Specific Pathogen-free Pigs Research Articles

Septicaemia and Arthritis in Pigs Experimentally Infected with Pasteurella multocida Capsular Serotype A

Twenty-five caesarean-derived, colostrum-deprived (CDCD) pigs and 18 specific pathogen-free pigs, aged 8 to 14 weeks, were inoculated intranasally or intratracheally with Pasteurella multocida capsular serotype A, isolated from a severe pneumonic lesion in a growing pig. The pigs were killed for necropsy on day 6 or 14 post-inoculation (PI) or, in the case of the only fatally infected animal, examined at the time of death. One CDCD pig, inoculated intratracheally with 5 ml of a bacterial suspension containing 1.7×10 9 colony-forming-units/ml, died of septicaemia on day 1 PI. Histological lesions such as severe pleuropneumonia, thrombi in glomerular capillaries, haemorrhage of the spleen, and abscesses in the tonsillar crypts were observed. The organism was recovered from a number of sites and its antigens were detected immunohistochemically in the pneumonic lesions, blood vessels of the tissues, and tonsillar crypts in the dead pig. Pneumonia, pleural adhesions and suppurative arthritis in the extremital joints were observed grossly in 3/29, 8/29 and 7/29 intratracheally inoculated pigs, respectively. In intranasally inoculated pigs, no macroscopical abnormalities were seen; histologically, however, exudative bronchopneumonia and fibrinous pleurisy were observed in 9/14 and 4/14 pigs, respectively. No significant changes were seen in the tissues of uninfected control pigs. The organism was recovered from the lesions and P. multocida type A antigen was demonstrated immunohistochemically. The organism was rarely recovered from the liver, spleen or lymph nodes (bronchopulmonary or mesenteric). The results suggest that P. multocida capsular serotype A alone can cause not only pneumonia in pigs but also septicaemia or arthritis.

Interleukin 6, serum amyloid A and haptoglobin as markers of treatment efficacy in pigs experimentally infected with Actinobacillus pleuropneumoniae

The possibility to use acute phase proteins to monitor the elimination of a bacterial infection in pigs would facilitate an objective assessment of treatment with various antimicrobial substances. To examine this possibility, the acute phase response (IL-6, serum amyloid A (SAA), and haptoglobin) elicited by Actinobacillus pleuropneumoniae and its reduction on treatment with various antibiotics was studied in serum from specific pathogen free (SPF) pigs. Pigs were infected intranasally with A. pleuropneumoniae serotype 2, and either left as non-treated control pigs or treated with different antibiotics intramuscularly at onset of respiratory disease (20 h post-infection). Pigs responded to the infection with prominent increases in activity and concentrations of IL-6, SAA, and haptoglobin. These responses were to a certain extent overlapping and covered the time span from a few hours after infection until development of detectable levels of specific antibodies (7–10 days post-infection in untreated pigs). The haptoglobin response lasted until the end of the study on day 17 and thereby partly coincided with the antibody response. Treatment with antimicrobials that effectively reduced establishment of the infection with A. pleuropneumoniae also reduced the duration of all three acute phase responses, and reduced the concentration of serum haptoglobin. In contrast, less efficacious treatments did not reduce these acute phase responses. Thus, acute phase reactants can be applied to monitor therapeutic effects of antimicrobial drugs in the pig and measurements of IL-6, SAA and haptoglobin could add valuable information about the stage of infection during a disease outbreak.

Pathogenicity of three isolates of porcine respiratory coronavirus in the USA

The pathogenicity of three isolates of porcine respiratory coronavirus (AR310, LEPP and 1894) from the USA was assessed in specific pathogen-free pigs. Pigs inoculated with 1894 developed mild respiratory disease...

Pathogenicity of Cryptosporidium parvum--evaluation of an animal infection model.

