DIETARY REGIMEN, ROTAVIRUS, AND HEMOLYTIC ENTEROPATHOGENIC ESCHERICHIA COLI IN WEANLING DIARRHEA OF PIGS

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1 DIETARY REGIMEN, ROTAVIRUS, AND HEMOLYTIC ENTEROPATHOGENIC ESCHERICHIA COLI IN WEANLING DIARRHEA OF PIGS James G. Lecce To cite this version: James G. Lecce. DIETARY REGIMEN, ROTAVIRUS, AND HEMOLYTIC ENTEROPATHOGENIC ESCHERICHIA COLI IN WEANLING DIARRHEA OF PIGS. Annales de Recherches Vétérinaires, INRA Editions, 1983, 14 (4), pp <hal > HAL Id: hal Submitted on 1 Jan 1983 HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

2 DIETARY REGIMEN, ROTAVIRUS, AND HEMOLYTIC ENTEROPATHOGENIC ESCHERICHIA COLI IN WEANLING DIARRHEA OF PIGS James G. LECCE Department of Animal Science, North Carolina State University, Raleigh, North Carolina 27650, USA. Abstract Previously, we induced weanling diarrhea in piglets by infecting them first with rotavirus followed by a hemolytic enteropathogenic Escherichia coli. We postulated that rotavirus, by damaging the epithelium of the small intestines, produced an entero-environment which favored the selection and growth of the enteropathogenic E. coli. Furthermore, diet might affect the entero-environment and influence the course of the disease. To test this, newly weaned 3-week-old piglets were assigned to one of four dietary regimens and infected with rotavirus followed 24 h later with an enteropathogenic E.!coli. The course of the disease was followed by monitoring the severity of diarrhea, and the fecal shedding of rotavirus and the enteropathogenic. E. coli in these dually infected piglets. The dietary regimen designed to tax the digestive and absorptive capacity of the piglet (high nutrient intake fed 3 times a day) produced the most prolonged diarrhea, colonization of gut by the hemolytic enteropathogenic E. coli and persistent shedding of rotavirus. The same nutrient intake divided into 24 equal increments and fed hourly produced a less severe response. The least response was seen in piglets fed one-third the nutrient intake either hourly or 3 times a day. We conclude that dietary regimen plays an important role in rotavirus-escherichia coli induced weanling diarrhea. Since the turn of the century scientists have searched for the cause of weanling diarrhea (Barnum et al., 1967; Gordon et al, 1963; Nielson et a/., 1968; Wilson, 1981). Certain strains of Escherichia coli are often implicated as the causal agent. These strains usually are hemolytic, produce enterotoxins, and possess pili which mediate specific adherence of the E. coli to enterocytes (Chopra et al., 1964; Ellis, 1978; Gaastra and Geraaf, 1982; Kenworthy and Allen, 1966; Tzipori et al., 1980; Wilson, 1981). These strains are called hemolytic enterotoxigenic E. coli. We found hemolytic enterotoxigenic E. coli adhering to enterocytes of piglets experiencing weanling diarrhea in North Carolina (Lecce et al., We noted, however, that at the onset of the diarrhea piglets shed rotavirus in their feces (Lecce and King, 1980; Lecce and King, 1978; Lecce et a/., And, it was a day or two later before the hemolytic enterotoxigenic E. coli were isolated from diarrhetic fluid (Lecce et al., Rotavirus multiplies in and destroys the absorptive cells of the small intestines. Because absorptive surface is lost in a rotavirus infection, some digested food is not absorbed. This leads to a malabsorption-type diarrhea, dehydration and death in the very young piglet. We reasoned that at weaning, because piglets were not nursing, they were no longer being protected by the antibodies to rotavirus that is present in sow s milk. Then, the ubiquitous rotavirus infects the newly weaned piglet and destroys some of the

3 - called absorptive cells, resulting in the accumulation of nutrients of the lumen of the gut. This &dquo; malabsorbed&dquo; entero-environment favors the selection and growth of hemolytic enteropathogenic E. coli. The rapidly growing hemolytic E. coli attach to intestinal cells for about seven days and produce enterotoxin. Without a prior rotavirus infection, the selective entero-environment is absent and so are the hemolytic enteropathogenic E. coli. And, the disease is less severe. We tested the idea that rotavirus makes the piglet s gut vulnerable to hemolytic E. coli (Lecce et al., 1 982). We did this by infecting newly weaned piglets with rotavirus alone, hemolytic E. coli alone, and with rotavirus followed one day later with hemolytic E. coli. Piglets infected with hemolytic E. coli alone did not develop diarrhea and it was not possible to reisolate the hemolytic E. coli. Piglets infected with rotavirus alone experienced a mild diarrhea, and rotavirus was reisolated. Piglets infected with rotavirus followed one day later with hemolytic E. coli developed a severe diarrhea and about 10 % died. Millions of the rotavirus particles and hemolytic E. coli were detected in the diarrhetic fluids. Because of the above results, we wondered whether dietary regimen could influence the amount