Epidemiological studies of piglet diarrhoea in intensively managed Danish sow herds. IV. Pathogenicity of porcine rotavirus

Colostrum-deprived piglets inoculated with rotavirus 24 h after birth developed a profuse diarrhoea that spread to non-inoculated, colostrum-deprived litter mates and, occassionally, to colostrum-fed piglets. Case fatality rates in these 3 categories of piglets were 63.2%, 35.7% and 8.3%, respectively. Surviving piglets recovered in 1-2 weeks, but shedded virus via the faeces for up to 3 weeks p.i. The D-xylose test revealed severe malabsorption, with extremely flat absorption curves for up to 3-4 weeks p.i. Malabsorption was more marked in piglets with a long-lasting faecal virus excretion than in piglets where virus disappeared from the faeces within 10 days p.i. Infected piglets (colostrum-fed and colostrum-deprived) had decreased weight gains and were 5 days older at a bodyweight of 25 kg than non-inoculated controls. It is concluded that rotavirus is probably of significance in diarrhoeal syndromes in suckling piglets, alone or in combination with E. coli or other pathogens.     

Single and mixed infections of neonatal pigs with rotaviruses and enteroviruses: virological studies.

Three rotaviruses and three enteroviruses were isolated from pigs with diarrhea. The three enteroviruses and one of the rotaviruses were recovered from pigs infected with both viruses. Separation of rotaviruses and enteroviruses from tissues containing both viruses was effected by pancreatin treatment, terminal dilution, and inoculation onto different cell lines. The three rotaviruses were group A serotype 1, and the enteroviruses were serotypes 2, 3 and 7. Cell culture preparations of these six viruses were inoculated into colostrum-deprived neonatal pigs. All of the rotavirus and enterovirus isolates established intestinal and systemic infection and were shed in the feces after oral inoculation. Concurrent infection with both viruses resulted in only minor alteration of systemic distribution and did not alter fecal shedding of either virus.     

Response of colostrum-deprived cynomolgus monkeys to intragastric challenge exposure with simian rotavirus strain SA11.

The infectivity and pathogenic potential of a cell culture-adapted simian rotavirus was evaluated in colostrum-deprived newborn and infant cynomolgus macaques (Macaca fascicularis). Intragastric challenge exposure with the simian rotavirus strain SA11 on postpartum day 2 induced diarrhea in 5 of 5 colostrum-deprived newborn monkeys. Compared with sham-inoculated controls, 3 of the 5 inoculated monkeys also manifested reduced body weight gain during the initial 5 days after challenge exposure. Rotavirus was detected in feces of 3 challenge-exposed monkeys for up to 2 days after inoculation. Evaluation of antibody response after rotavirus inoculation was obscured by high but variable prechallenge-exposure serum titers of rotavirus-specific antibody. Preexisting serum titer of neutralizing antibody in newborn monkeys was not predictive of clinical response to inoculation with rotavirus SA11. Two 90-day-old infant monkeys with low serum neutralizing antibody titer did not have diarrhea, reduced weight gain, or antibody response after oral inoculation with rotavirus SA11. Results of these challenge-exposure studies in newborn cynomolgus monkeys were consistent with a heterologous host-rotavirus model and indicate that neonatal serum antibody of maternal origin may not be associated with resistance to rotavirus-induced disease.     

ENTERITIS IN FOALS INDUCED BY ROTAVIRUS AND ENTEROTOXIGENIC ESCHERICHIA COLI

Colostrum-deprived, colostrum-fed or suckling foals were orally inoculated with foal rotavirus and enterotoxigenic Escherichia coli derived from a calf. Neither agent given alone caused diarrhoea in foals aged 1 or 2 days, although with rotavirus, 2 of the 3 inoculated foals became depressed 3 days after inoculation and all 3 were excreting rotavirus in the faeces. Inoculation of both agents induced diarrhoea in colostrum-deprived, colostrum-fed or suckling foals aged up to 16 days. There was an apparent age-related resistance to diarrhoea which developed between 2 and 3 weeks of age. It was related to failure of rotavirus to establish apparent infection in older foals and was independent of preinoculation maternal antibody.     

Studies on the age resistance of swine to group A rotavirus infection.

