Efficacy of postoperative oral metronidazole for haemorrhoidectomy pain: a randomized double-blind, placebo-controlled trial.

AIM: The aim of this work was to examine the efficacy of oral metronidazole in reducing posthaemorrhoidectomy pain versus placebo. METHOD: Forty patients were randomized to either metronidazole and standard care or placebo and standard care (21 metronidazole, 19 placebo) in a double-blinded, randomized controlled trial. The main outcome measure was posthaemorrhoidectomy pain scores over 21 days, measured on a 10-point Likert scale. RESULTS: There were no significant differences between groups with regards to age, gender, smoking status, self-reported general health or quality of life, haemorrhoid-related pain, haemorrhoid-related impact on quality of life, reported satisfaction with surgery, experience of surgery, median overall pain score or likelihood of recommending surgery to others. For reported median worst pain scores and defaecation-related pain, a trend to significance was identified between groups on days 16 and 18-21, with the metronidazole group reporting less pain. However, these differences were not significant when prespecified Bonferroni correction criteria were used. Using multilevel mixed effects modelling, the impact of time on median worst pain score was identified to be highly significant (P < 0.0001) whereas treatment allocation (placebo versus metronidazole) did not significantly affect the improvement in patients' reported pain (P = 0.8837). CONCLUSION: Our data do not support the hypothesis that postoperative metronidazole has a clinically meaningful effect on posthaemorrhoidectomy pain. This study adds to the previous literature, and implies that it should not be routinely used as an adjunct to analgesia.

Immune response phenotype induced by controlled immunization of neonatal pigs varies in type 1:type 2 bias.

Immune response (IR) of pigs varies by litter and by individual such that ratios of type-1 and type-2 IR differ. Estimates of heritability for antibody and cell-mediated IR suggest that genotype and the environment contribute approximately 20% and 80% to this variation. It is hypothesized that the IR phenotype of outbred neonatal pigs is immature and variable progressing with age from type-2 bias to a more balanced phenotype. To test this, pigs were IR phenotyped by a standardized protocol using two intramuscular injections of the combined type-1 and type-2 antigens (Ag) Candida albicans (CA) and hen egg white lysozyme (HEWL). Immune response was measured by wheal and flare reaction to HEWL and double skin fold thickness (DSFT) response to each Ag injected intradermally at 35 days of age. Blood was collected at 14 and 35 days of age to measure immunoglobulin IgG(1), IgG(2) and IgE isotype-relatedness of antibody (Ab) to CA and HEWL. Comparison was made between two different groups of pigs (A) and (B), from the same herd tested separately at an interval of two and a half years. An unexpected group difference in IR bias was observed. Bias in IR was not consistently toward type-2. Increase in DSFT to CA, an indicator of type-1 IR, was greater in A while frequency of wheal and flare to injection of HEWL, a type-2 IR correlate, was greater in B. Frequency of individuals with positive serum Ab activity to both Ags was greater in B than A for most isotypes. Ratios of Ab activity by type-1 and 2 isotypes and DSFT to type-1 and 2 Ags indicate diminished type-1 relative to type-2 biased IR response in B. We conclude that in normal neonatal pigs under standard husbandry IR bias is not invariably toward type-2. Phenotype varied between groups in type-1:type-2 bias with implications for protective and immunopathogenic IR. While the etiology was not pursued it is possible that unidentified environmental variables may have induced this change in IR phenotype.

Prophylaxis of experimentally induced ovomucoid allergy in neonatal pigs using Lactococcus lactis.

