Synergism between porcine reproductive and respiratory syndrome virus (PRRSV) and Salmonella choleraesuis in swine

Porcine reproductive and respiratory syndrome virus (PRRSV) and Salmonella choleraesuis are two leading causes of economic loss in the swine industry. While respiratory disease is common in both S. choleraesuis and PRRSV infections, the factors that contribute to its development remain largely undefined. We investigated the interaction of PRRSV, S. choleraesuis, and stress in 5-week-old swine. All combinations of three factors (inoculation with S. choleraesuis on Day 0, PRRSV on Day 3, and treatment with dexamethasone on Days 3-7) were used to produce eight treatment groups in two independent trials. Fecal samples, tonsil and nasal swabs, serum samples and postmortem tissues were collected for bacteriologic and virologic examinations. No clinical signs were observed in pigs inoculated with only PRRSV or only S. choleraesuis. In contrast, pigs which were dually infected with S. choleraesuis and PRRSV exhibited unthriftiness, rough hair coats, dyspnea, and diarrhea. The pigs which received all three treatment factors were the most severely affected and 43% (three of seven) of the animals in this group died. Individuals in this group shed significantly higher quantities of S. choleraesuis in feces and had significantly higher serum PRRSV titers compared to other treatments (p < or = 0.05). In addition, S. choleraesuis and PRRSV were shed longer and by more pigs in this group than other groups and S. choleraesuis was recovered from more tissues in this group on Day 21 post inoculation. These results suggested that PRRSV, S. choleraesuis, and dexamethasone acted synergistically to produce a syndrome similar to that observed in the field.     

Intranasal vaccination of pigs against Aujeszky's disease: comparison with one or two doses of attenuated vaccines in pigs with high maternal antibody titres

Ten-week-old pigs with high levels of maternally derived antibody (MDA) against Aujeszky's disease virus (ADV) were given either a single intranasal vaccination or one or two doses (with an interval of three weeks) of commercially available attenuated ADV vaccines intramuscularly. The pigs did not produce a clear neutralising antibody response to ADV. However, pigs vaccinated intranasally and pigs given two doses of attenuated ADV vaccines were protected against intranasal challenge with virulent ADV two months after the first vaccination. Pigs given one parenteral dose of attenuated ADV vaccine were insufficiently protected. Protection was shown by shorter periods of growth arrest and fever and a greater reduction of virulent virus shedding after challenge in vaccinated pigs than in unvaccinated control pigs. Although intranasal vaccination conferred protection comparable to two parenteral doses of attenuated vaccines, it reduced shedding of virulent virus much more effectively. These results, together with those of other studies, show that intranasal vaccination confers better protection against Aujeszky's disease in pigs with MDA than parenteral vaccination. However, the efficacy of intranasal vaccination also decreases with increasing levels of MDA at the time of vaccination.     

Intranasal vaccination of pigs against Aujeszky's disease: protective immunity at 2 weeks, 2 months and 4 months after vaccination in pigs with maternal antibodies.

The purpose of the study was to evaluate the short- and long-term immunity after intranasal vaccination in pigs with maternally derived antibodies (MDA). In two experiments, 10-week-old pigs with moderate MDA titres against Aujeszky's disease virus (ADV) were vaccinated intranasally with the Bartha strain of ADV to evaluate the protective immunity conferred at 2 weeks, 2 months and 4 months after vaccination. Protection was evaluated on the basis of severity of clinical signs, periods of fever and growth arrest, and duration and amount of virus excreted after challenge with a virulent ADV. During the first 2-3 weeks after vaccination, antibodies to ADV continued to decline as in unvaccinated control pigs. After that, antibody titres stabilized or gradually increased. At 2 weeks, 2 months and 4 months after vaccination, vaccinated pigs were significantly better protected than unvaccinated controls. The vaccinated pigs challenged 2 weeks after vaccination hardly developed any sign of disease. Mild signs of Aujeszky's disease and a growth arrest period of 5 days were observed in vaccinated pigs challenged 2 months after vaccination, whereas vaccinated pigs challenged 4 months after vaccination developed severe signs of disease and a growth arrest period of 13 days. Vaccinated pigs challenged 2 weeks after vaccination did not excrete challenge virus, and pigs challenged 2 or 4 months after vaccination excreted far less virus than unvaccinated controls. The results demonstrate that intranasal ADV vaccination of pigs with moderate MDA titres protected them from 2 weeks to at least 4 months after vaccination. Immunity steadily declined, however, after vaccination.     