With the intention of developing a standardised method for assessment of pathogenicity of Cryptosporidium parvum, the CPB-0 isolate was studied by propagation in 1-day-old calves followed by inoculation into specific pathogen free (SPF) piglets. The experiment was repeated. Diarrhoea and shedding of oocysts were seen in all animals infected with the CPB-0 isolate. Clinical signs included depression, inappetence, vomiting (exclusively in the piglets), and death. Histological examination at 17 and 19 days post-infection revealed parasitic stages and microscopic changes primarily restricted to colon and rectum.The unintended presence of rotavirus in some of the experimental animals revealed an additive or synergistic effect between rotavirus and C. parvum as indicated by prolonged diarrhoea, increased oocyst shedding, decreased weight gain and elevated levels of serum haptoglobin and serum amyloid A (SAA) in piglets infected simultaneously with both pathogens. The difference in daily weight gain between infected and control animals was significant only for piglets co-infected with rotavirus. The acute phase response of haptoglobin and SAA was characterised by a large individual variation. In piglets, co-infected with rotavirus, the levels of serum haptoglobin were 3.5 and 4.6 times higher in the infected versus the controls 6 and 9 dpi, respectively (mean values: 2411 μg/ml±S.D. 2023 and 1840 μg/ml±S.D. 1697). In the controls infected with rotavirus, peak haptoglobin concentration was seen 3 dpi (mean: 1022 μg/ml±S.D. 425). Elevated levels of SAA were seen in 1 of 6 piglets infected with C. parvum, and in 5 of 6 piglets co-infected with rotavirus. Tumour necrosis factor alpha (TNFα) was undetectable in all serum samples from piglets.The obvious advantages of the SPF pig model are the naturally acquired intestinal microflora, the development of distinct clinical signs similar to cryptosporidiosis in humans and calves, the size of the animals, and the accessibility of individuals born within a short time span. This makes the model ideal for dose–response studies, evaluation of therapeutic agents as well as for assessment of differences in the clinical response to isolates of diverse genetic background. In conclusion, it was shown that the CPB-0 isolate was pathogenic to calves and piglets at a dose of 2.5×105 oocysts, and that the clinical signs could be replicated during separate experiments. Moreover, diarrhoea, oocyst shedding, body weight changes, histological alterations, and the acute phase response of haptoglobin and SAA were identified as useful parameters for discrimination of isolate-specific differences of pathogenicity.

Ways to reduce nitrogen pollution from Swedish pork production

The aim of this paper is to examine measures in Swedish pork production to improve N-use efficiency and to reduce N-pollution of water and air. The entire chain of feed production, animal feed conversion and the use of animal manure is included, as well as the spatial allocation of pork production. A calculation model is developed using experimental data. It is simple and possible to use in practical environmental control. Using catch crops and halving N-input in feed grain production reduces N-leaching up to 25% per hectare. These measures have considerably less effect on leaching per kilo of produced animal growth and NH3-losses than on leaching per hectare. Reduced protein level in feed and Specific Pathogen Free pig production reduce both leaching and volatilisation, especially per kilo of animal growth. These animal-centred measures can also be profitable for many producers. Spreading all manure in spring and rapid incorporation is an effective way to reduce N-losses. However, as with catch crops and reduced fertilization, it is associated with considerable costs for the producers. A combination of best manure handling, catch crops and low protein feeding can reduce N-leaching and volatilisation up to 50%. Leaching from pig production on clay soil in Central Sweden is only one third of that on sandy soil in southwest Sweden. Spatial allocation of pork production also influences the environmental effects of N losses. These effects can be more negative in coastal zones with high N-deposition and low N-retention before the water reaches the sea, than in interior regions of Sweden with low original deposition. The results indicate that improved animal protein conversion, and especially suitable spatial allocation of pig production, are more cost-effective than additional measures in feed production and manure handling.

Attempts to Separate Female Ascaris suum Antigen and to Investigate its Partial Characterization

The location and separation of Ascaris suum antigen for serological testing was investigated. The antigenic constituent was rich in the ovary of the adult worm and was obtained by dialysis with 50% ammonium sulphate saturated solution. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotting analysis demonstrated that the heat labile antigenic preparation showed one major and seven faint bands. The major band seemed also to be a glycoprotein. The sera from pigs with/without hepatic milk spot showed relatively high precipitation titres, while, those from the specific pathogen free pigs manifested low titres.