Probiotic Lactococcus lactis (LL) is immunomodulatory and may prevent allergy by biasing from type-2 to a type-1 immune response. We hypothesized that newborn pigs pre-treated orally with LL are protected against allergy to ovomucoid (Ovm). Pigs were assigned to two treatment groups. Piglets were pretreated orally on days of age 1-7, 10, 12, 14, 21, 28 and 35 with LL (n=30) or medium (control, n=32) and sensitized to Ovm by intraperitoneal injection together with cholera toxin on days 14, 21 and 35. Pigs were orally challenged with egg white (day 46) and assigned scores for allergic signs. Outcomes were measured as direct skin tests, serum antibody to Ovm [IgG (H+L); IgE; IgG(1) and IgG(2)] and cytokine production by mitogen-stimulated blood mononuclear cells (BMC). Clinical signs and skin test positivity were less frequent in the LL group (p ≤ 0.0001). Serum antibody associated with IgG (H and L), IgE, IgG(1) or IgG(2) was significantly increased on day 46 (post-sensitization) compared to day 14 (pre-sensitization) (p ≤ 0.0001). The LL-treated pigs had more IgE and IgG(2)-related antibody activity and lower IgG(1)/IgG(2) and IgE/IgG(2) ratios indicating a type-1 bias in immune response (p ≤ 0.05). Concentration of type-2 cytokines interleukin IL-4 and IL-10 were significantly lower in supernatants of stimulated BMC of LL-treated pigs (p ≤ 0.0001). Interferon-γ, TGF-ß and IL-13 were not detected in control or treated animals. Thus, oral treatment of neonatal pigs with LL significantly reduced subsequent frequency of allergy to Ovm associated with reduced type-2 immune response correlates hence supporting the "hygiene hypothesis" and potential use of LL as a neonatal immunoregulator.

Control of cytokine gene expression using small RNA interference: blockade of interleukin-10 and interferon-gamma gene expression in pig cells.

The ability of small RNA interference (RNAi) to reduce specific gene expression was tested using interleukin-10 (IL-10) and interferon-gamma (IFN-gamma) production by cultured swine blood mononuclear cells stimulated by Escherichia coli lipopolysaccharide or concanavalin A. Antisense (AS) phosphorothioate oligodeoxynucleotides (ODNs) corresponding to a sequence in the region of the AUG initiation codon of swine IL-10 or IFN-gamma mRNA inhibited production of IL-10 (>or=93.5%) and IFN-gamma (>or=99%) mRNAs. Interleukin-10 and IFN-gamma protein production was inhibited more than 95% by the AS ODNs. Scrambled and sense ODNs RNAi used as negative controls did not alter mRNA expression for either cytokine but slightly reduced IL-10 protein production. Cytokine-specific and control RNAi did not inhibit beta(2)-microglobulin mRNA expression in mitogen-stimulated blood mononuclear cells. Thus AS ODNs RNAi specifically inhibit expression of pig IL-10 and IFN-gamma mRNAs by cultured, mitogen-stimulated blood mononuclear cells and may be an attractive alternative method for studying cytokine function.

Virulence-associated protein-specific serum immunoglobulin G-isotype expression in young foals protected against Rhodococcus equi pneumonia by oral immunization with virulent R. equi.

The purpose of this study was to determine whether foals immunized orally from 2 days of age with virulent Rhodococcus equi developed a protective pulmonary immune response and to characterise the antibody response of the immunized foals to the virulence-associated proteins (Vaps) of the bacterium. Two groups of foals were used. One (n=4) was given live R. equi ATCC 33701 orally at 2, 7, and 14 days of age. The second group comprised three non-immunized foals age-matched to the vaccinates. At 3 weeks of age, 1 week after the final immunization, both groups were challenged intrabronchially with virulent R. equi ATCC 33701 and observed for 2 weeks post-challenge. Unvaccinated foals became clinically pneumonic and had high fever with increased heart and respiratory rates and severe pneumonia evident at necropsy. Foals of the immunized group remained healthy and lung lesions were not found post-mortem. Thus, it is possible to immunize young foals orally to protect them by 3 weeks of age against lung challenge with R. equi, even in the presence of maternal antibodies. The antibody response of the immunized foals confirmed that VapA and VapC are highly immunogenic. The immunoglobulin G isotype-related serum antibody response of immunized compared to non-immunized foals had an IgGT bias and a relatively low IgGa:IgGb ratio, both features different from what has been previously observed in immune adults and immune foals. This suggests that the serum IgG isotype profile of antibody cannot be used as a measure of evidence of protection against R. equi pneumonia.

Construction, characterization, and evaluation of the vaccine potential of three genetically defined mutants of avian pathogenic Escherichia coli.