To determine whether swine become naturally age resistant to group A rotavirus infection, colostrum-deprived, rotavirus-naive newborn pigs that were raised in isolation (n = 34) were studied. Neonatal pigs and pigs 1, 2, 4, 6, 8, 10, and 12 weeks of age were inoculated orally with group A porcine rotavirus or mock inoculum and euthanatized at 24, 31, or 48 hours post-infection. Nine sections of small intestine, cecum, and colon were harvested and immunohistochemically examined for evidence of rotavirus replication within enterocytes. Infectivity was semiquantified by intestinal segment, and a composite score was obtained for each animal. In pigs inoculated at 1 week of age, enterocyte infection was mild and scattered; all other pigs became infected regardless of age or region of intestine, and older animals that became infected had infectivity scores similar to those of younger animals. In a second more limited study, pigs raised in the same isolation environment (n = 11) but previously exposed to virus and demonstrating rotavirus serum antibody had a much lower degree of enterocyte infection at 8, 10, and 12 weeks of age (2, 4, and 6 weeks, respectively, after initial exposure to virus). Age resistance to clinical rotavirus disease in swine is due to factors other than an age-dependent development of resistance of enterocytes to infection, at least through 12 weeks of age.     

Prolonged excretion and failure of cross-protection between distinct serotypes of bovine rotavirus

Four newborn calves were experimentally infected with two distinct serotypes of bovine rotavirus (BRV-1 and BRV-2). Initially, three colostrum-deprived calves were inoculated orally with either BRV-1 or BRV-2; all developed severe diarrhea and produced serotype-specific neutralizing antibodies. Fecal virus was first demonstrated by immunofluorescence the day after inoculation. The virus titers reached a maximum of 10(5.2)-10(6.6) fluorescent focus forming units g-1 of feces 2-5 days after inoculation and then decreased. Fecal virus was detected in low titers beyond 28 days after inoculation despite the development of serum neutralizing antibodies. One calf, which had acquired specific active immunity against BRV-1 following oral infection, was further infected orally with BRV-2 4 weeks later. The calf again manifested diarrhea, excreted BRV-2 and showed an increase in serum neutralizing antibody against BRV-2. These results indicated that calves infected with either BRV-1 or BRV-2 do not have cross-protection to infection with heterologous BRV, and that recurrence of the disease can occur. The possible mechanisms of the persistence of BRV in calves and its role in the epidemiology of this infection are discussed.     

Evaluation of killed and modified live porcine rotavirus vaccines in cesarean derived colostrum deprived pigs

Twenty-eight cesarean derived, colostrum deprived (CDCD) piglets were used to evaluate the efficacy of killed and modified live rotavirus (MLV) vaccines against challenge with virulent A-1 and A-2 rotaviruses. Two killed rotavirus vaccines were evaluated: an experimental vaccine and a commercially available vaccine. Efficacy parameters included: average daily weight gains, rotavirus shedding in feces, morbidity incidence and duration, and rotavirus serum antibody conversion post-vaccination and post-challenge. Piglets vaccinated orally/intramuscularly with the modified live vaccine were completely protected from A-1 and A-2 virulent rotavirus challenge. Nonvaccinated control piglets and piglets receiving killed rotavirus vaccines developed diarrhea, shed virus and exhibited reduced weight gains post-challenge. Only the MLV rotavirus vaccine was able to prevent virus shedding in feces after virulent challenge. Both controls and pigs which received killed vaccines intraperitoneally, orally or intramuscularly shed virus in the feces for 7 days post-challenge and virus peak titers approached 10(7) fluorescent antibody infectious dose (FAID)50/g feces. These studies clearly reflected the inability of killed rotavirus vaccines to induce active local immunity to rotaviral diarrhea in piglets.     

