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Vet Pathol 12 434 445 1975 Age Dependent Resistance to Transmissible Gastroenteritis of Swine 111 Effects of Epithelial Cell Kinetics on Coronavirus Production and on Atrophy of Intestinal Villi H W MOON L J KEMENY G LAMBERT S L STARK and G D BOOTH U S Department of Agriculture Agricultural Research Service National Animal Disease Center Ames Iowa Abstract Coronavirus titers in small intestine degree of villous atrophy and apparent rates of regeneration of intestinal villi were compared in newborn 3 week old and adult pigs for 1 week after they were exposed to the transmissible gastroenteritis virus of swine The response within the newborn group was homogeneous resulting in high virus titers maximal villous atrophy and comparatively slow regeneration In general virus titers were lower villous atrophy was less severe and regeneration more rapid in both older groups than in the newborn pigs However the response varied greatly in the older groups The 3 week old group was divided into two populations The major population had low virus titers and developed partial villous atrophy whereas the minor population had marked villous atrophy and virus titers comparable to those of the newborn pigs These observa tions support the hypothesis that the accelerated replacement of villous epithelium in the small intestine of pigs during the first 3 weeks contributes to the innate age dependent resistance to transmissible gastroenteritis The accelerated replacement of villous epithelial cells in older pigs contributes to resistance in two ways The increased proliferative capac ity of crypt epithelium results in a more rapid regeneration of atrophic villi and the com paratively young villous absorptive cells resulting from accelerated replacement produce less virus per cell than the older ones of the newborn pig Transmissible gastroenteritis of swine is an acute diarrheal disease caused by destruction of absorptive epithelial cells of the intestinal villi by a specific coronavirus The resultant lesion is called villous atrophy Crypt epithelium is not affected directly by the virus and intestinal villi rapidly regenerate in pigs that survive the acute diarrheal phase of the disease 16 Acute viral diseases of the small intestine in several species follow a similar sequence of selective cell destruction leading to variable degrees of villous atrophy and diarrhea followed by rapid regeneration of villi and the return of normal function Epizootic diarrhea of infant mice 11 lethal intestinal virus infec MOON et al 435 tion of mice 3 and diseases caused by a coronavirus 12 and a reovirus like agent ll in young calves follow this pattern as does the human disease caused by Norwalk agent 2 Pigs of all ages are susceptible however the frequency severity and dura tion of diarrhea in newborn pigs is greater than in 3 week old pigs exposed to the same virus 16 The fatality rate is high in newborn pigs approxi mately 100 and low usually less than 5 in those older than 3 weeks Because the signs and lesions result from the loss of villous absorptive cells in the small intestine and because 3 week old pigs normally replace these cells more rapidly than newborn pigs 13 we hypothesized that age dependent resistance was caused by more rapid regeneration of villi in the older pigs In a test of this hypothesis 16 comparison of age groups accord ing to regeneration rate was frustrated by the heterogeneity among 3 week old pigs This age group seemed to show two populations one with and the other without villous atrophy Interpretation further was complicated be cause virus titers were higher in newborns than in 3 week old pigs 17 Therefore our initial hypothesis was modified accelerated replacement of villous epithelium occurring in the small intestine of pigs during the first 3 weeks results in decreased virus production in the small intestine plus a more rapid regeneration of villi contributing to the innate age dependent resistance to transmissible gastroenteritis that develops during this period The major objective of this study was to compare coronavirus titers in small intestine degree of villous atrophy and apparent rates of regeneration in newborn 3 week old and adult pigs for 1 week after exposure to the transmissible gastroenteritis virus Secondary objectives were to determine if in contrast to newborn pigs there are two populations of 3 week old pigs according to villous atrophy and virus titer after exposure and if there are differences in virus titer between jejunum and ileum Materials and Methods Pigs Sixty nine hysterectomy derived colostrum deprived newborn pigs were randomly assigned to two groups One group was designated newborn 31 pigs and the other desig nated 3 week old 38 pigs Ten hysterectomy derived colostrum deprived pigs at least 1 year old were kept in isolation until used Principals and controls were selected randomly from each age group All pigs were raised in the laboratory in isolation