The delta galE, delta purA, and delta aroA derivatives of avian septicemic Escherichia coli EC99 strain (O78 serogroup) were constructed with a suicide vector containing the pir-dependent R6K replicon and the sacB gene of Bacillus subtilis. The resultant isogenic mutants were stable and lacked approximately 45%, 36%, and 52% of the genes for galE, purA, and aroA, respectively. The delta purA and delta aroA mutants did not grow on minimal medium, whereas the delta galE mutant grew on minimal medium but was sensitive to galactose-induced lysis. The reversion frequencies of all three mutants were <10(-12). The mutants were highly attenuated for virulence as determined by administration of approximately 10(7) colony-forming units of each mutant to 1-day-old chicks by the subcutaneous route. Chickens were vaccinated with the mutants by spray (droplet size approximately 20 microm) at 1 and 14 days of age to determine safety, immunogenicity, and efficacy. The mutants were found to be safe. Seven days after a second vaccination, immunoglobulin (Ig)Y antibodies to E. coli in serum and air sac washings were detected by enzyme-linked immunosorbent assay. In both serum and air sac washings, IgY antibodies were significantly higher in chickens vaccinated with the mutants as compared with phosphate-buffered saline-treated controls but were significantly lower compared with chickens that were vaccinated with the parent strain. In serum, but not in air sac washings, IgY antibodies were significantly lower in chickens vaccinated with the mutants compared with the parent strain. The vaccinated chickens were given infectious bronchitis virus intranasally at 17 days of age and were challenged with homologous (EC99 strain) or heterologous (O2 serogroup) E. coli 4 days later. Chickens that received wild-type EC99 strain or its mutant derivatives were protected from homologous but not from heterologous challenge. This study indicates that the delta galE, delta purA, and delta aroA mutants are safe and moderately immunogenic but the protection conferred by the mutants is serogroup specific.

Resistance of broiler chickens to Escherichia coli respiratory tract infection induced by passively transferred egg-yolk antibodies.

Egg-yolk antibodies induced by immunizing hens with selected Escherichia coli antigens were evaluated for their ability to protect broiler chickens against respiratory/septicemic disease caused by avian pathogenic E. coli (APEC). Seven groups of broiler breeder hens were vaccinated three times, 1 week apart with live E. coli, killed E. coli, E. coli antigens [lipopolysaccharide (LPS), type 1 pilus adhesin (FimH), P pilus adhesin (PapG), aerobactin outer membrane receptor (IutA)] or phosphate buffered saline (PBS). An O78 APEC strain was used for preparation of all the antigens. Egg yolk immunoglobulins (IgY) were purified from eggs of each group and antibody activity in serum and purified IgY was determined by enzyme-linked immunosorbent assay (ELISA). IgY (100mg) was injected intramuscularly into 11-day-old broiler chickens, which were challenged 3 days later with homologous (O78) or heterologous (O1 or O2) E. coli by the intra-air sac route. Mortality was recorded and surviving chickens were euthanized 1 week after the challenge and examined for macroscopic lesions. Passive antibodies against all antigens except FimH were protective (90-100%) against the homologous challenge, but only anti-PapG and anti-IutA were effective against heterologous challenge. Anti-PapG IgY provided the greatest protection against the three serogroups of E. coli used for challenge. Hence vaccination of broiler breeders to induce anti-PapG and anti-IutA antibodies may provide passive protection of progeny chicks against respiratory/septicemic disease caused by APEC.

Porcine Ig isotypes: function and molecular characteristics.

In pigs, protection against the toxigenic extra-cellular bacterium Actinobacillus pleuropneumoniae was correlated with an increased IgG(1):IgG(2) ratio of haemolytic toxin-specific antibodies. In all species so far studied, IgG isotype expression is controlled by Type 1 (IFN-gamma, IL-12) and Type 2 (IL-4, IL-10) cytokines which dictate immune response polarization to cell-mediated (CMI) or antibody-mediated immunity (AMI), respectively. Thus, immunoglobulin (Ig) isotypes reflect Type 1 or Type 2 immune responses. Immunoglobulin isotype production by porcine B-cells cultured in the presence of recombinant porcine (rp) cytokines varies by individual, however pigs tend to generate a high IgG(1):IgG(2) ratio in response to rp IL-10 and the inverse in response to rp IFN-gamma or rp IL-12. Differential Ig isotype production should favor an isotype with a functional advantage to control the inciting infection and disease. However, functions of porcine Ig isotypes have not been described. To compare function of porcine IgM, IgG(1) and IgG(2) of defined specificity for hen eggwhite lysozyme (HEWL), Ig isotypes were affinity purified from serum by HEWL specificity and by isotype-specific mouse monoclonal antibodies. Their ability to activate complement (C') and to opsonize was tested in vitro. Porcine IgG(2) had greater guinea pig C' activating ability than did IgG(1). Neither isotype opsonized HEWL-conjugated sheep erythrocytes in vitro. Amino acid sequence analysis of IgG isotypes revealed that all subclasses have putative C' binding sites but that IgG(2a), IgG(2b) and IgG(4) were more flexible in the middle hinge region than IgG(1) and IgG(3) and would likely activate C' more efficiently. Thus, porcine IgG isotypes associated with resistance and susceptibility to disease also differ in their actual and predicted biological functions.