Development of a classical swine fever subunit marker vaccine and companion diagnostic test

The development of a classical swine fever (CSF) subunit marker vaccine, based on viral envelope glycoprotein E2, and a companion diagnostic test, based on a second viral envelope glycoprotein E(RNS), will be described. Important properties of the vaccine, such as onset and duration of immunity, and prevention of horizontal and vertical transmission of virus were evaluated. A single dose of the vaccine protected pigs against clinical signs of CSF, following intranasal challenge with 100LD(50) of virulent classical swine fever virus (CSFV) at 2 weeks after vaccination. However, challenge virus transmission to unvaccinated sentinels was not always completely inhibited at this time point. From 3 weeks up to 6 months after vaccination, pigs were protected against clinical signs of CSF, and no longer transmitted challenge virus to unvaccinated sentinels. In contrast, unvaccinated control pigs died within 2 weeks after challenge. We also evaluated transmission of challenge virus in a setup enabling determination of the reproduction ratio (R value) of the virus. In such an experiment, transmission of challenge virus is determined in a fully vaccinated population at different time points after vaccination. Pigs challenged at 1 week after immunization died of CSF, whereas the vaccinated sentinels became infected, seroconverted for E(RNS) antibodies, but survived. At 2 weeks after vaccination, the challenged pigs seroconverted for E(RNS) antibodies, but none of the vaccinated sentinels did. Thus, at 1 week after vaccination, R1, and at 2 weeks, R=0, implying no control or control of an outbreak, respectively. Vertical transmission of CSFV to the immune-incompetent fetus may lead to the birth of highly viraemic, persistently infected piglets which are one of the major sources of virus spread. Protection against transplacental transmission of CSFV in vaccinated sows was, therefore, tested in once and twice vaccinated sows. Only one out of nine once-vaccinated sows transmitted challenge virus to the fetus, whereas none of the nine twice-vaccinated sows did. Finally, our data show that the E(RNS) test detects CSFV-specific antibodies in vaccinated or unvaccinated pigs as early as 14 days after infection with a virulent CSF strain. This indicates that the E2 vaccine and companion test fully comply with the marker vaccine concept. This concept implies the possibility of detecting infected animals within a vaccinated population.     

Experimental and natural infection of early weaned pigs with Salmonella choleraesuis

A model for experimental and natural infection of early weaned pigs with Salmonella choleraesuis, the aetiologic agent of swine paratyphoid, has been developed. An oral dose of 10⁸ colony forming units (cfu) of S choleraesuis caused 100 per cent infection of 10 pigs inoculated, as indicated by recovery of the challenge organism from ileocolic lymph nodes collected at necropsy seven days post challenge. Seven of the pigs were observed shedding S choleraesuis at least once post S choleraesuis challenge. The cumulative incidence of shedding was 30 per cent and was sufficient to infect four of 10 pigs exposed naturally. Oral challenges with less than 10⁸ cfu S choleraesuis were less effective in infecting early weaned pigs and did not result in natural transmission.     

Duration of the protection of an E2 subunit marker vaccine against classical swine fever after a single vaccination

The period during which pigs are protected after vaccination is important for the successful usage of a marker vaccine against classical swine fever virus (CSFV) in an eradication programme. In four animal experiments with different vaccination-challenge intervals we determined the duration of protection of an E2 subunit marker vaccine in pigs after a single vaccination. Unvaccinated pigs were included in each group to detect transmission of the challenge virus.Three groups of six pigs were vaccinated once and subsequently inoculated with the virulent CSFV strain Brescia after a vaccination-challenge interval of 3, 51/2, 6 or 13 months. All vaccinated pigs, 16 out of 18, with neutralising antibodies against CSFV at the moment of challenge, 3, 51/2, 6 or 13 months later, survived, whereas unvaccinated control pigs died from acute CSF or were killed being moribund. A proportion of the vaccinated pigs did however develop fever or cytopenia after challenge and two vaccinated pigs were viremic after challenge. Virus transmission of vaccinated and challenged pigs to unvaccinated sentinel pigs did not occur in groups of pigs which were challenged 3 or 6 months after a single vaccination. Two out of eight vaccinated pigs that were found negative for CSFV neutralising antibody at 13 months after vaccination died after subsequent challenge.The findings in this study demonstrate that pigs can be protected against a lethal challenge of CSFV for up to 13 months after a single vaccination with an E2 subunit marker vaccine.     

