The objective of this study was to determine whether host genetics play a role in susceptibility to the respiratory disease in growing pigs caused by the porcine reproductive and respiratory syndrome virus (PRRSV). Based on a previous study, 2 genetically diverse commercial lines of pigs that also were divergent in the susceptibility of monocyte-derived macrophages to PRRSV infection in vitro were selected for an in vivo challenge study. Based on the average percentage of infected macrophages for each line, a line derived from the Large White breed was characterized as fluorescence-activated cell sorting(hi) (FACS(hi)), and a line derived from Duroc and Pietrain breeds was characterized as FACS(lo). Pigs from each line were challenged at 6 wk of age with PRRSV VR-2385 and necropsied at 10 or 21 d after infection. Data collected included clinical evaluation of disease, virus titration in serum and lung lavage fluid, macroscopic lung lesion scores, and microscopic lung lesion scores. The FACS(lo) line had consistently more severe clinical disease compared with the FACS(hi) line in the early stages of infection. Differences between line means were significant (P < 0.05) at 10 d after infection for all variables just described, and the FACS(lo) line showed more severe signs of disease. By 21 d after infection, clinical signs and lesions were resolving, and the differences between lines were significant (P < 0.04) only for microscopic lung lesion scores but approached significance (P < 0.08) for virus titer in serum. At 21 d after infection, the relationship between the lines reversed; the FACShi line had higher serum virus titers than the FACS(lo) line. This report provides evidence that strongly suggests the existence of a host genetic component in disease susceptibility to PRRSV and indicates that further study is warranted to define the cellular mechanisms that affect disease susceptibility.

Objectives: To determine the efficacy of a chlortetracycline (CTC) feed additive on pneumonia and clinical signs induced by Mycoplasma hyopneumoniae in an experimental challenge model.Methods: Three groups of pigs (12 pigs per group) were challenged with M hyopneumoniae (Day 0). Two groups received feed containing CTC at 550 g per tonne (500 g per ton; 22 mg per kg of bodyweight) for 14 days, starting either on Day -3 (prior to challenge) or at onset of clinical signs (Day 10). Pigs were evaluated daily for clinical disease (coughing), and all were necropsied on Day 29. Percentage of lung affected by pneumonia, number of organisms isolated from lung tissue, and serum antibodies (ELISA) were measured.Results: Pigs fed CTC starting before inoculation had significantly fewer coughing days and less pneumonia than either of the other groups. Pigs in both CTC-medicated groups had significantly fewer M hyopneumoniae organisms at necropsy than non-medicated pigs. At necropsy, 50% of control pigs were seropositive for M hyopneumoniae antibodies, while none of the CTC-treated pigs had seroconverted.Implications: Under the conditions of this study, in pigs challenged with M hyopneumoniae, less severe clinical signs and pneumonia Occur and fewer organisms may be isolated from lung tissue when treatment with CTC begins before challenge rather than after the onset of clinical signs. In addition, fewer organisms may be isolated From lung tissue of pigs treated with CTC beginning with the onset of clinical signs, compared to untreated pigs. In-feed CTC may be effective against mycoplasmal pneumonia.

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be responsible for financial losses in the swine industry worldwide. It remains undetermined whether genetic variability of the host in susceptibility to PRRSV exists and if this variability can be exploited to help control this important disease. The objective of this study was to determine if an in vitro flow cytometry (FACS) assay that detects the percentage of monocyte-derived macrophages (MDM) infected with PRRSV could be utilized to demonstrate genetic variability in the susceptibility between distinct lines of pigs. Over 400 growing pigs from six genetic lines maintained in a single commercial breeding herd were screened using an in vitro FACS assay. From this initial screening, two genetically diverse lines of pigs that were also divergent in their FACS results were selected for further study. An additional 264 pigs from these two lines were subsequently tested for in vitro susceptibility to PRRSV. As in the preliminary screening, the Large White line had significantly higher average percent positive MDM over the Duroc-Pietrain synthetic line. This report suggests a genetic component for susceptibility to PRRSV exists and that the in vitro assay may be useful in predicting the relative susceptibility to PRRSV in large groups of animals.