Characterization of naturally processed and presented peptides associated with bovine major histocompatibility complex (BoLA) class II DR molecules.

Differential regulation of genetic resistance to infectious disease may partially be explained by variation in the binding affinity and the repertoire of pathogen-derived antigenic peptides associated with major histocompatibility complex (MHC) molecules. In this study, we investigated characteristics of peptides that bind to the bovine MHC allele BoLA-DRB3*2703, which is associated with occurrence of clinical mastitis in Holstein dairy cattle, and assigned a putative peptide-binding motif to this allele. This was achieved by in vitro expression of allele *2703 as well as a control allele, BoLA-DRB3*1201 which is present at high frequency in Holsteins. Transfected cell lines alone (for allele *1201) or in combination with blood mononuclear cells from an animal homozygous for allele *2703 were used as the source of naturally processed and presented peptides. Subsequent to elution of peptides from BoLA-DR+ cells, their sequences were determined by electrospray ionization mass spectrometry. Eluted peptides were between 13 and 20 amino acids long and the majority were in sets of overlapping sequences. These peptides were derived from intra- and extracellular proteins, as well as foreign proteins present in the culture medium. Some peptides had originated from molecular chaperones present in the endoplasmic reticulum, such as ER-60 and GRP78, pointing to some degree of overlap and cross-sampling between MHC class I and class II antigen presentation pathways. Consistent with reports of human and mouse MHC class II-associated peptides, putative peptide-binding motifs could be assigned to alleles *2703 and *1201, comprising a hydrophobic or an aromatic residue at relative position 1, a hydrophobic residue at position 4 and a small residue at position 6 of the eluted peptides. These findings provide the foundation for future studies of molecular mechanisms of MHC-disease associations of cattle.

The relationship between milk production and antibody response to ovalbumin during the peripartum period.

Suboptimal innate and immune mechanisms of host resistance during the peripartum period may contribute to increased incidence of mastitis. To evaluate associations between antibody response to ovalbumin and milk production during the peripartum period, 136 Holstein cows and heifers from three herds with known antibody response profiles, were evaluated for projected 305-d milk, protein, and fat yield. Using a previously described index (Wagter et al., 2000), cows were quantitatively classified based on their profile of antibody response to ovalbumin into high, average, or low antibody response groups. The single-effect antibody response group contributed significantly to variation in fat and protein yield, but not milk yield. The interaction between antibody response and parity significantly contributed to the variation in milk, fat, and protein yields; therefore the effects of group were reported on a within-parity basis. Among first-parity cows, low responders had a higher fat and protein yield than high or average antibody responder animals. Among older cows (parity 3 or greater) milk yield was significantly higher for those in the high antibody response group compared with average and low response groups. However, no significant differences in fat or protein yields were observed between high and low antibody response groups. These results suggest the possibility to select cows for enhanced immune response with no adverse effects on yield. That first-parity cows with low antibody response produce more fat and protein may be offset by the fact that mastitis occurrence was highest in this group in two out of three herds investigated. Selection for high immune response may prove beneficial to herd life by maintaining optimal yield, yet minimizing occurrence of disease.

Control of immunoglobulin isotype production by porcine B-cells cultured with cytokines.