In vivo isolation of Salmonella choleraesuis from porcine neutrophils.

Seventy-five pigs from 4 facilities were examined for Salmonella choleraesuis by use of bacteriologic culture of feces, blood, WBC (buffy coat), mononuclear leukocytes, and neutrophils. The organism was isolated from 0 of 75 fecal samples, compared with isolation from 39 of 75 purified neutrophil preparations. Of the pigs that did not have Salmonella isolated from feces or blood, but had S choleraesuis isolated from neutrophils, 6 were further examined. These pigs from 2 groups again had culture performed at least 3 successive times to test for repeatability and to determine optimal number of neutrophils required for Salmonella isolation. These same pigs were euthanatized and necropsied. Nineteen tissue specimens from each pig were obtained for culture, but S choleraesuis was isolated only from neutrophil samples. Results indicate that neutrophils may contribute to the carrier state in pigs and should be cultured when attempting to identify S choleraesuis carrier swine.     

Characterization and immunogenicity of an apxIA mutant of Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae is the aetiological agent of porcine pleuropneumonia, a highly contagious and often fatal disease. A candidate live vaccine strain, potentially capable of cross-serovar protection, was constructed by deleting the section of the apxIA gene coding for the C-terminal segment of ApxI toxin of the A. pleuropneumoniae serovar 10 reference strain (D13039) and inserting a chloramphenicol resistance gene cassette. The mutant strain (termed D13039A(-)Chl(r)) produced an approximately 48kDa protein corresponding to the N-terminus of the ApxI toxin, and exhibited no haemolytic activity and lower virulence in mice compared with the parental strain. The mutant was evaluated in a vaccination-challenge trial in which pigs were given two intra-nasal doses of the mutant at 14 days intervals and then challenged 14 days after the last vaccination with either A. pleuropneumoniae serovar 1 (4074) or serovar 2 (S1536) or serovar 10 (D13039) reference strains. The haemolysin neutralisation titres of the pre-challenge sera were significantly higher in the vaccinated pigs than in the unvaccinated pigs. The mortalities, clinical signs and lung lesion scores in the vaccinated pigs were significantly lower than those in the unvaccinated pigs for the serovar 1 challenge. A significantly lower lung lesion score was also observed in the vaccinated pigs, compared with unvaccinated pigs, for serovar 2 challenge. Our work suggests that the mutant strain offers potential as a live attenuated pleuropneumonia vaccine that can provide cross-serovar protection.     

Galactose epimeraseless mutants of Salmonella typhimurium as live vaccines for calves.

The purpose of the study was to evaluate the safety and efficacy of a galactose epimeraseless mutant of Salmonella typhimurium administered as an oral vaccine to one week old calves and to investigate properties of galactose epimeraseless mutants which affect their virulence and immunogenicity. The galactose epimeraseless mutant S. typhimurium strain G30D caused diarrhea and fever in three calves to which it was administered orally at a dose of 10(10) organisms; all three calves died following challenge with virulent S. typhimurium ten days postvaccination. Mild illness developed in four calves vaccinated with a dose of 9 X 10(6) organisms and one of these calves survived challenge. Three unvaccinated calves died following challenge. The vaccine organism persisted in tissues and was shed for a prolonged period by calves which received 10(10) organisms. Studies of characteristics of galactose epimeraseless mutants of S. typhimurium showed that, in the presence of galactose, there is selection for secondary mutants which are galactose resistant. The studies indicate that galactose epimeraseless mutants of S. typhimurium are not good candidate live vaccine organisms for use in calves.     

