Brad J. Thacker

Increased Production of Proinflammatory Cytokines following Infection with Porcine Reproductive and Respiratory Syndrome Virus and Mycoplasma hyopneumoniae

ABSTRACT Induction of the proinflammatory cytokines interleukin-1 (IL-1) (α and β), IL-6, IL-8, IL-10, IL-12, and tumor necrosis factor alpha (TNF-α) in pulmonary alveolar macrophages (PAMs) was assessed following experimental infection with porcine reproductive and respiratory syndrome virus (PRRSV) and/or Mycoplasma hyopneumoniae by using in vivo and in vitro models. The in vivo model consisted of pigs infected with PRRSV and/or M. hyopneumoniae and necropsied at 10, 28, or 42 days postinfection. Pigs infected with both pathogens had a greater percentage of macroscopic lung lesions, increased clinical disease, and slower viral clearance than pigs infected with either pathogen alone. The pigs infected with both PRRSV and M. hyopneumoniae had significantly increased levels of mRNA for many proinflammatory cytokines in PAMs collected by bronchoalveolar lavage (BAL) at all necropsy dates compared to those in uninfected control pigs. Increased levels of IL-1β, IL-8, IL-10, and TNF-α proteins in BAL fluid, as measured by enzyme-linked immunosorbent assay, confirmed the increased cytokine induction induced by the pathogens. An in vitro model consisted of M. hyopneumoniae-inoculated tracheal ring explants cultured with PRRSV-infected PAMs. PAMs were harvested at 6 or 15 h postinfection with either or both pathogens. The in vitro study detected increased IL-10 and IL-12 mRNA levels in PAMs infected with PRRSV at all time periods. In addition, IL-10 protein levels were significantly elevated in the culture supernatants in the presence of M. hyopneumoniae-inoculated tracheal ring explants. The increased production of proinflammatory cytokines in vivo and in vitro associated with concurrent M. hyopneumoniae and PRRSV infection may play a role in the increased rates of pneumonia associated with PRRSV infection. The increased levels of IL-10 may be a possible mechanism that PRRSV and M. hyopneumoniae use to exacerbate the severity and duration of pneumonia induced by PRRSV and modulate the respiratory immune response.

Interaction between Mycoplasma hyopneumoniae and Swine Influenza Virus

ABSTRACT An experimental respiratory model was used to investigate the interaction between Mycoplasma hyopneumoniae and swine influenza virus (SIV) in the induction of pneumonia in susceptible swine. Previous studies demonstrated that M. hyopneumoniae, which produces a chronic bronchopneumonia in swine, potentiates a viral pneumonia induced by the porcine reproductive and respiratory syndrome virus (PRRSV). In this study, pigs were inoculated with M. hyopneumoniae 21 days prior to inoculation with SIV. Clinical disease as characterized by the severity of cough and fever was evaluated daily. Percentages of lung tissue with visual lesions and microscopic lesions were assessed upon necropsy at 3, 7, 14, and 21 days following SIV inoculation. Clinical observations revealed that pigs infected with both SIV and M. hyopneumoniae coughed significantly more than pigs inoculated with a single agent. Macroscopic pneumonia on necropsy at days 3 and 7 was greatest in both SIV-infected groups, with minimal levels of pneumonia in the M. hyopneumoniae-only-infected pigs. At 14 days post-SIV inoculation, pneumonia was significantly more severe in pigs infected with both pathogens. However, by 21 days postinoculation, the level of pneumonia in the dual-infected pigs was similar to that of the M. hyopneumoniae-only-infected group, and the pneumonia in the pigs inoculated with only SIV was nearly resolved. Microscopically, there was no apparent increase in the severity of pneumonia in pigs infected with both agents compared to that of single-agent-challenged pigs. The results of this study found that while pigs infected with both agents exhibited more severe clinical disease, the relationship between the two pathogens lacked the profound potentiation found with dual infection with M. hyopneumoniae and PRRSV. These findings demonstrate that the relationship between mycoplasmas and viruses varies with the individual agent.

Effect of vaccination on the potentiation of porcine reproductive and respiratory syndrome virus (PRRSV)-induced pneumonia by Mycoplasma hyopneumoniae.

Porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae are frequently isolated pathogens from pigs with respiratory disease. A previous study conducted in our laboratory found that infection with M. hyopneumoniae increased the duration and severity of respiratory disease induced by PRRSV. The purpose of this experiment was to determine whether vaccination against M. hyopneumoniae and/or PRRSV decreased the enhancement of PRRSV-induced pneumonia. Both M. hyopneumoniae bacterin and PRRSV vaccine decreased the severity of clinical respiratory disease. Infection or vaccination with PRRSV appeared to decrease the efficacy of the M. hyopneumoniae bacterin. Vaccination with M. hyopneumoniae bacterin decreased the potentiation of PRRSV-induced pneumonia observed in the dual infected pigs. However, PRRSV vaccination in combination with M. hyopneumoniae bacterin eliminated this benefit and the amount of pneumonia induced by PRRSV increased. PRRSV vaccine alone did not decrease the potentiation of PRRSV pneumonia by M. hyopneumoniae.