to prevent inad vertent exposure to the virus Principals were isolated from controls before exposure Exposure The virus preparation was from the same stock previously used 16 211 Principals in the newborn group had a mean body weight of 1 16 kg at 3 days of age when 436 MOON et al they each were given intragastrically 2 0 ml of a lo dilution in phosphate buffered saline Principals in the 3 week old group had a mean body weight of 2 77 kg at 21 days of age when they each were given 4 76 ml of a lo dilution intragastrically Principals in the adult group had a mean weight of approximately 200 kg when they each were given 20 ml of a 10p dilution orally Four pigs in each of the newborn and 3 week old groups and three pigs in the adult group were unexposed controls Necropsy Randomly selected pigs were killed by parenteral overdose of either sodium pentobarbital or succinyl choline and necropsied at once Three principals from the new born group were killed at each 72 120 or 168 hour interval after exposure Three prin cipals from the 3 week old group were killed at each 12 hour interval from 24 to 120 h and at each 24 hour interval from 120 to 168 h Principals from the adult group were killed at intervals from 24 to 168 h Some of the pigs that died were examined in the same manner as those that were killed However data from those two groups were separated Data from pigs that died were not used in the figures in this report Histologic Examination Segments from four sites equidistant along the entire course of jejunum and ileum in each pig were removed at necropsy fixed in formalin embedded in paraffin cut into sections 7 pm thick stained with hematoxylin and eosin and examined with a light microscope equipped with an ocular micrometer Five well oriented villi from each of the four sites in each pig were measured and the results combined into a single value for mean villous length for each pig Age time interval and exposure status of the pigs were not known by the examiner Virus Titration The five separate parts of small intestine remaining after removal of segments for histologic examination were labeled from one to five These parts were homo genized individually to make a 10 suspension in phosphate buffered saline and the virus titer of each was determined I71 by a plaque assay in tissue culture using an agar overlay system Titer was expressed as the number of plaque forming units per gram of homogenized intestine In addition mesenteric lymph nodes from exposed pigs in the 3 week old age group were examined for coronavirus Part of mesenteric lymph node taken at necropsy from each pig was teased free of connective tissue finely minced in phosphate buffered saline filtered through a Millipore pad 0 22 jm and inoculated into an established line of swine testis cells in Earle s liquid medium with Eagle s additives After 1 week s incubation at 37 C under 5 CO cultures with characteristic cytopathology were recorded as con taining coronavirus Results Controls in all three age groups remained clinically normal Twenty six of the 27 newborn principals had diarrhea by day 3 after inoculation and 25 of the pigs in this group died by day 5 In comparison only two of 21 3 week old and one of five adult pigs had diarrhea by day 3 and none of the pigs in these two groups died Virus titer did not vary consistently from site to site in the intestine Pigs with high titers tended to have high titers in all five parts of the intestine For Transmissible Gastroenteritis 1 100 900 700 431 I rn 0 0 A of488 0 8 3 ho IIIIII1 24 48 72 96 120 144 168 Hours post exposure 500 1 5 0 0 8 3001 Y I I I I UI 0 B 100 s 0 24 48 72 96 120 144 168 Hours post exposure 2 0 700 0 6001 0 00 0 8 500 X 4001 e0 X 24 48 72 96 120 144 168 3 Hours post exposure Fig 1 Mean coronavirus titer of jejunum and ileum of pigs in three age groups exposed to coronavirus of transmissible gastroenteritis Each mark represents results from one pig Titer is the number of plaque forming units per gram in a continuous line of swine testes cells in culture W 3 day old group 0 3 week old group A adults Fig 2 Mean length of villi in jejunum and ileum of pigs Principals 0 were exposed to virus when 3 days old controls 0 were the same age but not exposed Each mark represents results from one pig Fig 3 Mean length of villi in jejunum and ileum of pigs Principals were exposed to virus when 3 weeks old controls 0 were the same age but not exposed Each mark represents results from one pig Principals are divided into those more X and less 0 than 10 plaque forming units of virus per gram of small intestine at necropsy 438 MOON et al Table I Coronavirus titers of segments of small intestine from each of three pigs in three different age groups killed 48 1 68 h after exposure to transmissible gastroenteritis virus Intestinal 48 h 72 h 120 h 168 h segment 3 day old group 1 NE NE NE 5x10 5xI0 6 lO 4x10 1x10 1x10 3X1O3 1 10 2 IO 2 NE NE NE 1x107 7x10 7x10 3 10 4x10 1 10 3x10 7 10 2x105 3 NE NE NE 2x10 