Serum-haptoglobin concentration in Danish slaughter pigs of different health status

A cross-sectional study was conducted with 617 finishing pigs aged 10–25 weeks in 11 commercial herds of different health statuses as defined by the Danish monitoring program for specific-pathogen-free (SPF) herds. A standard clinical examination was performed and a blood sample was obtained from each pig for determination of haptoglobin concentration in serum. Pigs aged 10–14, 15–19 and 20–25 weeks in conventional herds had higher haptoglobin concentrations than high-health SPF (SPF-x) pigs of the same age. There was no significant difference between SPF-x pigs of different ages. Conventional pigs aged 15–19 and 20–25 weeks had higher haptoglobin concentrations than conventional pigs aged 10–14 weeks. Herd influenced the haptoglobin concentration. Lame pigs and pigs with tail or ear bite had elevated haptoglobin concentrations. No significant effect of respiratory symptoms or umbilical hernia was found.

Microchimerism and transmission of porcine endogenous retrovirus from a pig cell line or specific pathogen-free pig islets to mouse tissues and human cells during xenografts in nude mice.

Pig islets could transmit porcine endogenous retroviruses (PERV) to diabetic patients. Our previous work showed that pig islets expressed low levels of PERV mRNA and were not likely to transmit PERV to human cells in vitro. The real risk of infection during pig tissue xenografts can only be evaluated by in vivo experiments. Nude mice bearing tumours containing human 293 cells were grafted with specific pathogen-free pig islets or PERV-producing pig PK15 cells to determine whether pig cells could transmit PERV to mouse and human cells in vivo. Infection was monitored by PCR, long PCR, RT-PCR and long RT-PCR. As detection of PERV sequences could be due to the presence of residual pig cells, we looked for pig mitochondrial (mt) DNA. Quantitative PCR for PERV and pig mt DNA was done to compare the PERV-to-pig mt (P-to-M) ratio of each sample with the reference ratio for grafted pig cells. Among 78 mouse tissues from PK15-grafted mice, 54 and 72 were positive for gag and pig mt DNA, respectively. Human tumours developed in these mice were positive for PERV (78%) and pig mt (89%). The P-to-M ratios for mouse tissues and PERV-positive human tumours from PK15-grafted mice were higher than the ratio in PK15 cells. Among 41 tissues from pig islet cell-grafted mice, 7 were positive for PERV (3 lymph nodes, 1 kidney, 2 salivary glands, 1 ovary), and 14 were positive for pig mt DNA. Three of these samples (1 lymph node, 1 kidney and 1 salivary gland) were positive for gag DNA, but negative for pig mt DNA. One human tumour in these mice was positive for PERV DNA. P-to-M reference ratio in grafted islet cells was 0.05+/-0.03. The three PERV-positive lymph nodes contained 78 gag/3 mt copies (P-to-M: 26), 101 gag/3 mt copies (P-to-M: 34), and 4 gag/0 mt copies. The two PERV-positive salivary glands contained 14 gag/1 mt copies, and 28 gag/0 mt copies. The ovary and the kidney contained 46 gag/3 mt and 69 gag/0 mt copies, respectively. The PERV-positive human tumour contained 47 gag/3 mt copies. Microchimerism and PERV transmission were frequently observed in both mouse and human tissues during grafting of pig PK15 cells into nude mice bearing human tumours, and sometimes during pig islet xenograft in this model. This strengthens the notion that there is a risk of transmitting PERV during pig islet xenograft.

Long-term follow-up failed to detect in vitro transmission of full-length porcine endogenous retroviruses from specific pathogen-free pig islets to human cells.

Islets from specific pathogen-free (SPF) pigs could prevent the transmission of conventional zoonosis, but not endogenous retroviruses (PERV), from pigs to diabetic patients. We previously reported that the pancreas showed the lowest expression of PERV mRNA among pig tissues intended for grafting. This study aimed to determine whether PERV from pig islets infect human cells during co-incubation. Human cells (including highly PERV-sensitive 293 cells) were incubated with SPF pig islet cells under conditions designed to increase contact (a high islet to human cell ratio, extended period of co-culture, and repeated contacts). PK15 and G2 retrovirus-producing pig cells were used in place of islet cells as "positive infection controls". Infection of human cells was monitored on cellular extracts and supernatants by PCR or long PCR, and RT-PCR or long RT-PCR, to detect PERV DNA and mRNA, respectively. Reverse-transcriptase activity was monitored by PERT. Despite the presence of all PERV sequences in pig islet cells, including full-length inserts, no DNA or RNA for gag, pol, and the 3 env sub-types were detected in any human cell line or blood mononuclear cells incubated with pig islet cells, during an 18-week follow-up period. No PERV sequences or RT activity were detected in supernatants. PERV signals were negative even when the pig islet to human cell ratio was increased to 100:1, the time of co-culture was extended to 5 days and two sequential co-incubations were done. By contrast, all PERV DNA and mRNA were detected in all human cells co-incubated with PK15 or G2 cells. Depending on human cell types, productive or non-productive infections were obtained: full-length PERV RNA and RT activity in supernatants were detected or not; and PERV sequences to previously unexposed human cells by PERV-infected human cells were transmitted or not. Some human cells were not productively infected by PK15 cells but became productively infected after co-incubation with PERV-infected 293 cells. SPF pig islet cells, even with PERV inserts and transcripts, have very little probability of transmitting PERV to human cells during co-incubation. The sensitivity of human cells to stable and productive infection by PERV depends on the cell type. Human adaptation of PERV was observed.

