Activation of natural killer cells in newborn piglets by interferon induction

Natural killer (NK) cell activity in the peripheral blood lymphocytes (PBL) of newborn piglets, normally negligible, was stimulated by in vitro treatment with porcine type I interferon (IFN), and the NK activity of PBL from weaned piglets was augmented by the same treatment. Binding of the PBL to the PK-15 targets used in the single cell cytotoxicity assay for NK activity was not affected by age or by IFN treatment. When newborn piglets were treated with a single intravenous dose at 2 days of age of 0.5 mg/kg of polyinosinic:polycytidylic acid complexed with poly-L-lysine and carboxymethylcellulose (poly ICLC), a synthetic IFN inducer, their IFN levels peaked at 6 h post-induction, and NK activity in their PBL peaked at 24 h post-induction at the level normally found in weaned piglets. The NK activity then declined until 7 days post-induction, when it increased again in a similar manner to that in untreated control piglets. Target-binding of the PBL was not affected by poly ICLC treatment of the piglets. Newborn piglets treated with poly ICLC and subsequently exposed to infection with transmissible gastroenteritis (TGE) virus showed a delay in onset of clinical signs of TGE compared with untreated control piglets. It was concluded that NK cells in newborn piglets can be activated by treatment of the piglets with poly ICLC, and that the presence of active NK cells is associated with some increase in resistance to challenge with TGE virus.     

Interferon induction with polyinosinic:polycytidylic acid in the newborn piglet.

Newborn piglets were treated with various doses of polyinosinic:polycytidylic acid intravenously and their serum interferon responses determined by a plaque reduction assay with vesicular stomatitis virus in Madin-Darby bovine kidney cells. A single dose of 5 mg of polyinosinic:polycytidylic acid was found consistently to induce detectable levels of interferon in serum, while the response to lower doses was inconsistent and higher doses produced clinical signs of toxicity. Piglets receiving 5 mg of polyinosinic:polycytidylic acid had maximum serum interferon titers between four and eight hours after treatment, and interferon was no longer detected at 72 hours after treatment. Following treatment with polyinosinic:polycytidylic acid leukopenia was observed, coincident with peak serum interferon titers. Elevated levels of serum glutamic oxaloacetic transaminase and blood urea, indicative of hepatic and renal dysfunction respectively, were also observed following interferon induction with polyinosinic:polycytidylic acid. Piglets treated with polyinosinic:polycytidylic acid also demonstrated antiviral activity in their intestinal mucosal tissues and intestinal washes, but the antiviral activity in the intestinal wash was not characterizable as interferon. A factor in the intestinal washes from newborn piglets was found to antagonize the antiviral effects of interferon by enhancing the plaque forming ability of vesicular stomatitis virus.     

Sows infected in pregnancy with porcine respiratory coronavirus show no evidence of protecting their sucking piglets against transmissible gastroenteritis

Eighteen litters of sucking piglets were challenged with one of two strains of transmissible gastroenteritis virus (TGEV). During pregnancy, their seronegative dams had been either inoculated intranasally with porcine respiratory coronavirus (PRCV), inoculated orally with TGEV or left untreated. On the basis of weight gain, clinical signs and survival, no differences in response to challenge was detected when piglets suckled by PRCV inoculated sows were compared with those suckled by uninoculated sows. Such a difference was evident when the litters of sows successfully pre-immunized with TGEV were compared with those of unicoculated or PRCV-inoculated sows. The possibility of transplacental transmission of PRCV was investigated in two litters born to sows that had been inoculated with this virus in late pregnancy. All sixteen live-born piglets were seronegative for the virus at birth and PRCV was not isolated from tissues taken from two stillborn piglets.     

The effect of interferon induction in newborn piglets on the humoral immune response to oral vaccination with transmissible gastroenteritis virus.

