Vol. 14, No. 3 Printed in U.S.A.
INFECTION AND IMMUNITY, Sept. 1976, p. 816-825 Copyright ©D 1976 American Society for Microbiology
Reovirus-Like Agent Associated with Fatal Diarrhea in Neonatal Pigs' J. G. LECCE,* M. W. KING,
AND
R. MOCK2
Department of Animal Science and Microbiology, North Carolina State University, Raleigh, North Carolina 27607 Received for publication 6 April 1976
Large numbers of a reovirus-like agent were visualized with electron microsin bacteria-free gut homogenates obtained from piglets with a fatal diarrhea resembling transmissible gastroenteritis. The syndrome, of vomiting, diarrhea, dehydration, and death, was reproduced in piglets artificially infected with these bacteria-free gut homogenates. Reovirus-like particles persisted in serial piglet passage and none was seen in uninfected, asymptomatic controls. Hyperimmune sera (made in recovered piglets) aggregated the reovirus-like particles, as judged by immunoelectron microscopy, and neutralized the infectious agent. The cytoplasm in enterocytes on infected intestinal epithelium fluoresced when this hyperimmune sera was used in an indirect fluorescent antibody test. Feeding cow colostrum or diets containing porcine gamma globulin protected infected piglets. No cytopathogenic effect was noted in infected tissue cultures, nor did this agent affect neonatal guinea pigs, hamsters, mice, and rats. The agent did not agglutinate human 0 or A erythrocytes. copy
In 1972 we reported on an infectious abacterial enteritis resembling transmissible gastroenteritis (TGE) of swine (J. G. Lecce and J. A. Coalson, Abstr. Annu. Meet. Am. Soc. Microbiol. 1972, V292, p. 234; J. G. Lecce, J. A. Coalson, and R. Mock, Abstr. J. Anim. Sci. 35:219, 1972). Both are characterized by an initial short period of vomiting, followed by a profuse, watery diarrhea, dehydration, and often death (5, 7, 12, 21, 28, 36). Piglets under 1 week old are severely affected, and the younger the piglet, the less likely its survival. Consistent with TGE, systemically or orally administered antibacterial drugs did not stem the course of this disease. Attempts by three diagnostic laboratories and our own to demonstrate TGE viral specific antigens in these diseased piglets' jejunum or ileum using a standard fluorescent antibody technique (30) were negative (W. G. Pearson, personal communication; J. Black, personal communication; and W. C. Stewart, personal communication). Enteropathogenic Escherichia coli (16) are suspect in a syndrome such as this, especially when it is difficult to establish a diagnosis for TGE via fluorescent antibody tests; often a clinical diagnosis of colibacillosis is made. The purpose of this paper is to report the 1 Journal series paper no. 4965 of the North Carolina Agricultural Experiment Station, Raleigh. 2 Present address: School of Veterinary Medicine, University of Illinois, Urbana, Ill. 61801.