Detection of transmissible gastroenteritis virus in feces from pigs by reversed passive hemagglutination

A reversed passive hemagglutination (RPHA) method was developed for the detection of transmissible gastroenteritis (TGE) virus in the fecal specimens from pigs. Ovine erythrocytes fixed with glutaraldehyde and treated with tannic acid were coated with anti-TGE virus swine antibodies, which were purified by affinity chromatographic technique linked with purified TGE virus. The RPHA test was done by the Microtiter method. Erythrocytes coated with purified specific antibodies were agglutinated by TGE virus, but not by porcine rotavirus or porcine enterovirus. The reaction was specifically inhibited by antiserum against TGE virus, confirming the specificity of the reaction. A litter of seven 3-day-old pigs was orally inoculated with TGE virus, and fecal specimens were obtained once a day and serum was obtained every 4th day. With the RPHA test, TGE virus was detected in the diarrheal feces; all of the inoculated pigs developed virus-neutralization antibody for the TGE virus. The RPHA test detected TGE virus in feces from pigs with naturally occurring diarrhea. The RPHA test detected TGE virus in 5 of 6 fecal specimens (80%), whereas the positive rate was only 50% (3/6) for the immunofluorescent staining of primary cultures of porcine kidney cells inoculated with the specimens. The advantages of the RPHA method are simplicity, high sensitivity, and rapid to do.     

Experimental Klebsiella and Salmonella infection in neonatal swine.

Twenty colostrum-fed piglets from three sows were separated from the sows 24 hours after birth and were randomly divided into five groups of four piglets each. Every piglet in each of four test groups was orally inoculated with about 10(10) colony forming units of Salmonella typhimurium, Salmonella choleraesuis var Kunzendorf or one of two isolates of Klebsiella pneumoniae. One group served as uninoculated controls. Piglets infected with K. pneumoniae developed severe diarrhea beginning about 12 hours after inoculation. They became dehydrated and weak but continued to drink. There were no morphological alterations in intestinal mucosa when piglets were killed and necropsied 48 or 72 hours after inoculation. Klebseilla pneumoniae was isolated from intestine and feces but not from liver or spleen. Piglets inoculated with S. choleraesuis became lethargic and disinterested in food by 24 hours after inoculation. Diarrhea developed by 48 hours after inoculation. Lesions at necropsy 60 or 72 hours postinoculation were subcutaneous edema, mesenteric lymphadenitis, diffuse intestinal superficial mucosal necrosis with villous atrophy, and focal deep ulceration in the ileum. Salmonella choleraesuis was isolated from all segments of intestine and from feces, liver and spleen. Piglets inoculated with S. typhimurium developed a relatively mild diarrheal disease with lesions similar to those with S. choleraesuis infection but less severe. The inoculated organism was recovered from all areas of intestine and from feces, liver and spleen. Serum from infected and control piglets had high (greater than 1:256) agglutinating titres against S. typhimurium but low titres (0 to 1:8) against S. choleraesuis. The agglutinins were assumed to originate from colostral antibodies.     

Experimental rotavirus infection in three-week-old pigs

Thirteen 3-week-old pigs that had been allowed to nurse for the first 16 to 18 hours after birth were orally inoculated with 1 x 10(6.5) TCID50 of porcine rotavirus. All developed diarrhea, anorexia, and vomiting by postinoculation (PI) hour 30. These signs had abated by PI day 6. Villus blunting in the small intestine was most severe in the jejunum and ileum of pigs euthanatized between PI days 3 and 5. Villi had returned to nearly normal length by PI day 6, although fused villi were seen in a few locations in the distal portion of the jejunum and in the ileum. Virus was detected in the feces of inoculated pigs by isolation in cell cultures and by electron microscopy during the 7-day course of the experiment. There was 1 extraintestinal virus isolation from the lung of 1 pig at PI day 2. Infection and disease developed in the presence of serum-neutralizing antibody obtained by nursing seropositive sows. There was no significant change in neutralizing antibody titers in the 3-week-old pigs over the course of the experiment. In this experimental work, a model to study rotavirus infection in 3-week-old pigs has been developed.     

Intestinal changes associated with rotavirus and enterotoxigenic Escherichia coli infection in calves

Newborn calves inoculated with rotavirus, enterotoxigenic Escherichia coli (ETEC) serotype 020:K' x 106':K99:HNM, either alone or in combination, became depressed, anorectic, diarrhoeic and dehydrated. ETEC did not adhere to the intestine although there was extensive proliferation in the lumen. Only slight mucosal changes were induced by ETEC and the activity of membrane bound lactase remained normal. More severe mucosal damage and a decrease in lactase activity were found in newborn calves inoculated with either rotavirus or rotavirus and ETEC in combination. The most severe clinical illness was found in calves inoculated with both rotavirus and ETEC. Calves inoculated at 1 week of age with either rotavirus or ETEC remained clinically normal. Rotavirus infection produced slight mucosal changes and a reduction of lactase activity. In contrast, colostrum-fed or suckling calves up to 2 weeks old inoculated with both rotavirus and ETEC became clinically affected, showed severe mucosal damage and decreased lactase activity. There was no bacterial adhesion to the intestinal mucosa as observed by immunofluorescent labelling and light microscopy.     