In vitro recognition and impairment of pig islet cells by baboon immune cells: similarity to human cellular reactions.

Grafting pig islets into patients with type 1 diabetes requires control of the strong cellular xenogeneic rejection. This in vitro study compared the cellular reaction of baboons and humans to pig islet cells (PICs) to confirm the validity of using these animals for further in vivo preclinical trials. Baboon or human peripheral blood mononuclear cells (PBMCs) or subsets were co-incubated with PICs from specific pathogen-free adult pigs for 7 days to determine the mechanisms and intensity of PBMC proliferation. Interleukin (IL) 10 and interferon (IFN) gamma secretion were assessed by enzyme-linked immunosorbent assay. Because proliferation was not indicative of aggression, a test based on perifusion analysis of the alteration of basal and stimulated insulin releases from PIC incubated with different baboon and human cells was developed. Baboon PBMCs strongly proliferated in response to PICs (stimulation index [SI]=24.8+/-6.9 [n=8] vs. 23.9+/-3.4 [n=34] for human PBMCs), showing considerable variation in intensity among animals (2.3<SI<63) and humans (1.8<SI<97). PBMC proliferation was inhibited in baboons and humans by anti-CD4 (% inhibition of SI: 71+/-10% and 75+/-7%, respectively) and anti-DR (75+/-35% and 80+/-6%) monoclonal antibodies (MoAbs) or by depletion of MHC class II+ cells (99+/-1% and 90+/-6%). Blocking by anti-CD8 or anti-CD16 MoAbs was weaker and variable among both animals and humans. IL-10 production by baboon and human PBMCs in response to PICs increased more than IFN-gamma production after 2 days of coculture, but the IL-10/IFN-gamma ratio was inverted after 5 days of coculture. After 7 days (and even after only 2 days) of coculture with baboon (n=8) or human (n=18) PBMCs, basal and glucose-stimulated insulin secretions from PICs were almost completely abolished (P<0.0001). The drop in insulin release could have mainly resulted from lysis of PICs, because the number of PICs decreased by 78% after 7 days of co-incubation with PBMCs. A decrease of insulin release by PBMCs was reproduced with plastic-adherent cells and was abolished by depletion of MHC class II+ cells or by addition of 100 microg/ml gadolinium (which inhibits macrophages), but not by cyclosporine. In baboons, as in humans, insulin release was also decreased after coculture of PICs with enriched T lymphocytes remixed with antigen-presenting cells (APCs). This study provides the first data on in vitro comparison of baboon and human cell-mediated recognition and impairment of PICs. Proliferation of PBMCs against PICs involves mainly CD4 T cells, with indirect recognition mediated by baboon or human MHC class II+ APCs. The Th2/Th1 profile of cytokines secreted in response to PICs was similar in baboon and human PBMCs. The model based on alteration of insulin secretion indicates that PIC impairment by whole mononuclear cells was strong and rapid and that a crucial role was played by MHC class II+ and plastic-adherent cells. Two mechanisms appear to be responsible for the role of these cells: (1) early and strong direct effect, which is potentially involved in vivo in primary nonfunction of islets aggressed by monocytes and macrophages; and (2) presentation of PIC xenoantigens, which leads to impairment by T lymphocytes possibly involved in in vivo-specific cellular rejection. The mechanisms and intensity of baboon cellular reactions to PICs in vitro were similar to those observed in humans, which suggests that the baboon is a suitable model for the study of cellular mechanisms during preclinical trials of pig islet xenografts.