Efficacy of an intranasal immunization with gEgC and gEgI double-deletion mutants of Aujeszky's disease virus in maternally immune pigs and the effects of a successive intramuscular booster with commercial vaccines

In this study, an intranasal immunization strategy was set up in maternally immune pigs in order to protect them not only clinically but also virologically. Two genetically engineered Aujeszky's disease virus (ADV) strains, Kaplan gE-gI- and Kaplan gE-gC-, were used for intranasal immunization. Both strains were safe for 4-week-old pigs. A single intranasal inoculation of 10(6.0) TCID50 of Kaplan gE-gI- and Kaplan gE-gC- at 4 weeks of age in the presence of moderate titres of maternally derived antibodies (SN titres: 12-16) reduced the amount of weight loss, fever and virus excretion upon challenge 6 weeks later. In a second experiment, the effect of an additional intramuscular booster with three different commercial vaccines (containing attenuated Bartha or NIA3-783 or inactivated Phylaxia; all suspended in an oil-in-water emulsion) at 10 weeks of age was evaluated. One month after the last intramuscular booster, between five and seven pigs from each group were selected for challenge. All intranasally/intramuscularly immunized pigs showed a significantly better clinical and virological protection after challenge than the single intranasally immunized pigs. In the double immunized group, the protection was better when Kaplan gE-gC- was used for the intranasal priming (only two of 14 pigs excreted virus with a duration of 4 days) than when Kaplan gE-gI- was used (13 of 18 pigs excreted virus with a duration ranging from 1 to 4 days). The virological protection was not influenced by the type of vaccine used for booster vaccination. Because the intranasal/intramuscular immunization approach is very compatible with current pig movements on farms and pigs with moderate levels of maternally derived antibodies can effectively be immunized, it can be considered as a good alternative to intramuscular/intramuscular vaccinations especially in regions with a high ADV prevalence.     

Pseudorabies virus latency and reactivation in vaccinated swine

Latency and reactivation of pseudorabies virus in swine was studied. Thirty-one pigs were assigned to 5 groups and were given 1 of 4 vaccines; 10 remained unvaccinated controls. All pigs were then challenge exposed with a sublethal dose of virulent pseudorabies virus. One hundred one days after challenge exposure, all pigs were treated with dexamethasone to reactivate the virus. Virus-positive tonsil and nasal mucus isolates were recovered from 29 of the 31 pigs over a 12-day period. Frequency and duration of virus-positivity were significantly (P less than 0.05) and consistently lower among vaccinated pigs than among the unvaccinated controls. It was concluded that vaccination before challenge exposure had little or no effect on the rate of establishment of virus latency, but that vaccination reduced shedding after subsequent reactivation of the virus.     

一种A型魏氏梭状芽孢杆菌氢氧化铝灭活疫苗及其控制猪“猝死症”的效果

Ａ型魏氏梭状芽孢杆菌ＸＰ５株分离自典型猝死症病猪，将其制成氢氧化铝灭活疫苗。疫苗０．６ｍｌ、２ｍｌ和４ｍｌ的剂量可以分别保护小鼠、兔和猪抵抗标准Ａ型魏氏梭状芽孢杆菌毒素１ＭＬＤ的攻击。在两个疫病流行区应用了疫苗，１２１５头接种疫苗猪中死于猝死症的有１２头，死亡率０．９９％，８１５２头未接种疫苗的对照猪中，死于猝死症的有２８４头，死亡率３．４８％，二者显著差异（Ｘ２＝１２．２３，Ｐ＜０．０１）。     

Mucosal vaccination with formalin-inactivated avian metapneumovirus subtype C does not protect turkeys following intranasal challenge

Studies were performed to determine if mucosal vaccination with inactivated avian metapneumovirus (aMPV) subtype C protected turkey poults from clinical disease and virus replication following mucosal challenge. Decreases in clinical disease were not observed in vaccinated groups, and the vaccine failed to inhibit virus replication in the tracheas of 96% of vaccinated birds. Histopathologically, enhancement of pulmonary lesions following virus challenge was associated with birds receiving the inactivated aMPV vaccine compared to unvaccinated birds. As determined by an enzyme-linked immunosorbent assay (ELISA), all virus-challenged groups increased serum immunoglobulin (Ig) G and IgA antibody production against the virus following challenge; however, the unvaccinated aMPV-challenged group displayed the highest increases in virus-neutralizing antibody. On the basis of these results it is concluded that intranasal vaccination with inactivated aMPV does not induce protective immunity, reduce virus shedding, or result in decreased histopathologic lesions.     