of malabsorbed nutrients and in turn the course of weanling diarrhea. To test this notion, newly weaned three-week-old piglets were infected with rotavirus and hemolytic enteropathogenic E. coli and assigned to one of four liquid dietary regimens (Lecce et al., 1983): 1. a diet containing high solids (18 % dry matter) fed three times a day; 2. the high solids diet fed hourly; 3. the high solids diet diluted with water to 6 % dry matter and fed three times a day (low solids), and the low solids diet fed hourly. Results of these experiments show that at least three factors play an important role in weanling diarrhea. First is an infection with rotavirus, followed by an infection with enteropathogenic E. coli and a third factor is a dietary regimen that burdens the digestive capacity of the gut (high solids fed three times a day). Materials and Methods Animals &dquo; Sanitary&dquo; piglets were farrowed in an intensive care farrowing facility where they nursed for three weeks (Lecce et al., 1982; Lecce and King, 1980). After weaning, piglets were individually caged and fed hourly or three times a day. Diets. Diets consisted mainly of non-fat cows milk solids, fat, vitamins and minerals. Thirty percent of the dry matter was protein, 40 % lactose and 20 % animal fat. The basic diet contained 18% solids (dry matter). This was called the high solids diet. When this diet was diluted with water to 6 % total solids, it was called the low solids diet. During the course of the experiment, the volume of the diets fed in a 24 h period was calculated to be 30 % of the piglet s body weight (e.g., 6 kg piglet was fed ml). The daily volume was divided into 24 equal increments or and fed hourly or three equal increments and fed three times a day (at 9, 13, 19h). Piglets were fed according to four dietary regimens: 1. high solids, three times a day; 2. high solids, hourly; 3. low solids, three times a day; and 4. low solids, hourly. Thus, piglets fed high solids received, proportionalto their weight, the same total solids and volume either in 3 or 24 equal increments. Piglets assigned to the low solids regimens were fed similarly except that the daily intake of solids (dispensed in either 3 or 24 equal increments) was one-third that of the high solids. Experimental At weaning. piglets were randomly selected from 12 sows and assigned to one of the four dietary regimens. This design was repeated through three farrowing groups. Statistics. The degree of diarrhea, and shedding of rotavirus and hemolytic E. coli-7 were compared between the piglets on the four dietary regimens by computing the average number of days each piglet had diarrhea, shed rotavirus, and hemolytic E. coli- 7. These averages were analyzed for significance. Microbiology After piglets had been fed according to their respective dietary regimens for seven days, they were infected per os with approximately 109 rotaviral particles and 24 h later with approximately 109 hemolytic, enteropathogenic E. coli (0157:K :NM) E. coli-7. This was day 1 and 2 respectively of the experiment (see figures). To determine whether piglets developed resistance to the dual infection, they were reinfected 13 days after the initial infection with the same dose of rotavirus and E. coli-7 and in the same sequence &horbar; except for two groups of five piglets, one fed high solids three times a day and the other fed low solids three times a day (fig. 5). The purpose of not reinfecting these two groups of piglets was to determine the length of persistence of diarrhea, fecal shedding of rotavirus and E. coli-7 after an initial infection. Both the E. coli-7 and pool of rotavirus were used in a previous study (Lecce et al., 1982). Fecal shedding patterns of rotavirus and E. coli-

4 7 were monitored as before, using an enzyme linked immunosorbant assay (ELISA) for detecting rotavirus and appropriate bacteriological and serological techniques for isolating and identifying E. coli-7 (Lecce et al., The small intestines of piglets that died or were killed in extremis were examined by immunofluorescence and scanning electron microscopy for adhering E. coli (Lecce et a/., 1982 ; Lecce et al., 1976). Results Diarrhea High solids, three times a day. There was a marked and significant difference (P < 0.01) in the severity of diarrhea between the piglets fed high solids three times a day and piglets fed according to the other dietary regimens. All of the piglets fed high solids three times a day had diarrhea from day 3 to 8 (fig. 11. This was followed by a gradual decline to 18 % by day 12. At day 13 they were reinfected with rotavirus followed by E. coli-7 on day 14. The diarrhea spiked to 100 / on day 1 5, and declined to 28 % by day 20. Four out of 1 5 pigs on this regimen died or were killed in extremis between day 5 and 7. E. coli-7 was found adhering to ileal villi in these piglets (fig. 2). High solids, 24 times a day. Piglets fed according to this dietary regimen experienced less diarrhea (P < 0.01) than the piglets