Polyclonal activation of B cells occurs in lymphoid organs from porcine reproductive and respiratory syndrome virus (PRRSV)-infected pigs

Porcine reproductive and respiratory syndrome virus (PRRSV) induces a persistent viral infection associated with an inefficient humoral immune response. A study of lymphoid B cells and specific humoral immune response was performed in blood and several lymphoid organs collected from PRRSV experimentally-infected pigs. Groups of specific pathogen-free (SPF) pigs were infected with the LHVA-93-3 isolate of PRRSV, and blood, tonsils, spleen and mediastinal lymph nodes (MLN) were collected at various times postinfection (p.i.) (3–60 days). Lymphoid cells were isolated, immunolabeled for cytofluorometric determination of B cell percentages, used for counting specific anti-PRRSV antibody secreting B cells by an ELISPOT assay, or cultured for metabolic activity. The presence of anti-PRRSV antibodies in the serum of infected pigs was determined using a commercial ELISA assay. Virus detection was performed in all tissues, including lungs, by virus isolation and RT-PCR. The results show that percentages of B cells increased in tonsils as soon as 3 days until 17 days p.i. in PRRSV-infected pigs while they increased in spleen at 3 days p.i. only, due to an increase of larger Ig high-producing B cells. Metabolic activity of lymphoid cells from blood and spleen increased at 3 days p.i. only while lymphoid cells from tonsils and MLN transiently decreased at that time and increased thereafter up to 60 days p.i. Anti-PRRSV antibody-secreting B cells occurred in tonsils after 10 days p.i. and strongly increased up to 60 days p.i. However, specific anti-PRRSV-secreting B cells were detected in blood and spleen after 17 days p.i and in MLN only after 45 days p.i. Specific antibodies were detectable in serum at 10 days p.i., reached the maximum level at 45 days and remained high up to 60 days p.i. Infectious virus was detected in lungs and MLN as soon as 3 days p.i., and remained detectable up to 45 days p.i. in tonsils of one pig while viral RNA was detected in most organs up to 60 days p.i. In vitro experiments revealed that inactivated virus induced a stimulation of lymphoid cells isolated from PRRSV-infected pigs while it was cytotoxic for lymphoid cells from control pigs. Taken together, these results indicate that viral infection induced simultaneously a polyclonal activation of B cells, mainly in tonsils, and an exaggerated and prolonged specific humoral immune response due to persistent viral infection in lymphoid organs.

Detection of Actinobacillus pleuropneumoniae in cultures from nasal and tonsillar swabs of pigs by a PCR assay based on the nucleotide sequence of a dsbE-like gene

A PCR assay for the detection of Actinobacillus pleuropneumoniae was developed based on the amplification of a dsbE-like gene. All of 157 field isolates of A. pleuropneumoniae reacted in the PCR by the amplification of a 342 bp product. No reaction was observed with related bacterial species or other bacterial species isolated from pigs, except for A. lignieresii. The lower detection limit of the PCR was 10 2 CFU per PCR test tube and was not affected by the addition of 10 6 CFU Escherichia coli. The PCR was evaluated on mixed bacterial cultures from nasal and tonsillar swabs as well as suspensions of nasal conchae and tonsils obtained from specific pathogen-free (SPF) pigs, experimentally infected pigs, and pigs from farrow-to-finish herds. The results of the new PCR were compared with a PCR based on the detection of the omlA gene coding for an outer membrane protein, with a commercially available PCR (Adiavet™APP, Adiagène, Saint-Brieuc, France), and with conventional culturing. No positive reactions were observed with any of the PCR methods in samples of SPF animals. In samples of the other animals, no or low significant differences between nasal swabs and suspensions as well as tonsillar swabs and suspensions were observed in any method. In general, more positive results were obtained from tonsillar samples in comparison to nasal samples. Interassay sensitivity and specificity values were assessed for each test by pair wise comparisons between assays. The agreement between tests was evaluated by calculating Cohen’s κ coefficient. From these analyses the three PCR assays showed a good agreement. The dsbE-based PCR proved to be highly sensitive (95 and 93%) and specific (82 and 74%) in comparison to the omlA-based PCR and the commercially available PCR, respectively. It was concluded that the dsbE-like gene-based PCR is a reliable diagnostic assay for demonstration of A. pleuropneumoniae. Furthermore, it was demonstrated that tonsillar swabs can be used for the detection of the pathogen in healthy carrier animals.