Inoculation of neonatal gnotobiotic dogs with a canine rotavirus

Neonatal gnotobiotic dogs were inoculated orally with a rotavirus isolated from a pup with fatal diarrhea, and in the gnotobiotic dogs, diarrhea was observed between postinoculation hours (PIH) 20 and 24. The diarrhea persisted through PIH 154, and inoculated pups had clinical signs of dehydration after PIH 24. Negative-contrast electron microscopy of the feces from inoculated pups revealed rotavirus particles from PIH 12 through 154. Using an indirect-fluorescent antibody test, serum rotavirus antibody was detected in inoculated pups by PIH 96. In the duodenum, jejunum, and ileum of inoculated pups, group-specific rotaviral antigen was observed within absorptive villus epithelial cells and mononuclear cells in the villus lamina propria with an indirect-fluorescent antibody test. Fluorescence was seen in the small intestine of inoculated pups killed by PIH 12 and was present in intestines of pups killed through PIH 154. Rotaviral antigen was also seen in the mesenteric lymph nodes of a few inoculated pups killed at PIH 48.     

Development of γδ T cell subset responses in gnotobiotic pigs infected with human rotaviruses and colonized with probiotic lactobacilli

γδ T cell responses are induced by various viral and bacterial infections. Different γδ T cells contribute to activation and regulation of the inflammatory response and to epithelial repair. How γδ T cells respond to rotavirus infection and how the colonization of probiotics influences the γδ T cell response were unknown. In this study, we evaluated by multicolor flow cytometry the frequencies and distribution of total γδ T cells and three major subsets (CD2-CD8-, CD2+CD8- and CD2+CD8+) in ileum, spleen and blood of gnotobiotic (Gn) pigs at early (3-5 days) and late phases (28 days) after rotavirus infection. The Gn pigs were inoculated with the virulent human rotavirus Wa strain and colonized with a mixture of two strains of probiotics Lactobacillus acidophilus and Lactobacillus reuteri. In na?ve pigs, the highest frequency of total γδ T cells was found in blood, followed by spleen and ileum at the early age (8-10 days old) whereas in older pigs (32 days of age) the highest frequency of total γδ T cells was found in ileum and spleen followed by blood. Rotavirus infection significantly increased frequencies of intestinal total γδ T cells and the putatively regulatory CD2+CD8+ γδ T cell subset and decreased frequencies of the putatively proinflammatory CD8- subsets in ileum, spleen and blood at post-infection days (PID) 3 or 5. The three γδ T cell subsets distributed and responded differently after rotavirus infection and/or lactobacilli colonization. The CD2+CD8+ subset contributed the most to the expansion of total γδ T cells after rotavirus infection in ileum because more than 77% of the total γδ T cells there were CD2+CD8+ cells. There was an additive effect between lactobacilli and rotavirus in inducing total γδ T cell expansion in ileum at PID 5. The overall effect of lactobacilli colonization versus rotavirus infection on frequencies of the CD2+CD8+ γδ T cell subset in ileum was similar; however, rotavirus-infected pigs maintained significantly higher frequencies of CD8- subsets in ileum than lactobacilli-colonized pigs. The dynamic γδ T cell responses suggest that γδ T cell subsets may play important roles in different stages of immune responses after rotavirus infection and probiotic colonization. The knowledge on the kinetics and distribution patterns of γδ T cell subsets in na?ve pigs and after rotavirus infection or lactobacilli colonization provides the foundation for further mechanistic studies of their functions.     