Objective: To compare the performance of three ELISAs in detecting Mycoplasma hyopneumoniae serum antibodies from M hyopneumoniae-naive and experimentally inoculated pigs. Methods: Archived serum samples from experimentally infected and known seronegative swine were tested using three M hyopneumoniae ELISAs, including a Tween-20 ELISA and two commercially available ELISA tests, the HerdChek Mycoplasma hyopneumoniae (Idexx Laboratories, Westbrook, Maine) and the DAKO Mycoplasma hyopneumoniae ELISA (DAKO Corporation, Carpenteria, California). Statistical analyses, including kappa coefficients, receiver operating characteristic curves, and covariance of tests, were used to compare the three assays. Results: The sensitivities of all three assays were lower than previously reported in the literature. The blocking ELISA was the most sensitive of these three assays. All three assays had excellent specificity. Using tests in combination increased sensitivity. Implications: Mycoplasma hyopneumoniae ELISA assays may be less sensitive than previously reported, especially for vaccinated animals and animals less than 21 days postinfection. These assays are inefficient at detecting serum antibodies in the early stages of infection; therefore, care should be exercised when interpreting results. Using a combination of tests to increase sensitivity may be valuable for the diagnosis of M hyopneumoniae infection.

Objectives: To evaluate the ability of needleless intradermal (ID) vaccines to induce protection against clinical disease and to compare the serological response of pigs to intramuscular (IM) and ID vaccination with Mycoplasma hyopneumoniae bacterins.Materials and methods: In each study, pigs seronegative for M hyopneumoniae were vaccinated either once or twice with M hyopneumoniae bacterins. The serological response to ID and IM vaccination was evaluated by the DAKO M hyopneumoniae blocking ELISA, and ID vaccine efficacy was tested by experimental respiratory challenge with a heterologous strain of virulent M hyopneumoniae.Results: Injection site reactions were minimal for all vaccines. Pigs vaccinated ID, either by needle or needleless injector, had significantly higher M hyopneumoniae antibody titers than did pigs vaccinated by IM injection. Pigs vaccinated by needleless ID injection had lower lung lesion scores and higher IgA and IgG titers in bronchoalveolar lavage fluid than did nonvaccinates following challenge. Protective immunity was established by 21 days postvaccination after a single ID dose of vaccine. Intradermal vaccination of swine for M hyopneumoniae by needleless injector was safe and efficacious, and did not require shaving or other preparation of the skin.Implications: Intradermal vaccination of swine with needleless injectors is feasible if vaccines are formulated for small doses (eg, 0.2 mL).

The objective of this study was to investigate the immune responses elicited by either a modified-live (MLV) or a killed virus (KV) porcine reproductive and respiratory syndrome virus (PRRSV) vaccine. Specifically, we investigated the effects of multiple vaccinations on antigen-specific cellular and antibody responses against PRRSV. Twelve sows were obtained from herds with either a history of repeated MLV or KV PRRSV vaccination and a non-vaccinated, PRRSV-negative herd. Within herd, sows were divided into three groups and vaccinated with MLV, KV, or injected with saline. On day 0, 27, and 38, recall responses of peripheral blood mononuclear cells (PBMC) to the parent strains of the vaccines (e.g., MLV-VR2332 or KV-ISUP) were examined. The concentrations of total PRRSV-specific and virus-neutralizing serum antibodies were determined by ELISA and serum neutralization assays. Following immunization, the antigen-specific proliferation of CD8alphabeta(+), CD4(+)CD8alphaalpha(+) T cells in the naive sows was greater than in sows repeatedly vaccinated with KV or MLV. This diminished lymphoproliferative responses of CD8alphabeta(+) and CD4(+)CD8alphaalpha(+) T cells could be partially overcome by heterologous immunization. However, B cell proliferation, PRRSV antibody concentrations and virus neutralizing antibody titers were not enhanced by heterologous immunization and only KV vaccination increased antibody levels in previously immunized (MLV or KV) sows.

A needle-free, transdermal injection device was evaluated for effectiveness of vaccine delivery and for injection site lesions in swine. A total of 130 pigs were vaccinated for pseudorabies virus (PRV) and Mycoplasma hyopneumoniae (M. hyopneumoniae). Pigs were divided into three groups; one group served as unvaccinated controls, the second group was vaccinated with conventional hypodermic needles and the third group was vaccinated with a needle-free, airpowered transdermal injection device. Blood samples collected for up to 36 days post-injection showed that both injection methods produced similar serological responses that were significantly greater than for unvaccinated controls. Injection sites, collected at slaughter from each carcass, showed minimal development of lesions and no carcass defects. The results show the needle-free, transdermal injection system to be effective and safe. Elimination of needles will prevent residual needle fragments in carcasses and associated carcass defects that develop from needle-induced injection-site lesions.