The effect of a bradykinin B2 receptor antagonist, NPC-567, on allergen-induced airway responses in a porcine model.

In order to assess the effect of selective blocking of the bradykinin (BK) B2 receptor in allergic airway reactions, the BK B2 receptor antagonist NPC-567 was administered to sensitized pigs before allergen challenge. Fourteen specific pathogen-free pigs sensitized to Ascaris suum were used. NPC-567 (2.5 mg, in 1 ml saline) was delivered as an aerosol twice to six pigs. Ascaris antigen (in 2 ml saline) was given as an aerosol to all pigs and airway mechanics were monitored for 8 h. NPC-567 (2.5 mg) was given at t = -30 min (in 1ml saline) and mixed with the antigen at t = 0 to six pigs. Allergen challenge caused an acute reaction with a rapid, significant increase in airways resistance from 4.1 +/- 0.5 cm H2O/l/s to a maximum of 16.2 +/- 3.0 cm H2O/l/s in the control pigs. In the NPC-567-treated pigs, the resistance only increased from 2.9 +/- 0.3 cm H2O/l/s to 6.5 +/- 0.9 cm H2O/l/s (p<0.005 compared to controls). There was also a higher reduction in dynamic lung compliance in the controls than in the treated animals upon allergen challenge. The histamine concentration in urine in the control pigs was markedly elevated after allergen challenge peaking at 15-30 min. This release was inhibited in the NPC-567-treated pigs. The BK B2 receptor antagonist NPC-567 seems to be effective in inhibiting the acute response to allergen in the pig airways, possibly due to inhibition of mast cell activation via indirect mechanisms. The late obstructive response was reduced as well, probably as a consequence of the reduced mediator release in the acute reaction.

Identification of an immunorelevant ORF2 epitope from porcine circovirus type 2 as a serological marker for experimental and natural infection.

Summary. Post-weaning multisystemic wasting syndrome (PMWS) is a recently identified disease of pigs linked to the emergence of a new porcine circovirus (PCV2). We report here the characterization of immunorelevant linear B-cell epitopes of the Open Reading Frame 2-encoded protein (Orf2) from PCV2 by an enzyme-linked immunosorbent assay (ELISA) using experimental antisera collected from pigs inoculated with a PCV2 isolate. Two epitopes spanning residues 69 to 83 and 117 to 131 were specific to PCV2. Antibodies to the 117 to 131 epitope (B-133) were detected in 22% and 100% of specific pathogen-free (SPF) pig sera 6 and 11 weeks post inoculation, respectively. Cross-sectional studies performed with field sera collected from PMWS-affected herds showed B-133 antibodies in 5% of 8 to 10 week-old pigs, 38% of 13–14 week-old pigs, 62% of 16 to 19 week-old pigs, 56% of 20 to 25 week-old pigs and 45% of 26 to 31 week-old pigs. All these data suggest that epitope B-133 is a serological marker of PCV2 infection that could be used for the detection of PCV2 antibody response.

Pseudorabies virus is transmitted among vaccinated conventional pigs, but not among vaccinated SPF pigs

Whereas the reproduction ratio ( R) of pseudorabies virus (PRV) in vaccinated specific pathogen free (SPF) pigs without maternally derived antibodies under experimental conditions has repeatedly been shown to be significantly below 1, R in vaccinated conventional pigs in the field with maternally derived antibodies was significantly above 1. To exclude the difference in husbandry conditions as a cause for this discrepancy, we quantified and compared the transmission of PRV in both groups under identical experimental conditions. Whereas none of the SPF sentinel pigs became infected ( R=0, significantly<1), all conventional sentinel pigs did become infected ( R=2.5, significantly>1). Moreover, only one SPF pigs shed virus in saliva, the mean cumulative titre being almost a 100-fold less than in conventional pigs (17 pigs, P=0.003). In addition, the mean proliferation of peripheral blood lymphocytes in response to PRV antigens was significantly higher in SPF pigs than in conventional pigs at all points studied ( P<0.0001). Moreover, the virus-neutralising antibody titre after vaccination was significantly higher in SPF pigs than in conventional pigs. We conclude that the discrepancy in transmission between vaccinated SPF pigs and vaccinated conventional pigs cannot be attributed to the experimental conditions.