猪血凝性脑脊髓炎病毒在人工感染仔猪体内侵染过程和分布规律的研究

为研究血凝性脑脊髓炎病毒(HEY)在人工感染仔猪体内的侵袭过程和分布规律,本实验采用滴鼻的方式感染5头1日龄未食初乳仔猪,并在感染后每隔24 h迫杀1头仔猪,采集鼻黏膜、气管、口腔黏膜、舌、食管、胃、肠、心肌、肝、脾、肺、肾、各段脊髓和脑等组织脏器制作切片,通过间接免疫组织化学方法检测HEV在仔猪体内的动态侵袭过程.此外,采用SYBR Green Ⅰ实时荧光定量PCR(Real-time PCR)方法检测病毒在各组织脏器中的分布情况.结果显示:感染病毒后2 d～3 d,仔猪出现精神沉郁、全身震颤等中枢神经系统症状,免疫组织化学检测可见病毒抗原首先出现于脑桥,并进一步蔓延至延髓和各段脊髓,最后进入小脑浦肯野氏细胞和大脑皮层锥体细胞中;Real-time PCR检测结果显示病毒广泛分布于大脑皮层、脑桥、延髓、脊髓和小脑等中枢神经系统的组织中,其中大脑皮层的检出率最高.     

Pathogenicity of Escherichia coli O115:K"V165" strains isolated from pigs with diarrhea

Eighteen strains of Escherichia coli serogroup O115:K"V165" isolated from 1- to 8-week-old pigs with diarrhea were tested for toxigenicity, pathogenicity in pigs and mice, serum resistance, mannose-resistant hemagglutination (MRHA), F165 and other surface antigens, colicin V (Col V), aerobactin, and biotype. Twelve strains were positive for heat-stable enterotoxin (STb), MRHA-negative, and F165-negative; 5 strains were enterotoxin-negative, MRHA-positive, and F165-positive; and 1 strain was MRHA-positive, but F165- and enterotoxin-negative. Six of the 12 STb-positive strains moderately colonized the ileum of newborn colostrum-deprived pigs within 24 hours after inoculation. Two of the colonizing strains were able to induce watery diarrhea. All 12 STb-positive strains were nonpathogenic for adult mice and were serum-sensitive; 11 of 12 were Col V-negative, 9 of 12 did not produce aerobactin, and 10 of 12 belonged to biotypes other than 1 or 2. All 6 enterotoxin-negative strains colonized the small and large intestines, associated with peritoneal serosal surfaces, and induced septicemia and polyserositis in newborn colostrum-deprived pigs 1 to 2 days after inoculation. In contrast, 3 STb-positive strains poorly colonized the intestines and did not induce septicemia in pigs at 3 days after inoculation. All 6 enterotoxin-negative strains were Col V-positive, produced aerobactin, and belonged to biotype 1 or 2. Of the 5 enterotoxin-negative, F165-positive strains, only 4 were pathogenic for intraperitoneally inoculated adult mice and were serum-resistant. The enterotoxin-negative, F165-negative strain was neither serum-resistant nor mouse-pathogenic.(ABSTRACT TRUNCATED AT 250 WORDS)     

Single and mixed infections of neonatal pigs with rotaviruses and enteroviruses: clinical signs and microscopic lesions.

Neonatal colostrum-deprived pigs were inoculated with cell-culture preparations of three rotaviruses and three enteroviruses, singly or in combination. The three enteroviruses established intestinal and systemic infection but did not induce diarrhea or intestinal lesions. The three rotaviruses produced severe enteric disease characterized by profuse watery diarrhea, dehydration and death. Villi were severely stunted. All three isolates were equally virulent. Inoculation with three different rotavirus-enterovirus combinations resulted in disease less severe than that produced by the rotaviruses alone. Intestinal lesions were less extensive and fewer pigs became moribund or died.     

Rotaviral diarrhea in pigs: brief review.

Rotavirus is a name given to a group of viruses that have similar characteristics and are generally capable of causing diarrhea in the young. Infection of pigs with porcine rotavirus is common and widespread and can result in diarrhea, especially in 1- to 4-week-old pigs. This virus is frequently associated with a diarrheal syndrome popularity known as "white scours," "milk scours," or "3-week-old scours." Pigs less than 1 week old are infrequently infected, presumably because of adequate passive immunity. The infection resembles enzootic transmissible gastroenteritis. Diagnosis can be made by immunofluorescent staining of mucosal scrappings from the small intestines.     