The effect of dietary Echinacea purpurea on performance, viremia, and ontogeny of the humoral antibody response against porcine reproductive and respiratory syndrome virus. (PRRSV) infection Was evaluated in weaned pigs. In three replicates, 120 weaned pigs (25 +/- 1 d of age; 8.46 +/- 0.48 kg of BW) from a PRRSV-naive herd were allotted randomly to one of eight pens (diets) in two separate rooms (four pens/room), with each pen containing five pigs. Pigs began one of four. dietary treatments (as-fed basis) 1 wk before;inoculation with PRRSV: 1) basal diet composed of corn, soybean meal, whey, and essential vitamins an. d minerals; 2) basal diet plus carbadox (0.055 g/kg of diet; as-fed basis); 3) basal diet plus Echinacea 2% (2% of the total diet); 4) basal diet plus Echinacea 4% (4% of the total diet). The diets were formulated to be isocaloric and isolysinic. Echinacea purpurea was purchased in powder form and determined by chemical analysis to contain L85% cichoric acid (as-fad basis). Seven days after starting the diets, all pigs in one room were intranasally inoculated with PRRSV isolate ATCC VR-2332 at a concentration of 104 tissue culture infectious dose(50)/mL. To monitor the effects of Echinacea and PRRSV challenge, BW and blood,samples were obtained from all pigs at 7-d intervals. Serum samples were analyzed for the presence of PRRSV and PRRSV-specific antibodies. All challenged pigs became infected with PRRSV, and all. unchallenged pigs remained free of infection. No differences (P > 0.10) in ADG, ADFI, or gain:feed (G:F) were observed in PRRSV-challenged compared with unchallenged animals. For PRRSV-challenged animals receiving diets supplemented with Echinacea At 2 or 4%, no differences (P > 0.10) were observed in ADG, ADFI, or G:F ratio. Among PRRSV-challenged pigs dietary Echinacea did not affect (P > 0.10) the rate or level of the ELISA-detectable antibody response, from d 7 to 42 or the level and duration of PRRSV in serum. For PRRSV-unchallenged animals receiving diets supplemented with Echinacea at 2 or 4%, no differences (P > 0.10) were observed in ADG, ADFI, and G:F ratio. Under the conditions of this study, dietary Echinacea did not enhance growth, exhibit antiviral effects to PRRSV, or show any evidence of immune enhancing properties.

To determine the effect of swine hepatitis E virus (HEV) infection on pregnant gilts, their fetuses, and offspring, 12 gilts were intravenously inoculated with swine HEV. Six gilts, who were not inoculated, served as controls. All inoculated gilts became actively infected and shed HEV in feces, but vertical transmission was not detected in the fetuses. There was no evidence of clinical disease in the gilts or their offspring. Mild multifocal lymphohistiocytic hepatitis was observed in 4 of 12 inoculated gilts. There was no significant effect of swine HEV on fetal size, fetal viability, or offspring birth weight or weight gain. The offspring acquired anti-HEV colostral antibodies but remained seronegative after the antibodies waned by 71 days of age. Swine HEV infection induced subclinical hepatitis in pregnant gilts, but had no effect on the gilts' reproductive performance, or the fetuses or offspring. Fulminant hepatitis associated with HEV infection was not reproduced in gilts.

Postweaning multisystemic wasting syndrome of swine associated with porcine circovirus (PCV) is a recently reported and economically important disease. Simple and reliable diagnostic methods are needed for detecting antibodies to PCV type 2 (PCV2) for monitoring of PCV infection. Here, we report the development of two modified indirect enzyme-linked immunosorbent assays (ELISAs): a PCV2 ELISA based on cell-culture-propagated PCV2 and an ORF2 ELISA based on recombinant major capsid protein. PCV2 and ORF2 ELISA detected antibodies to PCV2 and the capsid protein, respectively, in sera from pigs experimentally infected with PCV2 as early as 14 and 21 days postinoculation (dpi). The kinetics of the antibody response to PCV2 and the major capsid protein were similar. Repeatability tests revealed that the coefficients of variation of positive sera within and between runs for both assays were less than 30%. To validate the assays, PCV2 and ORF2 ELISAs were performed with 783 serum samples of young and adult pigs collected from different herds in the Midwestern United States and compared with an indirect immunofluorescent assay (IIF). Six out of 60 samples collected from nursery and growing pigs in 1987 were positive by both ELISA and IIF. Compared with IIF, the diagnostic sensitivity, specificity, and accuracy of PCV2 and ORF2 ELISAs were similar (>90%). The tests showed no cross-reactivity with antibodies to porcine parvovirus and porcine reproductive and respiratory syndrome virus. There was good agreement between the two ELISAs and between the ELISAs and IIF. The availability of the two ELISAs should accelerate our understanding of the host immune response to PCV2 and facilitate the development of prevention and control strategies by elucidating the ecology of PCV2 within swine populations.