Long-term effects of social stress on antiviral immunity in pigs

Mixing of unfamiliar pigs is common practice in intensive pig husbandry. Since pigs maintain a dominance hierarchy, mixing often leads to vigorous fighting. Apart from the negative impact that fighting has on welfare, there is evidence that the social stress associated with fighting suppresses immune function. In the present experiment, we investigated the impact of mixing on specific long-term immune responses and protection against challenge infection after vaccination with pseudorabies virus (PRV). Specific pathogen-free (SPF) pigs were mixed pairwise with an unfamiliar same-gender conspecific or left undisturbed with a same-gender littermate at 3 days after vaccination with PRV. Half of the pigs were females (gilts) and half were castrated males (barrows). Mixing increased agonistic behavior to the same degree in gilts and barrows. Cortisol concentrations in saliva and catecholamine excretion in urine were increased in mixed pigs, and these effects were independent of dominance status and gender. Subsequently, the effects of mixing, gender, dominance status and interactions between these factors on immune response parameters were studied. The main result was that mixed barrows showed suppressed immune responses after vaccination and increased clinical symptoms after challenge infection compared to control barrows. Mixed gilts however did not differ from control gilts. It also appeared that mixed dominants were more seriously affected than mixed subordinates were. We conclude that, in some pigs, social stress after mixing suppresses the immune response to a viral vaccine and consequently impairs protection against challenge infection.

Effects of an experimental infection with Actinobacillus pleuropneumoniae on the interferon-α and interleukin-6 producing capacity of porcine peripheral blood mononuclear cells stimulated with bacteria, virus or plasmid DNA

The effect of a bacterial infection on interferon-α (IFN-α) and interleukin-6 (IL-6) production by porcine cells was studied in specific pathogen-free (SPF) pigs, infected intranasally with Actinobacillus pleuropneumoniae serotype 2. Three experimental groups of five pigs were used: infected non-treated pigs, infected pigs that were treated with enrofloxacin at disease onset, and non-infected, non-treated control pigs. Blood samples were collected from all pigs on the day of infection and on days 1, 4, 7, 13 and 17 post-infection. Sera were analysed for presence of antibodies to A. pleuropneumoniae and for the cytokines IL-6 and IFN-α. Ability to produce these cytokines was tested in vitro using whole blood cultures stimulated with inactivated virus (Aujeszky’s disease virus infected porcine kidney cells (ADV/PK-15)), inactivated bacteria ( A. pleuropneumoniae) or bacterial plasmid (pcDNA3). All cytokine inducers were used neat or pre-incubated with the transfectious agent lipofectin. IL-6 appeared in the serum of all infected non-treated animals but no IFN-α was found in the serum of any of the experimental pigs. Accordingly, the bacteria induced a substantial IL-6 but hardly any IFN-α production when tested in vitro. However, following incubation with lipofectin, the inactivated bacteria as well as pcDNA3 became efficient inducers of IFN-α in whole blood cultures. The increased IFN-α production, previously recorded in vitro during the acute phase of infection with A. pleuropneumoniae, was confirmed using lipofected plasmid DNA and it was indicated that leukocytes obtained from infected but apparently cured animals also exhibited an increased production of IFN-α. Thus, even mild/sub-clinical bacterial infections may affect cytokine production in pigs.

Relationship between the amounts of antibodies to Actinobacillus pleuropneumoniae serotype 2 detected in blood serum and in fluids collected from muscles of pigs.

An indirect ELISA method, previously used to detect antibodies to Actinobacillus pleuropneumoniae serotype 2 in serum of pigs, was further developed aiming to measure antibodies to the microbe in muscle fluids. Serum and muscle fluid were collected from Specific Pathogen Free (SPF) pigs as well as from SPF pigs challenged with A. pleuropneumoniae which were either treated with effective antibiotics or left as infected controls. The antibody responses measured in serum correlated well to the clinical signs of respiratory disease observed and to pathological lesions found at necropsy performed 17 days post-infection. The amounts of antibodies monitored in serum and in muscle fluid collected from the diaphragm and the thigh, respectively, were compared. Higher concentrations of antibodies were assessed in serum than in diaphragm fluid, which in turn contained more antibodies per ml than fluid collected from the thigh. The amount of antibodies to A. pleuropneumoniae measured in fluid from the diaphragm diluted 1/50 correlated well with the quantity measured in serum diluted 1/1000 (r2 = 0.87; P < 0.001). When validated by using serum antibody responses as a standard, the specificity of the ELISA employed in fluid from the diaphragm was found to be 100%. The sensitivity was determined to be 88% when calculated on seropositive pigs (A450 = 0.3 in serum diluted 1/1000). That figure increased to 97% if calculated on pigs expressing pronounced amounts of serum antibodies (A450 > or = 0.5).