association of reovirus-like particles (21) with bacteria-free gut filtrates obtained from piglets with diarrhea. These filtrates are capable of reproducing (in serial piglet passage) the vomiting, diarrhea, dehydration, and death seen in the naturally diseased piglets. (This work was reported in part before the committee of Environmental Health and Livestock Production, Am. Soc. Anim. Sci., August 1973, Lincoln, Neb., and at a symposium on recent advances in bacterial and viral gastroenteritis, February 1976, Medical College of Virginia; Richmond, Va.) MATERIALS AND METHODS
Colostrum-free piglets. Piglets used for infectious studies were farrowed in a fumigated isolation room. At the time of farrowing, piglets were caught in sterile towels as they left the birth canal and carried to other isolation rooms and individually caged before infection (4, 5, 21, 23). Piglets were fed a liquid diet containing 20% milk solids four times a day (5, 22). These piglets are free of antibody and are referred to as colostrum free (19). Source of infectious material - 1972. In a swine herd, some nursing piglets (less than 2 weeks old) vomited (5, 21). This was followed by diarrhea; mortality was less than 10%. Twenty-four asymptomatic newborn piglets (after having nursed their sows for about 12 h) were brought to our isolation units, and 18 to 36 h later vomiting, diarrhea, dehydration, and death ensued. Part of a litter of colostrum-free piglets was 816
VOL. 14, 1976
PIGLET DIARRHEA AND REO-LIKE VIRUS
placed in cages contaminated by affected piglets, while the remainder of the litter was kept in isolation. Contaminated piglets developed the syndrome approximately 18 to 24 h later; controls remained normal. Infectious material was obtained from the colostrum-free piglets after they had diarrhea for about 2 days. Intestines were rapidly removed, homogenized (10% [wt/vol]) in physiologically balanced salt solution (BSS; NaCl, 8.0 g; KCl, 0.4 g; MgSO4 * 7H2O, 0.2 g; KH2PO4, 0.06 g; Na2HPO4 7H20, 0.39 g; in 1 liter of water, pH 7.4), pooled, and centrifuged at 4,000 x g, 4°C, for 10 min. The supernatant was filtered through a prefilter pad, a 0.8-,um filter, and finally a 0.45-,um filter (Millipore Corp., Bedford, Mass.). No bacteria or mycoplasma (18, 34) were demonstrated in the final filtrate (referred to hereafter as the 1972 bacteria-free gut homogenate), and it was stored at -20°C until used. This bacteria-free material was serially passed five times through at least four colostrum-free piglets per passage. Typical symptoms were produced at each passage. 1974. Two years later, a similar set of circumstances occurred in nursing piglets in another herd. Infectious material was obtained from piglets brought to our isolation facilities as described for the 1972 material (hereafter referred to as the 1974 bacteria-free gut homogenate). This material, passed three times through at least four colostrum-free pig'lets per passage, produced typical symptoms at each -
passage.
Later, infectious material was harvested directly from the gut fluid of piglets infected with the 1974 bacteria-free gut homogenate. This fluid, harvested and passed serially three times through at least four colostrum-free piglets per passage, produced typical symptoms. At the third passage, fluid was pooled from five pigs and used as a source of antigen for immunoelectron microscopy. Infectious dose. Unless noted to the contrary, colostrum-free piglets (less than 1.5 days old) were infected per os (via stomach tube) with 4 ml of 1:50 dilution of bacteria-free filtrates. Antiserum. Hyperimmune antisera were prepared to the 1972 and 1974 bacteria-free gut homogenates (hereafter referred to as 1972 and 1974 hyperimmune sera, respectively) from colostrum-free piglets that had recovered from an experimental infection at 5 days of age. These two groups of pigs were reinfected per os, producing no symptoms. At three weekly intervals, they were injected intramuscularly at four different sites (1 ml/site) with undiluted bacteria-free homogenate in Freund complete adjuvant, followed by intraperitoneal injections with 2 ml of undiluted homogenate. Serum used for immunoelectron microscopy was inactivated at 56°C for 20 min and clarified by spinning at 37,000 x g (17,500 rpm, Sorvall refrigerated centrifuge, Norwalk, Conn.) for 90 min. Electron microscopy. Initially, sediments resulting from centrifuging 4-ml samples of bacteria-free filtrates of gut fluid, or gut homogenates at 37,000 x g for 90 min, were examined (0.45-,im filtrate). Subsequently, gut fluid was filtered through cheesecloth and spun at 12,100 x g for 25 min, and 4 ml of this supernatant was centrifuged at 37,000 x g for 90