fed high solids three times a day but more than the piglets fed low solids 24 or three times a day (P < 0.01). All of the piglets fed high solids 24 times a day had diarrhea on day 3 and 4 (fig. 11. ). This was followed by a rapid decline to zero by day 10. These piglets did not experience diarrhea when reinforced at day 13 and 14 with rotavirus and E. coli-7, respectively. Low solids, three times a day. Seventy % of the piglets had diarrhea by day 4 (fig. 1) followed by a rapid decline to near zero by day 7. The amount of diarrhea in this group was not significantly different from the group fed low solids 24 times a day. Piglets fed low solids three times a day did not experience diarrhea when reinfected at day 13 and 14 with rotavirus and E. coli-7, respectively. Low solids, 24 times a day. These piglets experienced the least amount of diarrhea, i.e., 50 % by day 3, zero by day 7, and no diarrhea when reinfected at day 13 and 14 with rotavirus and E. coli-7, respectively. Rotavirus High solids, three times a day. Again, there was a marked and significant difference (P < 0.01) between piglets fed according to this dietary regimen and those fed according to the other 3 regimens. Forty to 80 % of the piglets shed rotavirus for the entire 21 days of observation (fig. 31. High solids, 24 times a day. Piglets fed according to this dietary regimen shed significantly more rotavirus (P < 0.01) than those in the group fed low solids 24 times a day but not more than the piglets fed the low solids three times a day. Between 40 to 70 % of the piglets fed high solids 24 times a day (fig. 3) shed rotavirus from day 2 to day 11. From 20 to 50 % of the piglets shed rotavirus for four days after they were reinfected at day 13 with rotavirus. Low solids, three times a day. About 90 % of the piglets shed rotavirus by day 6, declining rapidly to zero by day 9 (fig. 3). There was a negligible response to reinfection with

5 rotavirus at day 13 (10 % or less). There was a significant difference (P! 0.05) in the shedding pattern between this group and those fed low solids 24 times a day. Low solids, 24 times a day. A peak of 55 / of the piglets shed rotavirus by day 6 followed by rapid decline to zero by day 9 (fig. 3). There was a negligible response to reinfection with rotavirus at day 13. Hemolytic Enteropathogenic E. coli-7. High solids, 3 times a day. Again, there was a significant difference (P < 0.01 ) in the response of piglets fed according to this regimen as compared to piglets fed according to the other three regimens. About 90 % of piglets in this group shed E. coli-7 from day 3 to day 7, the percentage declined to zero by day 11 (fig. 4). Fifty % of the piglets shed E. coli-7 for one day when reinfected on day 14 with E. coli-7. High solids, 24 times a day; low solids, three times a day; low solids, 24 times a day. Piglets fed according to these three regimens (fig. 4) responded in a similar manner (not significantly different from each other) when infected with E. coli-7, i.e., about 40 to 90 % of the piglets shed E. coli-7 by day 3 declining to zero by day 9. About 20 % of the piglets fed high solids, and low solids 24 times per day shed hemolytic E. coli-7 for one day when reinfected with E. coli-7 on day 14. Persistence of rotavirus and diarrhea. Rotavirus and diarrhea (but not E. coli-7) persisted for at least 39 days in piglets fed high solids three times a day (fig. 5). In contrast (P < 0.01 ), no diarrhea and shedding of rotavirus was seen from day 22 to 39 in piglets that were fed low solids three times a day. Discussion We noted previously that orally inoculating newly weaned, 3-week-old piglets with hemolytic enteropathogenic E. coli (0 1 57:K.:N M) was of little consequence unless the piglets were concurrently infected with rotavirus (Lecce et al., 1982). Then the syndrome, weanling diarrhea (colibacillosis) as seen in the field and described by many investigators in the past, was reproduced (Buxton and Thomlinson, 1961 ; Chopra et al., 1964; Ellis, 1978; Kenworthy and Allen, 1966; Kenworthy and Crabb, Nielsen et al., 1968; Neilsen and Sautter, 1968; Stevens, 1963a, b; Tzipori et al., 1980; Wilson, 1981). Dually infected piglets experienced severe diarrhea accompanied by the ready colonization of the small intestines by adhering hemolytic enteropathogenic E. coli. One possibility considered by us was that an initial infection with rotavirus, a virus that multiplies in and destroys small intestinal epithelial cells, produced malabsorption which in turn provided a nutrient milieu that favored the selection and growth of enteropathogenic E. coli. Thus, we theorized that the dietary regimen might influence the course and severity of weaning diarrhea. Investigators have long felt that diet played a role in weanling diarrhea because there is a drastic change in dietary regimen at weaning which coincides with the initiation of diarrhea (Kenworthy and Allen, 1966; Kenworthy and Crabb, 1963; Stevens, 1963a, b; Tzipori et al., 1980).