Experimental Transmission of Porcine Circovirus Type 2 (PCV2) in Weaned Pigs:a Sequential Study

Weaned specific pathogen-free pigs were inoculated intranasally with porcine circovirus type 2 (PCV2) and killed in groups of two or three animals at 6, 13, 20, 27 and 34 days post-inoculation (dpi), together with appropriate uninfected controls, for examination by histopathological, immunohistochemical (immunogold silver staining; IGSS), polymerase chain reaction (PCR) and viral isolation techniques. Serum samples were also collected for detection of antibodies. No major clinical signs were observed in infected pigs, and gross lesions were essentially limited to the lungs and lymph nodes of some of the animals. Histologically, no lesions were seen at 6 dpi, but bronchointerstitial pneumonia was invariably noted from 13 dpi onwards. Granulomatous inflammation, with or without intracytoplasmic inclusions, was present in lymphoid tissues (e.glymph nodes, thymus, spleen and tonsil) from day 20 onwards, being most severe at days 20 and 27 dpi. Liver inflammation was present at days 13, 20 and 27 dpi. Virus was demonstrated in the tissues by isolation and PCR methods throughout the experiment. PCV2 antigens were detected by IGSS in bronchial and bronchiolar epithelial cells, in mononuclear cells and multinucleated giant cells within inflammatory lesions, and in mononuclear cells of apparently normal tissues (e.glamina propria of the small intestine and the bronchus-associated lymphoid tissue). The lesions were consistent with those of postweaning multisystemic wasting syndrome (PMWS), although not all previously reported PMWS lesions were seen. PCV2 antibodies were detected in infected pigs from day 13 onwards. The results demonstrated widespread distribution of PCV2 after infection and persistence of the virus in vivo for at least 34 days. It would appear that PCV2 can induce PMWS lesions in weaned pigs in the absence of porcine parvovirus and other common swine pathogens.

The first description of a (1→6)-β- d-glucan in prokaryotes: (1→6)-β- d-glucan is a common component of Actinobacillus suis and is the basis for a serotyping system

The chemical and antigenic properties of the cell-surface lipopolysaccharides (LPSs) and capsular polysaccharides (CPSs) of seven representative strains of Actinobacillus suis from healthy and diseased pigs were investigated. Four strains produced a linear (1→6)-β- d-glucan homopolymer, β- d-Glc p-(1-[→6)-β- d-Glc p-(1-] n →, as a LPS-O-chain (O1) and as a CPS (K1). Polyclonal antisera prepared against a (1→6)-β- d-glucan-containing strain showed a positive reaction against both LPSs and CPSs derived from the above strains (designated serotype O1/K1). One strain carried the (1→6)-β- d-glucan solely as a LPS-O-chain (serotype O1) and two strains did not express the (1→6)-β- d-glucan, but, instead, produced a different O-chain (designated serotype O2); these three strains expressed their own characteristic CPSs. (1→6)-β- d-Glucan structures are common cell wall components of yeast, fungi and lichens, but, to our knowledge, this is the first time a (1→6)-β- d-glucan has been described in a prokaryotic organism. Conformational and nuclear magnetic resonance analyses showed that the β- d-Glc p-(1→6)-β- d-Glc p linkage was flexible and two distinct glycosidic conformers are described. Cross-reactive antibodies to the A. suis (1→6)-β- d-glucan could be detected in sera from a variety of species and in sera from specific pathogen free pigs. This cross-reactivity may arise from immuno-stimulation of organisms present in the surrounding environment that contain (1→6)-β- d-glucan, which may also explain the high incidence of false positive results in previous serological tests for A. suis. In addition, these (1→6)-β- d-glucan background antibodies may be protective against A. suis infection. The characterization herein of (1→6)-β- d-glucan is the foundation for the development of a serotyping system for A. suis.