6x10 5 10 4x10 2x10 2x10 8x103 7x10 3x10 4 NE NE NE 5x10 9 10 3x10 IxlOE 8x10 1 10 4x10 6x10 lxlOE 5 NENE NE 6 10 4x10 3 10 6 lO 4 lO 7 10 1 10 4x10 8 10 3 week old group 1 0 4 10 9x10 0 0 1x10 0 0 3 10 0 0 0 2 0 2X10 2X10 0 0 1x1070 o 2x107 o o 4x10 3 0 3X1Op 6x10 0 0 1x10 0 0 9x10 0 0 6x lo5 4 o 5x101 2x107 0 o 5x10 0 0 7 10 0 0 6x105 5 0 0 3x10 0 0 8x10 0 0 2 10 0 0 8x105 Adults 1 0 NE NE 0 3X1O5 NE 0 NE NE 1x10 NE NE 3 0 NE NE 0 2x10 NE 0 NE NE 2X1O5 NE NE 4 0 NE NE 0 7x 103 NE o NE NE 3x10b NE NE 5 0 NE NE 0 2 10 NE 6x10 NE NE 3X1O5 NE NE 2 0 NE NE 0 3x104 NE o NE NE 9x104 NE NE NE Not examined 0 coronavirus not detectable Segment 1 is proximal jejunum segment 5 is distal ileum segments 2 3 and 4 are between 1 and 5 this reason a single arithmetic mean virus titer of all parts was calculated and plotted for each pig fig 1 however the titer of site 5 exceeded that of site 1 by more than 1 log in three of nine newborn pigs six of 33 pigs 3 weeks old and two of eight adults Conversely the titer of site 1 exceeded that of site 5 by a log or more in three of 33 pigs 3 weeks old Titers of individual sites for all pigs killed after 48 72 120 and 168 h are listed in table I Some of the variations mentioned above are apparent by comparing 3 week old pigs at 48 h after exposure to 3 day old pigs at 168 h Titers in the newborn pigs were uniformly high and tended to decrease slightly from 72 to 168 h Plaque forming virus titers in the intestine of three newborn pigs that died after 72 96 h were comparable 10s 107 g to those of pigs in this group that were killed during this interval In contrast titers of older pigs were not uniform Virus was not detected in small intestine of Transmissible Gastroenteritis 439 5 Fig 4 Normal mucosa from the small intestine of a 3 week old control pig Fig 5 Epithelium of a partly atrophic villous in the small intestine of a 3 week old pig with experimental infection The small intestine of this pig contained insufficient virus to be detected by the tissue culture plaque assay Epithelial degeneration flattening exag gerated sloughing and infiltration with inflammatory cells along with decreased villous length are evidence of viral induced mucosal damage most pigs from the 3 week old and adult groups a few had low titers and others had titers comparable to those of the newborn pigs fig 1 Virus was recovered from most of the 3 week old pigs by inoculation of mesenteric lymph node into liquid cell culture For example all six lymph nodes from 3 week old pigs taken 24 36 h yielded virus however none was detected by the intestinal titrations done in a different system and at a different con centration from these pigs fig 1 The degree of villous atrophy among newborn principals was uniform at each interval and villi regenerated slightly between 72 and 168 h fig 2 The lengths of the villi in five newborn principals that died from 72 to 96 h were comparable to those shown for the newborn principals that were killed Three week old principals differed from the newborn in that villous atrophy 440 MOON et at A AA A 300 24 48 72 96 120 166 168 Hours post exposure Fig 6 Mean length of villi in jejunum and ileum of adult swine Principals A were exposed to virus controls A were not Each mark represents results from one pig tended to be less severe and to be markedly varied among pigs at 48 144 h fig 3 Furthermore apparent regeneration of villi was more nearly com plete by 168 h in the 3 week old than in the newborn pigs The degree of villous atrophy in 3 week old principals from which virus was not recovered or was recovered in only comparatively low titer lo5 killed at the same intervals fig 3 An analysis of covariance for villous length was con ducted on all 3 week old principals virus titer was used as a covariate The analysis indicated that titer and villous length were significantly P O Ol related for example an increase in virus titer corresponds to a decrease in vil lous length An analysis t test of villous length in 3 week old principals from which virus was not recovered also indicated that villous length in these pigs did decrease significantly P O Ol from 24 to 60 h fig 1 3 5 Villous atrophy was detected in only one of the 10 adults fig 6 7 This was the only adult that developed diarrhea and was the adult from which virus was recovered at 72 h fig 1 Discussion HOOPER and HAELTERMAN 8 found that pig duodenum and jejunum produced more transmissible gastroenteritis virus than did the ileum We used a different experimental design and virus titers did not vary consistently Transmissible Gastroenteritis 44 1 Fig 7 Mucosa from the small intestine of an adult pig with experimental infection Villi are atrophic and covered by a degenerate flattened epithelium There is edema and increased inflammatory cell infiltration in the lamina propria Crypt epithelium is not affected directly but undergoes compensatory hyperplasia with site in the intestine In our study some virus contained in the ileum could have been produced