6 Experiments reported here were designed to manipulate the luminal nutrient milieu of the gut by using four markedly different liquid dietary regimens. The first dietary regimen was designed to tax digestion and absorption by presenting nutrient surges to the gut. This was done by feeding large amounts of a high caloric-high protein diet (high solids) in three equal increments within a short interval (9, 13, and 19 h). This diet is similar to sow s milk (Lecce and Coalson, 1976). The second dietary regimen was more natural to the pig in that it mimicked the dietary regimen of a sow nursing her piglets. In this case, piglets were fed the same total daily nutrient intake as those fed by the first regimen except it was presented in 24 equal increments every hour. This dietary regimen produces (as it does in nursing piglets) rapid, efficient rates of weight gain (Lecce and Coalson, Presumably, the rapid gain is sustained by a continuous flow of nutrients presented in a manner that maximizes assimilation of nutrients. The other two dietary regimens (low solids) served as controls. These piglets were fed the same volume and on the same schedules as the piglets fed on the high solids regimens except the total nutrient intake was one-third that of the high solids. Results of the experiments reported here show that the dietary regimen designed to tax the digestive and absorptive capacity of the piglet (high solids three times a day) produced the most prolonged diarrhea, colonization of the gut by hemolytic E. coli and persistent shedding of rotavirus in the feces of dually infected piglets (P < The same daily nutrient intake divided into 24 equal increments and fed hourly produced a less severe response (P < 0.01) with the least response (P < 0.01) seen in piglets fed one-third the nutrient intake either hourly or three times a day. Thus, in terms of the intent of these experiments and consistent with the above results, we offer the explanation that feeding high solids three times a day produced a nutrient surge which in concert with the rotaviral damaged epithelium overwhelmed the digestive and absorptive capacity of the piglet. Malabsorption and concomitant diarrhea ensued. This resulted in an entero-environment which favored for a limited time (^- 9 days) the colonization of the gut by hemolytic E. coll and for an unlimited time the persistent fecal shedding of rotavirus (at least 39 days). Fecal shedding of the hemolytic E. coli ceased about 9 days post-inoculation regardless of dietary regimen. Also, piglets resisted dual infection with hemolytic E. coli and rotavirus 13 days after the initial infection (figs. 3, 4) except for those fed high solids three times a day. Piglets fed high solids three times a day continued to have diarrhea and to shed rotavirus, whether or not they were reinfected, for as long as they were observed (39 days after the initial rotavirus infection) (figs. 3, 5). Perhaps the rotavirus persisted because the altered entero-environment increased the efficiency of infection of enterocytes by rotavirus. If this were so, then a cycle of rotavirus shedding, fueled by malabsorption, would be generated for as long as the dietary regimen contributed to the malabsorption. Conversely, as was observed, feeding piglets by a less burdensom dietary regimen (low solids) would limit the cycle. We reported previously on a persistent rotaviral infection associated with multiple episodes of diarrhea in piglets reared in isolation (Lecce and King, 1980). At the time we could not account for the chronicity, but in hindsight the dietary regimen used for those piglets was similar to that used here for the piglets fed high solids. We feel that diet plays an important role in the provocative events leading to weanling diarrhea. We envision the sequence from normal to abnormal as follows: 1. piglets ane weaned into an environment contaminated with enteropathogens; 2. the piglet s gut becomes susceptible to enteropathogens at weaning because the gut is no longer bathed by protective antibody coming from sow s milk (Lecce and King, 19 78); 3. the ubiquitous enteropathogen (rotavirus) infects and destroys absorptive epithelium, thereby, producing a malabsorption of nutrients; 4. this malabsorption creates an entero-environment which not only favors the colonization of the small intestines by enteropathogenic E. coli but also increases the efficiency of infection of enterocytes by rotavirus, and 5. the more burdensome the dietary regimen, the more efficient the reinfection by rotavirus, and the more persistent the diarrhea and shedding of rotavirus. Why rotavirus persisted and hemolytic E. coli-7 did