Protection of pigs against challenge with virulent Streptococcus suis serotype 2 strains by a muramidase-released protein and extracellular factor vaccine

The efficacy of a muramidase-released protein (MRP) and extracellular factor (EF) vaccine in preventing infection and disease in pigs challenged either with a homologous or a heterologous Streptococcus suis serotype 2 strain (MRP+EF+) was compared with the efficacy of a vaccine containing formalin-killed bacterin of S. suis serotype 2 (MRP+EF+). The enhancement of the immune response by different adjuvants (a water-in-oil emulsion [WO] and an aluminium hydroxide-based adjuvant [AH]) and their side effects were also studied. The MRP and EF were purified by affinity chromatography. Pigs were vaccinated twice at three weeks and six weeks of age and challenged intravenously with virulent S. suis serotype 2 strains (MRP+EF+) at eight weeks of age. At challenge, the pigs vaccinated with MRP+EF/WO had high anti-MRP and anti-EF titres and were protected as effectively as pigs vaccinated with WO-formulated vaccines with bacterin. Eight of the nine pigs survived the challenge and almost no clinical signs of disease were observed. The titres obtained with the MRP+EF/AH vaccine were low and only two of the five pigs were protected. Pigs vaccinated with either MRP or EF were less well protected; three of the four pigs died after challenge but the clinical signs of disease were significantly less severe than those observed in the placebo-vaccinated pigs. The protective capacity of the bacterin/AH vaccine was very low, and the mortality among these pigs was as high as in the placebo-vaccinated pigs (80 per cent). Postmortem histological examination revealed meningitis, polyserositis and arthritis in the clinically affected pigs. The results demonstrate that a subunit vaccine containing both MRP and EF, formulated with the WO adjuvant, protected pigs against challenge with virulent S. suis type 2 strains.     

Vaccination of calves with a diaminopimelic acid mutant of Salmonella typhimurium.

The purpose of this study was to evaluate the safety and efficacy of a diaminopimelic acid mutant of Salmonella typhimurium as a vaccine for calves. Transposon techniques were used to create a stable nonreverting diaminopimelic acid mutant of a virulent S. typhimurium strain. Calves were vaccinated at weekly intervals with the diaminopimelic acid mutant given as an oral dose of 10(10) organisms, followed by two subcutaneous doses of 10(9) organisms. The calves tolerated vaccination well and the vaccine strain was eliminated from the feces within four days. Of five vaccinated calves, three survived challenge with 5 X 10(9) organisms of the parent strain whereas all five unvaccinated calves that were challenged died. The surviving calves eliminated the challenge organism from the feces within three weeks.     

Studies on the pathogenesis of Salmonella typhimurium and Salmonella choleraesuis var kunzendorf infection in weanling pigs

Twenty-six 4-week-old pigs were randomly allotted to 4 groups: group 1--orally inoculated with Salmonella typhimurium; group 2--orally dosed with S choleraesuis; and groups 3 and 4, with surgically constructed intestinal loops--loops inoculated with either S typhimurium or S choleraesuis. One pig each from groups 1 and 2 was killed at 8, 12, 24, 48, 72, 96, and 120 hours after inoculation. One pig each from groups 3 and 4 was killed at 2, 4, 6, 8, 12, and 24 hours after intestinal loop inoculation. Inoculation of S typhimurium resulted in acute enterocolitis of variable severity, whereas inoculation of S choleraesuis resulted initially in septicemia followed by formation of large necrotic and ulcerative lesions in the colonic mucosa. The most consistent systemic lesion of S choleraesuis infection was interstitial pneumonia and multifocal hepatic necrosis. Salmonella typhimurium and S choleraesuis were ultrastructurally within enterocytes of ligated ileal loops. Intracellular bacteria were morphologically intact, occurred free in the cytoplasm and membrane bound, and caused no detectable cytotoxic effect to the cell. Both S typhimurium and S choleraesuis penetrated the intestinal mucosa and were isolated from mesenteric lymph nodes at 2 hours after inoculation.     