Groups of newborn piglets were vaccinated orally with a modified live transmissible gastroenteritis (TGE) virus vaccine at 3 days and 13 days of age, and treated with the synthetic interferon (IFN) inducer polyinosinic:polycytidylic acid (poly ICLC) at 2, 3 or 4 days of age. Control groups consisted of piglets which were vaccinated but not treated with poly ICLC, as well as piglets which were treated with poly ICLC but not vaccinated. Significantly higher mean IFN titres were produced in response to induction at 3 or 4 days of age than at 2 days, and the mean IFN titre of the vaccinated piglets treated with poly ICLC at 3 days of age was significantly higher than in the unvaccinated piglets which were treated at the same time. The mean TGE virus neutralizing antibody titres in the vaccinated piglets which were treated with poly ICLC on the day before vaccination were significantly lower than the mean titres in the untreated vaccinated piglets 10 and 14 days after the first dose of vaccine. The mean titres in the vaccinated piglets which were treated with poly ICLC at 3 or 4 days of age did not differ significantly from those in the untreated vaccinated piglets. The piglets which were treated with poly ICLC on the day after vaccination developed severe diarrhoea which persisted for 5-7 days.     

The interferon sensitivity of selected porcine viruses.

The objective of this study was to compare the sensitivity of 11 porcine viruses to the antiviral effects of porcine interferon-alpha in serum from piglets which had been infected 19 h previously with transmissible gastroenteritis virus, and of porcine interferon-beta prepared in PK-15 cells by induction with polyinosinic:polycytidylic acid, in yield reduction assays in pig kidney cells which were treated with interferon before virus challenge, and both before and after virus challenge. The most sensitive virus to both types of interferon was vesicular stomatitis. A porcine isolate of bovine herpesvirus type 1, hemagglutinating encephalomyelitis virus and porcine enterovirus types 1 and 2 were also highly sensitive to interferon-alpha. There was little reduction in the yield of porcine parvovirus or porcine rotavirus, while swinepox, swine influenza and transmissible gastroenteritis viruses were intermediate in their sensitivity to interferon-alpha. In addition to vesicular stomatitis virus, porcine adenovirus type 3, swine influenza, hemagglutinating encephalomyelitis and porcine rotavirus were highly sensitive to interferon-beta, while swinepox, bovine herpesvirus type 1, porcine parvovirus, transmissible gastroenteritis and porcine enteroviruses were less sensitive than the above viruses to interferon-beta, although all showed significant reductions in virus yield.     

The postulated role of feeder swine in the perpetuation of the transmissible gastroenteritis virus.

Clinical, immunofluorescence and histopathological observations were found to be an efficient approach for the confirmation of the diagnosis of transmissible gastroenteritis in feeder swine. Two cases are reported to exemplify how feeder swine exposed to points of concentration such as holding areas, sales barns and auctions can play an important role in the epizootiology of transmissible gastroenteritis. A third field case is reported as an example of an outbreak of transmissible gastroenteritis beginning in feeder swine and then spreading to baby pigs on the farm. All baby pigs died that were born during the acute phase of the outbreak in the feeder swine. Baby pigs born shortly after the clinical signs had abated in the herd, and from sows that had been exposed orally to virulent transmissible gastroenteritis virus and vaccinated with a commercial transmissible gastroenteritis vaccine ten days before farrowing, survived. This was explained by a combination of a decrease in the amount of virus shed in the environment and the immunity induced in the sows. These observations of field outbreaks of transmissible gastroenteritis combined with recently reported experimental studies lend strong support to the hypothesis of a reservoir for transmissible gastroenteritis virus in feeder pigs. This reservoir would be based principally on the transmission of the virus on a continuous basis from the feces of recently infected pigs to susceptible pigs. Clinical signs of transmissible gastroenteritis in such pigs are difficult to recognize or absent and this contributes to the importance of the reservoir in the field.     

Mild transmissible gastroenteritis in pigs suckling vaccinated sows

A strain of transmissible gastroenteritis (TGE) virus of low virulence was isolated from 14-day-old pigs suckling sows vaccinated with an attenuated TGE vaccine. Diarrhea developed in suckling pigs approximately 14 days after farrowing in 4 farrowings; however, none of these pigs died from diarrhea. Diarrhea ceased after the 4th farrowing, when vaccination of sows was discontinued. Experimentally, both the field isolate and the vaccine strain were infective and in some instances lethal for 2-day-old pigs exposed orally; however, neither strain was as virulent as the Purdue strain.     

