinally with 0.1-ml volumes of the vaccine diluted to 100, 10-1, 10-2, 10-3, and 10-4. For each dilution, 3-6 animals possessing no specific antibody were used. When sacrificed 28 days later, the monkeys showed no clinical signs of the disease. Histological preparations were made from 16 standard areas of * From the Institute of Poliomyelitis and Virus Encephalitides of the USSR Academy of Medical Sciences, 142782 Moscow, USSR. ' Chief of the Laboratory. 2 Senior Research Worker. the brain and spinal cord of each animal: motor cortex (1 section), thalamus (1 section), midbrain (1 section), medulla (2 sections at different levels), segments 2-7 of the cervical cord (1 section of each), segments 2-5 of the lumbar cord (1 section of each). Measurement of the percentage of affected squares (PAS) Each of the 16 preparations from each animal was examined under a microscope at a magnification of 7 x 9 with a measuring grid inserted in the eyepiece. The grid consisted of 16 squares of similar size (1). The preparation was moved in such a way that the border of the part of the field already measured was immediately adjacent to the border of the next part to be measured. In each field of vision, only those squares were counted in which any lesions specific of poliomyelitis were seen (neuronophagy nodules, cytolysis, gliosis, vascular infiltrates, etc). Simul- taneously, the number of measured fields of vision were counted. Multiplying this number by 16 gave the number of grid squares in the area of the section examined. The number of grid squares showing pathological changes was divided by the total number of grid squares covering the entire area of a given section, and expressed as the "percentage of affected squares" (PAS). In the absence of pathological changes, the PAS was equal to 0. In order to determine the reproducibility of the results obtained, the measurements were repeated 5 times with the same section. 3859 - 815- A. V. TYUFANOV & S. B. STEFANOV Calculated values based on the PAS The mean PAS value for each vaccine dilution was calculated by dividing the total PAS for all animals receiving this dilution by the total number of animals (Table 1). The number of squares showing pathological changes induced by this dilution was taken as 100% and divided by the number of squares showing pathological changes in each part of the CNS; in this way the contribution of each part of the CNS to the total number of lesions was ex- pressed as a percentage (Table 2). Table 1. Percentage of affected squares (PAS) in the CNS of monkeys inoculated with live poliomyelitis vaccine Vaccine lot and dilution Calculation Lot 102 Lot 106 10-1 10-2 10-3 10-4 100 10-1 10-2 10-3 10-4 PAS in 0.2 0 0.7 0 0.5 0.7 0 0 0 individual monkeys 2.5 0.6 0.6 0 4.0 0.4 2.5 0 0 inoculated with dilution 0 3.2 1.1 0 0 0.4 0.7 0 0 shown 0.8 3.3 0.4 2.6 0.8 0 0 0 4.0 8.2 1.8 6.0 8.6 0 0 1.7 1.0 1.1 0.2 0 0 Mean PAS per dilution 1.5 3.0 0.97 1.6 1.5 0.5 2.0 0 0 Mean PAS per vaccine 1.72 0.78 Table 2. Percentage contribution of each part of the CNS to the total number of lesions in monkeys inocu- lated with live poliomyelitis vaccine Vaccine lot and dilution Part of CNS in which PAS Lot 102 Lot 106 counted 10-1 10-2 10-3 10-4 100 10-1 10-2 10-3 10-4 Brain 5.4 15.0 14.8 15.1 3.4 5.0 2.5 0 0 Cervical cord 21.0 42 6.5 46 23 1 21 0 0 Lumbar cord 74 43 79 39 74 94 75 0 0 RESULTS None of the monkeys had any clinical signs of poliomyelitis. Histological lesions were very mild or completely absent in many animals. PAS values varied from 0 to 8.6% (Table 1). In all dilutions, vaccine No. 102 produced no lesions in some animals and severe lesions in others. Vaccine No. 106 generally produced milder lesions and dilutions 10-3 and 10-4 no lesions at all. The highest PAS values were found in the lumbar cord, with lower values in the cervical cord, and the lowest values in the brain. The reproducibility of the results was found to lie within acceptable limits. DISCUSSION The morphometric assessment of lesions in the CNS gave detailed data for each animal and for each part of the