817
min, yielding a sediment for examination. Sediments were resuspended in 0.1 ml of distilled water, and 0.02 ml of this suspension was negatively stained by mixing with an equal volume of 3% sodium phosphotungstate (pH 7.0). Approximately 1.5 min later, a Formvar carbon-coated grid was floated on a drop of this mixture for 1.5 min, after which time the retrieved grid was dried of excess moisture by edging with filter paper. The grid was immediately placed in the vacuum chamber of an electron microscope (Siemens, Elmiskop 1A). At least 10 grid squares were examined before a preparation was scored negative (
INFECTION AND IMMUNITY, Sept. 1976, p. 816-825 Copyright ©D 1976 American Society for Microbiology
Reovirus-Like Agent Associated with Fatal Diarrhea in Neonatal Pigs' J. G. LECCE,* M. W. KING,
AND
R. MOCK2
Department of Animal Science and Microbiology, North Carolina State University, Raleigh, North Carolina 27607 Received for publication 6 April 1976
Large numbers of a reovirus-like agent were visualized with electron microsin bacteria-free gut homogenates obtained from piglets with a fatal diarrhea resembling transmissible gastroenteritis. The syndrome, of vomiting, diarrhea, dehydration, and death, was reproduced in piglets artificially infected with these bacteria-free gut homogenates. Reovirus-like particles persisted in serial piglet passage and none was seen in uninfected, asymptomatic controls. Hyperimmune sera (made in recovered piglets) aggregated the reovirus-like particles, as judged by immunoelectron microscopy, and neutralized the infectious agent. The cytoplasm in enterocytes on infected intestinal epithelium fluoresced when this hyperimmune sera was used in an indirect fluorescent antibody test. Feeding cow colostrum or diets containing porcine gamma globulin protected infected piglets. No cytopathogenic effect was noted in infected tissue cultures, nor did this agent affect neonatal guinea pigs, hamsters, mice, and rats. The agent did not agglutinate human 0 or A erythrocytes. copy
In 1972 we reported on an infectious abacterial enteritis resembling transmissible gastroenteritis (TGE) of swine (J. G. Lecce and J. A. Coalson, Abstr. Annu. Meet. Am. Soc. Microbiol. 1972, V292, p. 234; J. G. Lecce, J. A. Coalson, and R. Mock, Abstr. J. Anim. Sci. 35:219, 1972). Both are characterized by an initial short period of vomiting, followed by a profuse, watery diarrhea, dehydration, and often death (5, 7, 12, 21, 28, 36). Piglets under 1 week old are severely affected, and the younger the piglet, the less likely its survival. Consistent with TGE, systemically or orally administered antibacterial drugs did not stem the course of this disease. Attempts by three diagnostic laboratories and our own to demonstrate TGE viral specific antigens in these diseased piglets' jejunum or ileum using a standard fluorescent antibody technique (30) were negative (W. G. Pearson, personal communication; J. Black, personal communication; and W. C. Stewart, personal communication). Enteropathogenic Escherichia coli (16) are suspect in a syndrome such as this, especially when it is difficult to establish a diagnosis for TGE via fluorescent antibody tests; often a clinical diagnosis of colibacillosis is made. The purpose of this paper is to report the 1 Journal series paper no. 4965 of the North Carolina Agricultural Experiment Station, Raleigh. 2 Present address: School of Veterinary Medicine, University of Illinois, Urbana, Ill. 61801.