Upper respiratory infection of lactating sows with transmissible gastroenteritis virus following contact exposure to infected piglets.

Ten breeding sows were left in direct contact with their newborn piglets that had been experimentally infected with transmissible gastroenteritis (TGE) virus. All sows became infected with the virus. The sows developed fever and showed mild clinical signs of the disease for a few days. The sows excreted virus in the nasal secretion, feces, and milk during the acute febrile phase of illness. Virus was isolated from the nasal secretion of one sow as early as 20 hours after contact exposure to the infected piglets. At necropsy, the virus was more frequently isolated from the tissues of the upper respiratory tract than from small intestines; this finding indicated that the TGE coronavirus replicated in the upper respiratory tract and induced an acute respiratory infection in susceptible adult swine. Neutralizing antibody was present in the sera 8 sows after 12 to 36 days during the convalescent period. From these results, we conclude that susceptible sows in direct contact with ill piglets can become infected and by excreting virus can serve as a source of TGE virus for other susceptible pigs on the premises.     

Quantitative transmissible gastroenteritis virus shedding patterns in lactating sows.

To test the role of sows in spreading transmissible gastroenteritis (TGE), 11 sows were intravenously, intranasally, or intramammarily inoculated with virulent virus within 5 days of farrowing. Six of the sows were separated from their offspring, and 5 were allowed to nurse their litters. All sows became clinically ill with sign of anorexia, depression, and fever that persisted until postinoculation day 4 or 5. They shed virus through milk, nasal secretions, and feces, with individual variations occurring in degree and duration of shedding in the 1st week after inoculation. Of 40 pigs separately fed milk samples from the 6 inoculated sows, 19 pigs (47.5%) became sick in 24 to 40 hours, and virus was isolated from them at necropsy. Of 43 pigs in the 5 litters that nursed exposed dams, all became sick with typical signs of TGE, and 29 (67.4%) died in 2 to 9 days. Sows given the single intramammary inoculation of virus developed statistically significant higher levels of TGE virus-neutralizing antibodies than did sows inoculated intravenously or intranasally.     

Vaccination of pregnant sows against transmissible gastroenteritis with two attenuated virus strains and different inoculation routes

Summary Two attenuated transmissible gastro-enteritis (T. G. E.) virus strains were used for vaccination experiments in sows. Four different experiments were carried out (see Table 1). In each experiment, 9 sows were vaccinated during pregnancy and 3 sows served as controls. They were kept together in one farrowing house. The sows were due to farrow at about the same time. The sows and their litters were challenged shortly after farrowing by exposing 3 piglets of 2 control litters to virulent TGE virus. The following vaccination schedules were used (see Table 1): twice intramuscularly with TGE-vac (a commercially available TGE-vaccine), one oral administration followed by an intramuscular vaccination with an attenuated TGE Purdue (Pu) strain, twice orally with Pu strain in enteric coated capsules, and one direct intra intestinal administration followed by 2 intramuscular vaccinations or 3 intramuscular vaccinations with the Pu strain. All sows, except most of those treated with enteric coated capsules, seroconverted demonstrably (Table 2). The geometric mean seroneutralization (SN) titer log 2 varied from 4.1 to 7.5 after the first vaccination and from 7.6 to 10 after the second vaccination. None of the vaccination schedules resulted in an effective lactogenic immunity. The morbidity in the piglets was 100% within 3 to 5 days after challenge. The mortality rate varied from 44 to 80% in litters from vaccinated sows and from 71 to 100% in litters from control sows (see Table 3). Clinical signs were observed in 33,3% of the control sows and in 36% of the vaccinated sows. No correlation was found between the titer of SN antibodies in the sera of the piglets and their survival rate (Table 4). A rapid decrease in antibody concentration was observed, during the first week of lactation in milk samples collected from 4 orally and intramuscularly vaccinated sows (Table 5).     