Cytokines regulate immunoglobulin (Ig) isotype production following the Th1/Th2 paradigm, derived from studies of inbred mice. In pigs, it is not known which, if any, Ig isotypes may reflect a Th1/Th2 response. To evaluate this, purified porcine CD21(+) B-cells were co-cultured with Staphylococcus aureus Cowan strain 1 or Escherichia coli lipopolysaccharide as B-cell mitogens together with recombinant human IL-2, and recombinant porcine (rp) interferon (IFN)-gamma, IL-12 or IL-10. While the mitogens increased B-cell proliferation, cytokines had no additional effect. A quantitative competitive enzyme-immuno assay was used to measure concentrations of porcine IgM, IgG(1) and IgG(2) in B-cell culture supernatants. In vitro, porcine B-cells produced IgG(2), 106 +/- 17.3 microg/ml; IgG(1) 107 +/- 38.3 microg/ml and IgM 25.6 +/- 8.45 microg/ml. In some individuals, Th1 cytokines such as rpIFN-gamma and IL-12, enhanced IgG(2) in the face of low concentrations of IgG(1). Furthermore, individual responses, in some cases, tended to be diametrically opposed, reminiscent of previously documented categorical immune responses in pigs such that some individuals produced high concentrations of IgG(1) in response to the various doses of rp cytokines, while others produced lower concentrations. Pigs may generate a high IgG(1):IgG(2) ratio in response to rpIL-10, and possibly to other Th2-associated cytokines. However, B-cell response to rp cytokines in vitro exhibits marked variation by pig, a feature that is likely a function of highly variable individual genotypes and their interaction with complex environments.

Systemic and mucosal antibody responses to selected cell surface antigens of avian pathogenic Escherichia coli in experimentally infected chickens.

The immune response to four cell surface antigens of avian pathogenic Escherichia coli (APEC) was investigated as the first step in identifying vaccine candidates. F1 pilus adhesin, P pilus adhesin, aerobactin receptor protein, and lipopolysaccharide (LPS) from an O78 E. coli (strain EC99) were used as antigens. The proteins were purified as 6xhistidine-tagged recombinant proteins and LPS was purified from a phenol/water extract. Groups of 12 broiler chickens were vaccinated intranasally with the EC99 strain and challenged with the same strain 10 days later via the intra-air sac route. The chickens that survived were euthanatized 10 days postchallenge. Scores were assigned to infected chickens on the basis of lesions and recovery of the challenge E. coli. The immunoglobulin (Ig) IgG, IgA, and IgM antibodies to the four antigens were measured in serum and air sac washings in an enzyme-linked immunosorbent assay. Among the chickens that were not vaccinated prior to challenge, two died and three of the survivors were ill, whereas, of the chickens that were vaccinated prior to challenge, one died and one of the survivors became ill. After the intranasal vaccination, high antibody activity against all four antigens was associated with each Ig isotype in serum and air sac washings. IgG was the predominant isotype of Ig in air sac washings as detected by radial immunodiffusion. Chickens that were not ill after challenge had greater IgG, IgA, and IgM antibody activity against all four antigens in serum and air sac washings than did sick chickens. Thus, all of the antigens tested appear to be suitable candidates for a vaccine to protect chickens from respiratory tract infections caused by APEC.

Blood lymphocyte subsets in pigs vaccinated and challenged with Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae bacterins do not induce protection in pigs while infection with low doses of the CM5 strain of A. pleuropneumoniae given by aerosol induces complete protection. To evaluate possible correlates of protection in blood lymphocyte subset phenotypes, pigs were treated with a commercial bacterin given intramuscularly, low dose (10(5)cfu/ml) aerosol infection with CM5 or control treatments of the bacterin adjuvant or phosphate buffered saline. All pigs were challenged with a high dose (10(7)cfu/ml) of A. pleuropneumoniae. Lymphocytes and sera were collected prior to and following primary and secondary immunizations and challenge, for evaluation of B- and T-cell markers and antibody to four A. pleuropneumoniae antigens. IgM(micro)+ B-cells were increased following primary exposure to antigen in the bacterin-vaccinated group only. An increase in CD4+ cells in the LD aerosol-infected group was apparent following secondary exposure to antigen. These early changes suggest little difference in lymphocyte populations between treatment groups, however, greater differences were observed following high-dose challenge; CD4+ lymphocytes were increased significantly in both bacterin and LD-challenged groups (p<0.05) while CD8+ cells decreased in the LD-group at this time period. Consequently, there were significant differences (p<0.05) in the CD4:CD8 ratio after high-dose challenge compared to earlier time points and control groups. Variation in cellular expression of SLA-DR and DQ was observed but trends correlating to treatment group were not evident. Complete protection or lack of protection associated with LD challenge or immunisation resulted in significant differences in B-cell frequencies and CD4:CD8 ratio phenotypes in pigs, but only changes in CD4:CD8 ratios appeared relevant to protection.

Evaluation of equine immunoglobulin specific for Rhodococcus equi virulence-associated proteins A and C for use in protecting foals against Rhodococcus equi-induced pneumonia.