in the duodenum or jejunum Virus production and villous atrophy of 3 week old and adult pigs were less than in newborn pigs These age differences probably contribute significantly to the innate age dependent resistance to transmissible gastroenteritis We did not detect virus by the plaque titration assay of intestine from most of the 3 week old principals but most did yield virus after inoculation of undiluted suspension of mesenteric lymph nodes into cell cultures in liquid media Furthermore pigs from which intestinal virus was not recovered developed partial villous atrophy after 48 h The degree of villous atrophy in pigs from which virus was recovered was directly and significantly related to virus titer Presumably virus was present in low titer in the intestine of some pigs but not detected by plaque assay Three week old pigs consist of at least two populations The major population produces less virus than new born pigs resulting in only partial villous atrophy and little or no diarrhea On the other hand the minor population produces virus in amounts com parable to the newborn resulting in marked villous atrophy expressed clini cally as severe diarrhea 442 MOON et al The apparent rate of regeneration of villi in the minor population of 3 week old pigs was more rapid than in the newborn pigs fig 2 3 This is consistent with that part of the hypothesis predicting more rapid regenera tion in older pigs based on the observation that normal newborns replace villous epithelium in 7 10 days whereas 3 week old pigs do so in 2 4 days 131 Presumably regeneration was continuous and concomitant with des truction in both groups Thus comparatively rapid regeneration in 3 week old pigs may partially explain why maximal villous atrophy was less in this group than in newborn pigs The intestine of newborn pigs contained 102 105 times more virus per gram than that of most 3 week old pigs fig 1 The entire small intestine of a 3 week old pig 1 7 days after inoculation weighs only about five times more than that of a newborn pig 17 Thus there was considerably less virus in the intestine of most 3 week old principals than in newborn principals Virus is produced in villous epithelial cells 18 221 Villi of 3 week old pigs are 30 50 shorter than those in newborn pigs and thus contain fewer epithelial cells 13 16 241 Assuming the total number of intestinal villi in pigs is con stant from birth to adult as in some species 4 51 reduced villous length would result in fewer total productive cells and some reduction in titer How ever there is some evidence that the number of intestinal villi does increase during the neonatal period in pigs 24 The modest reduction in titer ex pected from the reduced length of villi even if villi are constant in number would be inadequate to explain the differences in virus titer between new born pigs and the major population of 3 week old pigs We speculate that the major reason for the differences in virus titer be tween age groups is related to differences in epithelial cell kinetics The nor mal life span of villous absorptive cells in 3 week old pigs 2 4 days is less than in newborn pigs 7 10 days 13 Virus production in the compara tively old cells of newborn pigs was greater than in the comparatively young cells of 3 week old pigs Other lines of evidence indicate a direct relationship between virus production and cell age In vivo this virus replicates in the mature differentiated nonproliferating villous epithelial cells and spares the juvenile proliferating epithelial cells of the crypts 18 221 Furthermore absorptive cells destroyed by the virus are rapidly replaced by juvenile epi thelial cells from the crypts 22 23 and these juvenile cells are comparatively resistant to virus production after they have migrated onto the villi 18 In vitro 5 day old monolayers of swine testis cells produce more than 10 fold as much virus as do 2 day old monolayers of the same cell line 20 This relationship between cell age and production of the coronavirus of Transmissible Gastroenteritis 443 transmissible gastroenteritis of swine appears to be in contrast to the parvo virus of feline panleukopenia infectious feline enteritis Panleukopenia virus is produced at high titer in young rapidly proliferating tissue culture cells but not by aged cells in culture 9 and directly affects crypt but not villous epithelium in the small intestine 6 101 The virus enters and accumulates in the apical tubular and vacuolar sys tem of villous absorptive cells of newborn pigs 25 from which it replicates by internal budding This tubular vacuolar system is extensive in newborn pigs but lacking or less well developed in pigs more than 3 weeks old 14 19 It has been suggested 25 that virus production by absorptive cells of older pigs is limited by the inadequate tubular vacuolar system thus limiting cell damage and contributing to age resistance The differences in virus titer and villous atrophy with age reported here are consistent with this Our hypoth