brain and spinal cord. The smallest contribution to the total PAS value was made by the brain, although the brain sections have the largest area. In the cervical cord, the PAS was greater than in the brain, but lower than in the lumbar cord; this is in agreement with classical views regarding the neurotropism of poliomyelitis virus. PAS values in individual animals varied from 0 to 8.6 %. As none of the 47 monkeys showed any clinical signs typical of poliomyelitis, it must be admitted that none of these PAS values corresponds to pathological changes sufficiently severe to cause clinical symp- toms. Of course, the PAS value does not reflect all the qualitative features of the lesions caused by vaccines nor does it show how these lesions differ from those caused by virulent viruses. It is sufficient to state here that the method used may be extended to permit the quantitative examination of lesions of different types: the morphometric grid can measure not only general histological changes in organs and tissues, but also subtle cytological changes. By developing the proposed method, and using the same measuring procedures, it is possible to obtain differential PAS values, instead of the total PAS, for any abnormal morphological process de- tectable by visual examination. The PAS may be. counted using any eyepiece measuring grid. The grid we have used, consisting of 16 squares, has proved to be the least tiring and most convenient for the study of rare phenomena in a large area of normal tissue. We counted the affected area by squares and not by the point-counting method used in classical morphometry. This was necessary because we had to measure both the area of the entire section and the area of the affected squares, which was very small. The proposed method at present takes a little longer than the traditional semiquantitative method of scoring. However, it is simple to apply and if used to supplement the conventional method would allow a comparison of the results of the large-scale ap- plication of both methods. 816 NEUROVIRULENCE OF POLIOMYELITIS VACCINE 817 RtSUM8 METHODE MORPHOMEBTRIQUE DE CONTROLE DE LA NEUROVIRULENCE DU VACCIN ANTIPOLIOMYEL1TIQUE VIVANT Les auteurs ont applique une methode strictement mor- phometrique a l'evaluation de la neurovirulence residuelle du vaccin antipoliomy6litique couramment effectuee au moyen de m6thodes semi-quantitatives. Sur chaque singe rh6sus vaccine en vue de l'etude, on a preleve et examine au microscope 28 jours apres la vaccination 16 6chantillons des diverses zones types du systeme nerveux cer6bro-spinal. La visee microscopique a eu lieu a travers une grille comportant 16 carres egaux et, dans chaque portion correspondante de la preparation, progressivement deplacee, le nombre des carres dans lesquels une alteration pathologique apparaissait etait note; la relation de ce nombre au nombre total de carr6s represent6s par l'echantillon constituait le pourcentage des carres affectes (PAS); ces operations de mesure ont ete repetees 5 fois pour chaque echantillon. Pour chacun des deux lots de vaccin prepares a partir d'une souche attenu6e mais presentant un degre different d'infectiosite, quatre ou cinq dilutions diverses ont e utilisees. Les dilutions 10-3 et 10-4 du vaccin N° 106 n'ont pas cause de lesions. Les valeurs de PAS les plus elevees ont ete enregistrees dans la corde lombaire et les plus basses dans le cerveau, ce qui correspond a l'opinion d6ja etablie sur le neurotropisme du virus poliomyelitique. Ces valeurs variaient, selon l'animal et la dilution de l'un ou l'autre vaccin, de 0 a 8,6%, mais aucun signe clinique de poliomyelite n'a ete releve. La m6thode employee permet de mesurer de tres faibles modifications cytologiques. REFERENCES 1. STEFANOV, S. B. Ocular insert for complete stereologic examinations. Cytology, 17: 1439-1441 (1974). 2. TYUFANOV, A. V. Pathomorphological study of the central nervous system of monkeys inoculated intra- cerebrally with Sabin attenuated poliomyelitis virus. In: International Symposium on Neurovirulence, Munich, 1965, vol. 2, pp. 287-290.