Vaccination of pregnant sows against transmissible gastroenteritis with two attenuated virus strains and different inoculation routes

Summary

Two attenuated transmissible gastro‐enteritis (T. G. E.) virus strains were used for vaccination experiments in sows.

Four different experiments were carried out (see Table 1). In each experiment, 9 sows were vaccinated during pregnancy and 3 sows served as controls. They were kept together in one farrowing house. The sows were due to farrow at about the same time. The sows and their litters were challenged shortly after farrowing by exposing 3 piglets of 2 control litters to virulent TGE virus.

The following vaccination schedules were used (see Table 1): twice intramuscularly with TGE‐vac (a commercially available TGE‐vaccine), one oral administration followed by an intramuscular vaccination with an attenuated TGE Purdue (Pu) strain, twice orally with Pu strain in enteric coated capsules, and one direct intra intestinal administration followed by 2 intramuscular vaccinations or 3 intramuscular vaccinations with the Pu strain.

All sows, except most of those treated with enteric coated capsules, seroconverted demonstrably (Table 2). The geometric mean seroneutralization (SN) titer log 2 varied from 4.1 to 7.5 after the first vaccination and from 7.6 to 10 after the second vaccination.

None of the vaccination schedules resulted in an effective lactogenic immunity. The morbidity in the piglets was 100% within 3 to 5 days after challenge. The mortality rate varied from 44 to 80% in litters from vaccinated sows and from 71 to 100% in litters from control sows (see Table 3). Clinical signs were observed in 33,3% of the control sows and in 36% of the vaccinated sows.

No correlation was found between the titer of SN antibodies in the sera of the piglets and their survival rate (Table 4).

A rapid decrease in antibody concentration was observed, during the first week of lactation in milk samples collected from 4 orally and intramuscularly vaccinated sows (Table 5).     

Induction of lactogenic immunity to transmissible gastroenteritis virus of swine using an attenuated coronavirus mutant able to survive in the physicochemical environment of the digestive tract.

A transmissible gastroenteritis (TGE) coronavirus mutant (188-SG), selected as attenuated and resistant to acidity and proteases of the digestive tract of adult pigs, was used as vaccine ("Nouzilly strain") in sows to protect suckling piglets against a challenge exposure carried out with a highly virulent TGEV strain. The pregnant sows were immunized once (42-49 days before farrowing) or twice (42-49 and 7-15 days before farrowing) by the oral, intramuscular or conjunctival route with the 188-SG strain. Sows exposed to virulent TGEV in the field and experimentally infected sows (two oral inoculations during pregnancy) were used as positive controls leading to high protection. The neutralizing antibody response to vaccination and/or infection was studied in serum and milk. No protection against mortality was observed in the litters of (1) the nine seronegative, susceptible sows, with piglet mortality of 65/70, (2) the seven once orally vaccinated sows, with mortality of 44/54, (3) the seven sows vaccinated twice by the conjunctival route, with mortality of 55/76. Moderate protection was observed in (1) the eight sows vaccinated intramuscularly twice with piglet mortality of 36/90, (2) the seven orally and intramuscularly vaccinated sows with piglet mortality of 31/51. In of 3 contrast, improved protection was observed in (1) the 10 sows vaccinated twice orally, with piglet mortality of 23/95, (2) the four naturally infected sows with piglet mortality of 6/41, (3) the six sows experimentally infected with virulent TGEV with piglet mortality of 1/59. No correlation was found between neutralizing antibodies titers in serum and milk and protection rate of the piglets. The results indicate that relative protective lactogenic immunity against TGEV is induced only by repeated ingestion of the attenuated 188-SG strain of TGEV.     

Intestinal defence of the neonatal pig: interrelationship of gut and mammary function providing surface immunity against colibacillosis.