OBJECTIVE: To determine whether purified equine immunoglobulin specific for Rhodococcus equi virulence-associated proteins A and C (VapA and VapC) can confer passive protection against R. equi-induced pneumonia in foals. ANIMALS: Twenty-eight 3-week-old mixed-breed pony foals. PROCEDURE: 7 foals received IV injections of equine hyperimmune plasma (HIP) against whole-cell R. equi, and 7 received purified equine immunoglobulin specific for VapA and VapC 1 day prior to intrabronchial infection with R. equi strain 103+. Eleven foals were not treated prior to infection, and 3 control foals were neither treated nor infected. Heart rate, respiratory rate, and rectal temperature were recorded twice daily, and serum fibrinogen concentration and WBC count were determined every other day following infection. Foals were euthanatized 14 days following infection, and lung lesions and concentration of R. equi in lungs were assessed. RESULTS: The onset of clinical signs of pneumonia was significantly delayed in the HIP- and immunoglobulin-treated groups, compared with the untreated infected group. Moreover, pulmonary lesions were less severe in the treated groups, and significantly fewer R. equi organisms were cultured from the lungs of treated foals. CONCLUSIONS AND CLINICAL RELEVANCE: Degree of protection against R. equi-induced pneumonia provided by purified immunoglobulin specific for VapA and VapC was similar to that provided by commercially available HIP. Results not only suggest that immunoglobulin is the primary component of HIP that confers protection against R. equi-induced pneumonia in foals but also indicate that antibodies against R. equi VapA and VapC are protective.

A quantitative approach to classifying Holstein cows based on antibody responsiveness and its relationship to peripartum mastitis occurrence.

A quantitative approach was developed to classify Holstein cows and heifers based on phenotypic variation of serum antibody response and to determine associations with peripartum mastitis. Using an index, 136 cows and heifers were classified into high (Group 1), average (Group 2), or low (Group 3) antibody groups following immunization with ovalbumin at wk -8, -3, and 0 relative to parturition. The ranking of groups based on the quantitative index of serum antibody response to ovalbumin were similar for sera and whey antibody such that Group 1 > Group 2 > Group 3. Animals were also vaccinated with Escherichia coli J5 (Rhône Mérieux, Lenexa, KS) at wk -8 and -3 relative to parturition. The ranking of groups for E. coli J5 was similar to that observed for serum and whey antibody to ovalbumin. Serum and whey IgG1 and IgG2 concentrations were measured at wk 0, 3, and 6 but differences between groups were not significant. There was no occurrence of mastitis for Group 1 animals in two of the herds. In contrast, Group 1 animals from the third herd had the highest occurrence of mastitis; however, these cases all occurred in first-parity heifers. According to pooled data across all herds, Group 3 animals had the highest occurrence of mastitis. Heritability estimates of serum antibody response to ovalbumin varied between 0.32 to 0.64 depending on week relative to parturition. Heritability estimates of serum antibody response to E. coli J5 also varied between 0.13 to 0.88 depending upon week relative to parturition. These results indicate that high peripartum antibody may be beneficial in some herds.

Cytokine mRNA expression in leukocytes of efferent lymph from stimulated lymph nodes in pigs.

To test the hypothesis that characteristic cytokine responses occur in stimulated porcine lymph nodes (LNs), lymph node efferent ducts were surgically cannulated. Efferent lymph (EL) leukocytes were collected before and after stimulation of LNs with mitogens [bacterial lipopolysaccharide (LPS) or phytohemagglutinin-P(PHA-P)] and antigens [hen egg white lysozyme (HEWL) or purified protein derivative of tuberculin (PPD)]. Cytokine mRNA expression was evaluated by quantitative reverse transcription polymerase chain reaction (Q-RT-PCR). Interleukin (IL)-1alpha was predominantly produced after all stimuli except for HEWL after which tumour necrosis factor (TNF)-alpha message was dominant. None of the stimuli induced message for IL-2, IL-4 or IL-8. Other cytokine mRNAs were produced in variable amounts and percentage of overall production of each cytokine message was in the following descending rank: LPS: IL-1alpha, TNF-alpha, interferon (IFN)-gamma, IL-10, IL-12-p35, IL-6, IL-12-p40 and TNF-beta; PHA-P: IL-1alpha, TNF-alpha, IL-10, IFN-gamma, IL-12-p40 and TNF-beta; HEWL: TNF-alpha, IL-1alpha, IFN-gamma, IL-10, IL-6, IL-12-p40, TNF-beta and IL-12-p35 and PPD: IL-1alpha, IFN-gamma, TNF-alpha and IL-10. Time course response of cytokines revealed early (IL-1alpha, 10, TNF-alpha) and intermediate (IL-12-p40, TNF-beta, IFN-gamma) responses for PHA-P and early (IL-1alpha, 6, 10, IL-12-p35, IL-12-p40, TNF-alpha), intermediate (TNF-beta, IFN-gamma) and late (IL-1alpha, 6) for LPS. Cytokine mRNA response induced by HEWL was early (IL-alpha, IFN-gamma), intermediate (IL-10, IL-12-p40, TNF-beta), late (IL-1alpha, IL-12-p35) and very late (IL-1alpha, 6, 10, IL-12-p40, TNF-alpha). In Bacillus Calmette-Guérin (BCG) sensitized pigs, stimulation of LNs with PPD induced message for IL-1alpha, 10, TNF-alpha and IFN-gamma which peaked at 24h. Cytokine mRNAs varied by stimulus and differed for antibody and cell-mediated immune response.