7 not, is currently under study. To this end, the effect of malabsorption produced by noninfectious agents on an infection by rotavirus and enteropathogenic E. coli, along with the immune response of the gut to these two enteropathogens is being investigated. EEC seminar on gastro-intestinal diseases in the young pig and calf, 1-3 December 1982, INRA, CRZV de Theix, Beaumont, France. References BARNUM D.A., GLANTZ P.J., MOON H.W., Colibacillosis. CIBA Veterinary Monograph Series N 2. CIBA Pharmaceutical Co. BUXTON A., THOMLINSON J.R., The detection of tissue-sensitizing antibodies to Escherichia coli in oedema disease, haemorrhagic gastro-enteritis and in normal pigs. Res. Vet. Sci., 2, 73. CHOPRA S.L., BLACKWOOD A.C., DALE D.G., Enteritis of early weaned pigs. I. Enteropathogenic Escherichia coli. Can. J. Camp. Med., 28, ELLIS R.P., Serologic and epidemiologic investigations of colibacillosis in pigs. Procee dinqs of the Second International Symposium on Neonatal Diarrhea. Veterinary Infectious Disease Organization. University of Saskatchewan, Saskatoon, Canada. pp GAASTRA W., GERAAF F.K., Host-specific fimbrial adhesions of non-invasive enterotoxigenic Escherichia coli strains. Microbiol. Rev., 46, GORDON J.E., CHITKARA LD., WYON J.G., Weanling diarrhea. Am. J. Med, Sci., 245, KENWORTHY R., ALLEN W.D., The significance of Escherichia coli to the young pig. J. Comp. Pathol., 76, KENWORTHY R., CRABB W.E., Intestinal flora of young pigs, with reference to early weaning, Escherichia coli, and scours. J. Comp. Pathol. Ther., 73, LECCE J.G., KING M.W., MOCK R., Reovirus-like agent associated with fatal diarrhea in neonatal pigs. lnfect. lmmun., 14, LECCE J.G., BALSBAUGH R.K., CLARE D.A., KING M.W., Rotavirus followed by hemolytic enteropathogenic Escherichia coli in weanling diarrhea of pigs. J. Clin. Microbiol., 16, LECCE J.G., CLARE D.A., BALSBAUGH R.K., COLLIER D.N., Effect of dietary on rotavirus. Escherichia coli weanling diarrhea of piglets. J. Clin. Microbiol., 17, LECCE J.G., COALSON J.A., Diets fort rearing colostrum-free piglets with an automatic feeding device. J. Anim. Sci, 42, LECCE J.G., KING M.W., Role of rotavirus (Reo-like) in weanling diarrhea of pigs. J. Clin. Microbiol., 8, LECCE J.G., KING M.W., Persistent rotaviral infection producing multiple episodes of diarrhea in weanling pigs reared in isolation. Proceedings of the Third International Symposium on Neonatal Diarrhea. Veterinary Infectious Disease Organization. University of Saskatchewan, Saskatoon, Canada. pp NIELSON N.O., MOON H.W., ROE W.E., Enteric colibacillosis in swine. J. Arrr. Vet. Med. Assoc., 153, NEILSEN N.O., SAUTTER J.H., Infection of ligated intestinal loops with hemolytic Escherichia coli in the pig. Can. Vet. J., 9, STEVENS A.J., 1963a. Symposium: Enteritis in pigs 1. Coliform infections in the young and a practical approach to the control of enteritis. Vet. Rec., 75, STEVENS A.J., 1963b. Enteritis in pigs-a working hypothesis. Br. Vet. J., 119, TZIPORI S., CHANDLER D., SMITH M., MAKIN T., HENNESSY D., Factors contributing to the postweaning diarrhea in a large intensive piggery. Aust. Vet. J., 56, WILSON M.R., Enteric colibacillosis diseases of swine. /n: A.D. Leman, R.D. Glock, W.L. Mengeling, R.H.C. Penny, E. Scholl, B. Straw (eds) Diseases of Swine. Iowa State University Press, Ames, Iowa p Question From Dr Vannier to OrLecce What effect does the way of diet presentation have on diarrhcea and E. coli and Rotavirus excretion 7 (solid or liquid, effects other than for the absorption). Answer The dietary regimen may produce poor absorption which in turn could provide a selective advantage for hemolytic enteropathogenic E. coli or the dietary regimen could change binding sites on the enterocytes that make the enterocytes more readily infected with rotavirus or more efficiently adhered to by E. coli.