The neonatal requirements for maternal passive immunity and the lactation immunobiology with regard to sow immunisation for neonatal protection are reviewed. A vaccination protocol which combines oral and parenteral antigen administration to produce antibody activity mediated mainly by IgM is described. Its efficacy in affording protection to neonatal piglets was tested against a lethal oral infection with a virulent strain of Escherichia coli "Abbottstown". Piglets suckled on vaccinated or non-vaccinated sows were exposed to an infective challenge in the gastrointestinal tract and the relative pathology in test and control groups observed over the neonatal period. Death ensued in 76 per cent of piglets suckled on control sows and 26 per cent of piglets suckled on sows vaccinated by two intramuscular injections. Litters suckled on orally vaccinated sows were able to resist a similar infective challenge, there being only one fatality out of 42 piglets.     

Interferon induction in piglets with polyinosinic:polycytidylic acid complexed with poly-L-lysine and carboxymethylcellulose

When newborn piglets were inoculated intravenously with 1.0, 0.5 or 0.25 mg kg-1 of polyinosinic:polycytidylic acid (poly I:C) complexed with poly-L-lysine and carboxymethylcellulose (poly ICLC), the highest serum interferon levels and the lowest white blood cell counts were found in response to a dose of 0.5 mg kg-1. Similar responses were observed in weaned piglets inoculated with 0.25 mg kg-1 of poly ICLC. Poly ICLC was a more effective interferon inducer than poly I:C, particularly in newborn piglets.     

Interferon induction in porcine leukocytes with transmissible gastroenteritis virus

Leukocytes were harvested from the peripheral blood, mesenteric lymph node and small intestinal lamina propria from groups of three piglets before, and 1,2 and 3 weeks after infection with virulent transmissible gastroenteritis virus (TGEV) at 2 weeks of age. The donor piglets developed clinical signs of transmissible gastroenteritis which persisted for up to 3 days, and they developed peak serum titres of TGEV-neutralizing antibodies 2 weeks post-infection. The leukocytes were cultured in the presence of pokeweed mitogen (PWM), various dilutions of purified TGEV, or control media for 3 or 5 days, and the culture supernatants were tested for antiviral activity in MDBK cells challenged with vesicular stomatitis virus. The antiviral activity was characterized as porcine interferon (IFN)- or porcine IFN-τ on the basis of its stability at pH 2.0 and neutralization by anti-human IFN- antibodies. Viability of the leukocytes in culture, determined by trypan blue exclusion, was highest for the peripheral blood leukocytes and lowest for the mesenteric lymph node leukocytes. There were no consistent differences in antiviral activity between cultures incubated for 3 or 5 days. Porcine IFN- was found in the supernatants of the leukocyte cultures stimulated with TGEV antigen, harvested before or after infection of the donor piglets with TGEV. Porcine IFN-τ was demonstrated in the supernatants of the leukocyte cultures stimulated with PWM, more frequently when the leukocytes were harvested post-infection. This was the first demonstration of IFN induction in vitro in leukocytes from porcine gut-associated lymphoid tissue.     

Elimination of strains of Streptococcus suis serotype 2 from the tonsils of carrier sows by combined medication and vaccination

The effect of vaccination with a killed whole-cell vaccine of extracellular factor-positive Streptococcus suis serotype 2 (S suis 2 EF(+)) combined with medication with amoxicillin on the presence of virulent S suis 2 EF(+) strains on the tonsils of sows and their offspring was evaluated. In two herds, 14 pregnant sows that carried these virulent strains, as detected by PCR in three consecutive tonsillar brush samples, were selected and randomly assigned to be treated or left untreated as controls. The treated sows were vaccinated at six and three weeks before the expected farrowing date and medicated from one week before expected farrowing until the end of the experiment. Two weeks before parturition, the sows were housed in individual isolation farrowing rooms, and the sow and its litter were sampled by using tonsil brushes and tonsil swabs, respectively. Approximately 27 days postpartum, the sows and their piglets were euthanased and their tonsils were collected and analysed by pcr. No S suis 2 EF(+) could be detected in the tonsils of the seven treated sows, but the tonsils of the seven untreated sows remained positive. Only one of the litters of the untreated sows became infected, five days after birth, and none of the litters of the treated sows became infected.     