Isolation of porcine epidemic diarrhea virus in porcine cell cultures and experimental infection of pigs of different ages

This paper describes the isolation of porcine epidemic diarrhea (PED) virus in Vero and porcine cell cultures, and the influence of age on disease in experimental infection. PED virus was isolated from the small intestine of piglets inoculated with PED samples and cultured in Vero, porcine bladder and kidney cells propagated in collagen-coated tissue culture plates in maintenance medium (MM) containing trypsin. In porcine bladder and kidney cell cultures inoculated with isolated PED virus, cytopathic effects (CPE) including cell fusion were detected. Specific brilliant fluorescence was observed in the cytoplasm of these cells. Two- and 7-day old, and 2-, 4-, 8- and 12-week old specific pathogen-free (SPF) pigs were orally inoculated with PED virus isolated from an outbreak. All 2- and 7-day old pigs inoculated developed severe watery diarrhea from post-inoculation day (PID) 1 and died between PID 3 and 4. Although three of five 2-week old pigs developed diarrhea on PID 1-4, they eventually recovered. In the 4-week old group, three of five pigs had mild diarrhea for 1-2 days. None of the 8- and 12-week old pigs showed any clinical signs. Antibodies against PED virus were detected in all surviving pigs by virus neutralization (VN) test and immunofluorescence assay (IFA). Therefore, there is an age-dependent resistance to pathogenic PED virus infection in pigs.     

Development of nasal, fecal and serum isotype-specific antibodies in calves challenged with bovine coronavirus or rotavirus

Unsuckled specific pathogen free calves were inoculated at 3-4 weeks of age, either intranasally (IN) or orally (O) with bovine coronavirus or O plus IN (O/IN) or O with bovine rotavirus. Shedding of virus in nasal or fecal samples, and virus-infected nasal epithelial cells were detected using immunofluorescent staining (IF), ELISA or immune electron microscopy (IEM). Isotype-specific antibody titers in sera, nasal and fecal samples were determined by ELISA. Calves inoculated with coronavirus shed virus in feces and virus was detected in nasal epithelial cells. Nasal shedding persisted longer in IN-inoculated calves than in O-inoculated calves and longer than fecal shedding in both IN and O-inoculated calves. Diarrhea occurred in all calves, but there were no signs of respiratory disease. Calves inoculated with rotavirus had similar patterns of diarrhea and fecal shedding, but generally of shorter duration than in coronavirus-inoculated calves. No nasal shedding of rotavirus was detected. Peak IgM antibody responses, in most calves, were detected in fecal and nasal speciments at 7-10 days post-exposure (DPE), preceeding peak IgA responses which occurred at 10-14 DPE. The nasal antibody responses occurred in all virus-inoculated calves even in the absence of nasal shedding of virus in rotavirus-inoculated calves. Calves inoculated with coronavirus had higher titers of IgM and IgA antibodies in fecal and nasal samples than rotavirus-inoculated calves. In most inoculated calves, maximal titers of IgM or IgA antibodies correlated with the cessation of fecal or nasal virus shedding. A similar sequence of appearance of IgM and IgA antibodies occurred in serum, but IgA antibodies persisted for a shorter period than in fecal or nasal samples. Serum IgG1 antibody responses generally preceeded IgG2 responses and were predominant in most calves after 14-21 DPE.     

The Response of Colostrum-Deprived, Specific Pathögen-Free Pigs to Experimental Infection with Teschen Disease Virus

The clinical response to Teschen disease and the excretion and rate of virus distribution in tissues of colostrum-deprived, specific pathogenfree pigs was determined. Severe, mild, and clinically inapparent responses to the disease were noticed following simultaneous intracranial and intranasal infections. Fourteen-day-old pigs reacted more severely to infection than 21-day-old pigs. The virus was detected in feces 2-3 days following infection but not in stools of surviving pigs 30 days after infection. The highest concentration of virus occurred during the incubation period and before onset of paralysis; the lowest concentrations were found during terminal disease stages. In tissues collected before or immediately after death of pigs, Teschen disease virus was found in several visceral organs but not in blood, urine or urinary bladder tissue. Virus yield was highest in brain and spinal cord tissues. Highest virus concentration was found in the cervical thoracic portions of the spinal cord, thalamus and cerebellum. Other aspects of the clinical disease are discussed.