Mucosal vaccination against influenza: protection of pigs immunized with inactivated virus and ether-split vaccine

Effective vaccinations against swine influenza reduce the economic loss of pig industries, and also may minimize the possibility of emergence of new pandemic viruses, since pigs are intermediate hosts to generate reassortant viruses among avian and mammalian influenza viruses. In this study, we showed that intranasal immunization of pigs with formalin-inactivated or ether-split influenza vaccine (A/Aichi/2/68) induced virus-specific IgG, IgM, and IgA antibodies in their nasal secretions and sera, resulting in complete protection from virus challenge. Antibody response to the challenge virus was not observed in the immunized pigs, suggesting that the replication of the virus in the primary targets, respiratory epithelial cells, was inhibited. The present results indicate that intranasal immunization of pigs with inactivated vaccines is effective to control swine influenza, and also provide a good model, as well as a mouse model, to evaluate an intranasal application of influenza vaccine for humans.     

Level of virulent virus excreted by infected pigs previously vaccinated with different glycoprotein deleted Aujeszky's disease vaccines.

Seven deleted Aujeszky's disease vaccines were compared for their ability to induce an immunity which suppresses virus excretion. For each vaccine, the levels of clinical protection and viral excretion were compared. Groups of eight pigs were vaccinated twice with attenuated deleted Aujeszky's disease vaccines (which do not express certain glycoproteins: gI, gX or gp63). Pigs were vaccinated at the beginning of the fattening period and challenge took place at the end of it when the pigs were 18-19 weeks old. Live virus vaccines were suspended in water or in an oil-in-water emulsion. The experiment was performed in three successive assays of two groups of eight pigs (except three groups for the first assay). At each assay, a control unvaccinated group of eight pigs was added to compare the effects of challenge between vaccinated and unvaccinated animals. In total, 80 pigs were involved in this experiment. All the vaccinated pigs excreted virus from 3 to 9 d after challenge. However the level of viral excretion and the duration of the period of excretion were reduced after vaccination and especially, when oil-in-water emulsion was used. There were obvious differences between vaccines. With some vaccines, when the level of viral excretion was low, the level of clinical protection was high. However, in other cases, the level of clinical protection could be good despite a higher level of viral excretion. The seroneutralizing titres were significantly and inversely related to a low level of viral excretion but not to the level of clinical protection.     

Experiences with a vaccine being developed for the control of swine dysentery

SUMMARY A prototype vaccine that is being developed for the control of swine dysentery (SD) was tested in two groups of experimental pigs. Vaccination induced high circulating antibody titres against the aetiological agent, Serpulina (Treponema) hyodysenteriae .
Pigs in the first trial were vaccinated twice before being challenged orally with the bacteria. Five of 6 unvaccinated animals developed dysentery within a fortnight of challenge, but only 1 of 6 vaccinated pigs showed signs of disease at this time. Unexpectedly, 1 mo after challenge, the surviving unvaccinated pig and 2 remaining healthy vaccinated animals succumbed to the disease. The reason for the development of this late-onset form of dysentery was not clear.
In the second trial, 8 pigs were vaccinated 3 times. Only 2 of these animals (25%) developed severe dysentery after being mixed with infected pigs, whereas 7 of 8 (88%) unvaccinated control pigs in the same pen became diseased. The late-onset form of dysentery was not observed.
The prototype vaccine for SD provided a useful level of protection, and could be used in programs to control the disease in Australia.     

Interaction of an intranasal combined feline viral rhinotracheitis, feline calicivirus vaccine and the FVR carrier state

Eight cats were vaccinated intranasally with a combined feline calicivirus/feline viral rhinotracheitis (FVR) virus commercial vaccine. Following intranasal challenge with a field strain of FVR virus and subsequent treatment with corticosteroid, no virus was recovered from any of the eight cats, while FVR virus was recovered following corticosteroid treatment from two of four unvaccinated and challenged controls. No evidence was found for the development of an FVR virus carrier state with the intranasal vaccine virus.