association of reovirus-like particles (21) with bacteria-free gut filtrates obtained from piglets with diarrhea. These filtrates are capable of reproducing (in serial piglet passage) the vomiting, diarrhea, dehydration, and death seen in the naturally diseased piglets. (This work was reported in part before the committee of Environmental Health and Livestock Production, Am. Soc. Anim. Sci., August 1973, Lincoln, Neb., and at a symposium on recent advances in bacterial and viral gastroenteritis, February 1976, Medical College of Virginia; Richmond, Va.) MATERIALS AND METHODS
Colostrum-free piglets. Piglets used for infectious studies were farrowed in a fumigated isolation room. At the time of farrowing, piglets were caught in sterile towels as they left the birth canal and carried to other isolation rooms and individually caged before infection (4, 5, 21, 23). Piglets were fed a liquid diet containing 20% milk solids four times a day (5, 22). These piglets are free of antibody and are referred to as colostrum free (19). Source of infectious material - 1972. In a swine herd, some nursing piglets (less than 2 weeks old) vomited (5, 21). This was followed by diarrhea; mortality was less than 10%. Twenty-four asymptomatic newborn piglets (after having nursed their sows for about 12 h) were brought to our isolation units, and 18 to 36 h later vomiting, diarrhea, dehydration, and death ensued. Part of a litter of colostrum-free piglets was 816
VOL. 14, 1976
PIGLET DIARRHEA AND REO-LIKE VIRUS
placed in cages contaminated by affected piglets, while the remainder of the litter was kept in isolation. Contaminated piglets developed the syndrome approximately 18 to 24 h later; controls remained normal. Infectious material was obtained from the colostrum-free piglets after they had diarrhea for about 2 days. Intestines were rapidly removed, homogenized (10% [wt/vol]) in physiologically balanced salt solution (BSS; NaCl, 8.0 g; KCl, 0.4 g; MgSO4 * 7H2O, 0.2 g; KH2PO4, 0.06 g; Na2HPO4 7H20, 0.39 g; in 1 liter of water, pH 7.4), pooled, and centrifuged at 4,000 x g, 4°C, for 10 min. The supernatant was filtered through a prefilter pad, a 0.8-,um filter, and finally a 0.45-,um filter (Millipore Corp., Bedford, Mass.). No bacteria or mycoplasma (18, 34) were demonstrated in the final filtrate (referred to hereafter as the 1972 bacteria-free gut homogenate), and it was stored at -20°C until used. This bacteria-free material was serially passed five times through at least four colostrum-free piglets per passage. Typical symptoms were produced at each passage. 1974. Two years later, a similar set of circumstances occurred in nursing piglets in another herd. Infectious material was obtained from piglets brought to our isolation facilities as described for the 1972 material (hereafter referred to as the 1974 bacteria-free gut homogenate). This material, passed three times through at least four colostrum-free pig'lets per passage, produced typical symptoms at each -
passage.
Later, infectious material was harvested directly from the gut fluid of piglets infected with the 1974 bacteria-free gut homogenate. This fluid, harvested and passed serially three times through at least four colostrum-free piglets per passage, produced typical symptoms. At the third passage, fluid was pooled from five pigs and used as a source of antigen for immunoelectron microscopy. Infectious dose. Unless noted to the contrary, colostrum-free piglets (less than 1.5 days old) were infected per os (via stomach tube) with 4 ml of 1:50 dilution of bacteria-free filtrates. Antiserum. Hyperimmune antisera were prepared to the 1972 and 1974 bacteria-free gut homogenates (hereafter referred to as 1972 and 1974 hyperimmune sera, respectively) from colostrum-free piglets that had recovered from an experimental infection at 5 days of age. These two groups of pigs were reinfected per os, producing no symptoms. At three weekly intervals, they were injected intramuscularly at four different sites (1 ml/site) with undiluted bacteria-free homogenate in Freund complete adjuvant, followed by intraperitoneal injections with 2 ml of undiluted homogenate. Serum used for immunoelectron microscopy was inactivated at 56°C for 20 min and clarified by spinning at 37,000 x g (17,500 rpm, Sorvall refrigerated centrifuge, Norwalk, Conn.) for 90 min. Electron microscopy. Initially, sediments resulting from centrifuging 4-ml samples of bacteria-free filtrates of gut fluid, or gut homogenates at 37,000 x g for 90 min, were examined (0.45-,im filtrate). Subsequently, gut fluid was filtered through cheesecloth and spun at 12,100 x g for 25 min, and 4 ml of this supernatant was centrifuged at 37,000 x g for 90
817
min, yielding a sediment for examination. Sediments were resuspended in 0.1 ml of distilled water, and 0.02 ml of this suspension was negatively stained by mixing with an equal volume of 3% sodium phosphotungstate (pH 7.0). Approximately 1.5 min later, a Formvar carbon-coated grid was floated on a drop of this mixture for 1.5 min, after which time the retrieved grid was dried of excess moisture by edging with filter paper. The grid was immediately placed in the vacuum chamber of an electron microscope (Siemens, Elmiskop 1A). At least 10 grid squares were examined before a preparation was scored negative (