Cytokines in Mycoplasma hyorhinis-induced arthritis in pigs bred selectively for high and low immune responses.

Yorkshire pigs were bred selectively for high and low immune responses (H and L pigs, respectively) based on multiple antibody (Ab) and cell-mediated immune response traits. In a previous experiment, generation 4 (G4) pigs of each line were infected with Mycoplasma hyorhinis. High responders had a more rapid and higher Ab response and less polyserositis, but arthritis was more severe in H pigs than in L pigs. To test the hypothesis that line differences were attributable to differential expression of cytokines, M. hyorhinis infection was induced in pigs of G8. Arthritis was more severe clinically (P,

Quantitation of porcine cytokine and beta 2-microglobulin mRNA expression by reverse transcription polymerase chain reaction.

A quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) method was developed to measure pig cytokine mRNA expression. The method utilized an internal control with primer sequences for interleukin (IL)-1alpha, 2, 4, 6, 8, 10, tumor necrosis factor (TNF)-alpha, TNF-beta, interferon (IFN)-gamma and beta-2 microglobulin (beta(2)-m). The control was modified by insertion of sequences for IL-12 (p35 and p40). Pig blood mononuclear cells (BMCs) were stimulated in vitro with phytohemagglutinin-P (PHA-P) or bacterial lipopolysaccharide and cytokine or beta(2)-m mRNA quantified. To evaluate method performance and the use of beta(2)-m as a housekeeping gene (HKG), beta(2)-m mRNA expression was examined. Quantitative analysis was achieved at up to threefold differences between control and target for beta(2)-m. Results were reproducible with coefficients of variations (CVs) ranging between 12.5% and 22.4%. There were no significant differences in beta(2)-m mRNA between treated and untreated cells or between untreated cells of three pigs (p>/=0.05) suggesting that beta(2)-m can be used as a HKG. The method allows quantitation of multiple cytokine mRNAs using a single internal control subjecting target and control to the same conditions throughout the Q-RT-PCR. The system is versatile since the control plasmid can be modified by insertion or deletion of sequences.

Selection for high immune response: an alternative approach to animal health maintenance?

To test the hypothesis that variation in ability to respond immunologically correlates with health, Yorkshire pigs were bred for high (HIR) and low (LIR) antibody (Ab) and cell-mediated immune response (CMI). Selection was based on standardized measures of Ab (secondary response to hen egg white lysozyme, serum IgG concentration) and CMI (cutaneous delayed-type hypersenstivity to purified protein derivative of tuberculin after immunization with bacillus Calmette-Guérin and in vitro lymphocyte response to Con-A). Differences in Ab and CMI by line were not restricted to the antigens used in the selection. Antibody response to vaccines was highest in HIR and non-responders were restricted to LIR pigs. The HIR pigs had the best rate of weight gain. After infection with Mycoplasma hyorhinis, HIR developed more severe arthritis and less polyserositis. Differences were associated with variation in cytokine message in joint-related cells. Following exposure to attenuated transmissible gastroenteritis virus, natural killer cells of the LIR pigs but not of HIR or control lines, were unresponsive. Genetic selection for Ab and CMI may provide health and productivity advantages and complement traditional health-maintenance methods.