A number of polymerase chain reaction (PCR)-based diagnostic tests have been developed for Mycoplasma hyopneumoniae, including one from this research group. This report presents further development, optimization, and standardization of a nested PCR test. Detection sensitivity was 1 fg of M. hyopneumoniae chromosomal DNA (approximately 1 organism). This exceeded the sensitivity of or compared favorably with other published PCR tests. Polymerase chain reaction primers to porcine β2-microglobulin were included as internal controls for amplifiable chromosomal DNA from porcine samples. To standardize the test, a number of samples from experimentally infected pigs, including nasal, tonsil, tracheobronchial swabs, lung tissue, bronchial alveolar lavage (BAL) fluid, and tracheobronchial brush samples, were examined by PCR. Samples obtained from BAL fluid and tracheobronchial sites were most predictive of infection, whereas nasal swabs and lung tissue were not reliable indicators of experimentally induced infection. In conclusion, the nested PCR developed for this study was found to be a highly sensitive and specific diagnostic tool for M. hyopneumoniae, but the enhanced sensitivity may be unnecessary if the proper sites are sampled.

Objective: To investigate the efficacy and level of immune response induced by a Mycoplasma hyopneumoniae (MH) bacterin in pigs previously vaccinated with a modified live virus (MLV) porcine reproductive and respiratory syndrome (PRRS) vaccine and subsequently challenged with MH.Methods: Pigs were vaccinated once with MLV PRRS vaccine (Day 0), twice with MH vaccine (Days 8 and 22), or both, and challenged with MH on Day 36. Necropsies were performed on Days 35 and 61-63. Efficacy of MH vaccine was determined by reduction in the percentage of lung affected by mycoplasmal pneumonia. Serum antibodies to PRRS virus (PRRSV) and MH antibodies to MH in bronchoalveolar lavage fluid (BAL), and production of MH-specific interferon-gamma- (IFN-gamma-) secreting cells in tissues and peripheral blood were measured on Days 0 and 14 and at necropsy. Immunological assays included ELISAs for PRRSV and MH antibodies and an enzyme-linked immunospot assay for MH-specific IFN-gamma-secreting cells.Results: Mycoplasma vaccine efficacy was not decreased by prior administration of PRRS vaccine. Serum MH antibody levels in pigs receiving both vaccines and challenged were numerically but not significantly higher than those in pigs receiving only MH vaccine and challenged. Pigs receiving both vaccines had significantly lower PRRSV sample:positive (S:P) ratios, but remained ELISA-positive (S:P>0.4). Levels of MH-specific IgG and IgA in BAL were significantly higher in both groups vaccinated for MH and challenged.Implications: Administration of an MLV PRRS vaccine to MH-free pigs prior to vaccination with MH vaccine did not interfere with vaccine efficacy or immune responses to MH infection.

An in vitro culture system was developed to investigate the induction of proinflammatory cytokines by Mycoplasma hyopneumoniae and porcine reproductive and respiratory syndrome virus (PRRSV). M. hyopneumoniae infected porcine tracheal ring explants were co-cultured with PRRSV infected pulmonary alveolar macrophages (PAMs) for 24 h to assess the cytokine production of each pathogen alone and the interaction between the two pathogens in vitro. Semiquantitative RT-PCR was used to measure interleukin (IL) 1α, IL1β, IL6, IL8, IL10, IL12 and tumor necrosis factor (TNF) α mRNA in PAMs. Commercial ELISAs were used to measure soluble IL1β, IL8, IL10 and TNF in the culture supernatant. In the dual infected group, mRNA expression of IL1α, IL1β, IL8 and TNF was increased. Both the M. hyopneumoniae- and PRRSV-infected only groups tended to have increased expression of IL1α, IL1β and IL8 mRNA, although no statistical difference was observed. Increased levels of IL1β, IL8 and IL10 were present in the supernatant of the dual infected group as measured by ELISA. No increase in soluble TNF was observed in any of the groups. IL8 levels appeared high in all groups independent of infection status. The cause of the elevated IL8 was unknown, however, it may have been a non-specific response by the cells to tissue damage during the harvesting of the tracheal rings. Correlation between mRNA expression and the soluble cytokine levels were similar in the dual infected groups with the exception of IL10 and TNF. Levels of mRNA and soluble protein levels in the single pathogen infected groups were not as consistent. The increased production of proinflammatory cytokines IL1α, IL1β, IL8 and TNF in the group infected with both M. hyopneumoniae and PRRSV suggests that cytokine induced inflammation may play an important role in the severe, chronic pneumonia induced by the concurrent infection of the two pathogens.