Experimental Immunization of Sows with Cell-cultured TGE Virus

Five sows were inoculated with a cell-cultured, cytopathic strain of the virus of transmissible gastroenteritis (TGE). Two sows were inoculated intramuscularly, and three by the intramammary route. The response was measured by the neutralizing antibody titers in the serum and the milk, and by the protection against experimental challenge of piglets nursing the sows. There were no marked differences in the serum or milk antibody titers resulting after the two methods of inoculation, although milk titers at the time of challenge were higher after intramammary inoculation. Piglets nursing sows inoculated by the intramammary route were protected to a greater extent than those nursing sows inoculated intramuscularly.     

Antiviral activity against transmissible gastroenteritis virus, and cytotoxicity, of natural porcine interferons alpha and beta.

Porcine interferon (POIFN)-alpha prepared in primed peripheral blood leukocyte cultures induced with Newcastle disease virus and POIFN-beta from PK-15 cell cultures induced with polyinosinic:polycytidylic acid were partially purified by precipitation with potassium thiocyanate and anion exchange chromatography. Mean purification factors in terms of units of POIFN per mg of protein, of 37 and 12 were obtained for POIFN-alpha and POIFN-beta respectively. In yield reduction assays in swine testis and pig kidney cell cultures, POIFN-alpha and POIFN-beta had greater antiviral activity against vesicular stomatitis virus than against transmissible gastroenteritis virus (TGEV). The antiviral effects were greater at higher concentrations of interferon (IFN), and when the IFN treatments were continued postinfection. Porcine interferon-beta showed greater antiviral activity against TGEV than POIFN-alpha, but this may have been partly due to cytotoxicity. There were no major differences in the antiviral activities of crude and partially purified IFN preparations. Both types of IFN showed antiviral activity against TGEV in yield reduction assays in porcine intestinal explant and intestinal epithelial cell cultures. Crude POIFN-beta was found to be rapidly cytotoxic, especially in porcine cells, and some fractions of partially purified POIFN-beta were also cytotoxic. The cytotoxicity of POIFN-beta was partially neutralized by antibodies against human IFN-beta, but human IFN-beta was not cytotoxic for porcine or bovine cells.     

Protective effect of immunoglobulins in serum and milk of sows exposed to transmissible gastroenteritis virus.

Experimental exposure of susceptible pregnant sows by various routes to the gut-origin transmissible gastroenteritis virus stimulated production of milk and serum antibodies. These antibodies neutralized the cytopathic effect of transmissible gastroenteritis virus propagated in cell culture. This in vitro neutralizing antibody resided in the IgG and IgA immunoglobulin classes. On the other hand, protection for baby pigs resided in the IgA class of milk immunoglobulin of sows exposed orally or intramammarily but not of sows exposed intramuscularly to the virus.     

Transmissible gastroenteritis in endemically infected breeding herds of pigs in East Anglia, 1981-85

Endemic infection was a common sequel to primary outbreaks of transmissible gastroenteritis in large breeding herds of pigs in East Anglia. The main clinical features of the disease were diarrhoea affecting sucking piglets aged about six days or more, diarrhoea among recently weaned pigs and brief episodes of overt clinical recrudescence in part of the herd. Post mortem and bacteriological findings were often more suggestive of colibacillosis than transmissible gastroenteritis. In some herds, endemic infection remained clinically mild or inapparent for long periods. Evidence of endemic transmissible gastroenteritis infection was found in 43 (50.6 per cent) of 85 herds of pigs studied prospectively between 1981 and 1983. There was a significant correlation with herd size; the disease recurred during the 12 months after primary outbreaks in 36 (65.5 per cent) of 55 herds with over 100 sows compared with seven (23.3 per cent) of 30 herds with less than 100 sows (P less than 0.001). In the larger herds it occurred more commonly where finishers were kept (P less than 0.05). Sow morbidity and management factors during the primary outbreak had no statistically significant effect on the incidence of recrudescence. Epidemiological aspects of the findings are discussed with emphasis on the difficulties associated with the diagnosis and control of endemic transmissible gastroenteritis infection.