Differential production of proinflammatory cytokines: in vitro PRRSV and Mycoplasma hyopneumoniae co-infection model

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 24h 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) 1alpha, IL1beta, IL6, IL8, IL10, IL12 and tumor necrosis factor (TNF) alpha mRNA in PAMs. Commercial ELISAs were used to measure soluble IL1beta, IL8, IL10 and TNF in the culture supernatant. In the dual infected group, mRNA expression of IL1alpha, IL1beta, IL8 and TNF was increased. Both the M. hyopneumoniae- and PRRSV-infected only groups tended to have increased expression of IL1alpha, IL1beta and IL8 mRNA, although no statistical difference was observed. Increased levels of IL1beta, 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 IL1alpha, IL1beta, 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.

Use of a Mycoplasma hyopneumoniae nested polymerase chain reaction test to determine the optimal sampling sites in swine

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.

Impact of immunizations with porcine reproductive and respiratory syndrome virus on lymphoproliferative recall responses of CD8+ T cells.

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.

Antigenic differences within Actinobacillus pleuropneumoniae serotype 1.

Two distinct antigenic subtypes of Actinobacillus pleuropneumoniae serotype 1 were identified via coagglutination (Co-A) and designated as 1A and 1B. The reference strains, ATCC 27088 (1A) and ISU 158 (1B), were used to prepare hyperimmune rabbit sera for Co-A reagents. Of 75 serotype 1 field isolates tested by Co-A, 35 isolates typed as 1A, 12 as 1B and 28 as 1A/1B. Significant cross-reactivity between the 2 subtypes was found in the Co-A and was eliminated in 20/28 1A/1B strains by using Co-A reagents prepared with rabbit sera absorbed with the heterologous reference strain. However, twelve isolates (5 1A and 7 1A/1B; 16%) showed no reaction with Co-A reagents prepared with absorbed sera. Immunoblots of outer membranes (OM) prepared from APP 1A or 1B reference strains and field isolates indicated that antigenic differences between subtypes 1A and 1B were located within the high molecular weight (MW) region of the gels (40-100 kDa). Hyperimmune rabbit sera against 1A or 1B and sera from pigs vaccinated with whole-cell, formalin inactivated 1A or 1B bacterins reacted with the high MW region only in strains of the homologous subtype. In contrast, 4 of 5 sera from 1B infected pigs and 2 of 5 sera from 1A infected pigs reacted with all APP serotype 1 strains regardless of subtype. Apparently, infection exposed cross-reactive antigenic determinants that were not exposed by immunization with killed bacteria preparations. SDS-PAGE gels with LPS purified from APP 1A, 1B, 9 and 11 showed that 1A, 9 and 11 LPS O-antigens had an identical smooth ladder pattern, while 1B LPS was distinctly different. In immunoblots with OM or LPS and in dot-immunobinding assays with LPS, rabbit antiserum against APP 1A reacted with 1A, 9 and 11. In contrast, rabbit antiserum against APP 1B only reacted with APP 1B and weakly with APP 9 in the OM immunoblot and with LPS from APP 1B, 9 and 11 in the LPS immunoblot and dot-immunobinding assay. We conclude that 2 subtypes of APP serotype 1 can be distinguished based on their antigenic differences. These differences are located, at least in part, within the LPS O-antigens. LPS O-antigens from APP 1B appear more antigenically similar to APP 9 LPS than to either APP 1A or APP 11 LPS. There may also be antigenic differences in the capsular polysaccharides of APP 1A and 1B strains.(ABSTRACT TRUNCATED AT 400 WORDS)

A genetically-defined riboflavin auxotroph of Actinobacillus pleuropneumoniae as a live attenuated vaccine

Actinobacillus pleuropneumoniae is a gram negative pleiomorphic rod that is the causative agent of a severe, highly infectious and often fatal pleuropneumonia in swine. We have previously reported the construction of genetically-defined stable riboflavin auxotrophs by replacement of a portion of the APP riboflavin biosynthetic operon (ribGBAH) with an antibiotic cassette encoding resistance to kanamycin, and have demonstrated that such riboflavin auxotrophs are avirulent. In this study, we evaluated riboflavin auxotrophs of A. pleuropneumoniae for their ability to stimulate protective immunity against pleuropneumonia. An initial challenge experiment demonstrated that intramuscular vaccination with a live attenuated serotype 1A rib mutant, in a vaccine formulation that included a limiting amount of exogenous riboflavin, provided better protection against challenge with virulent A. pleuropneumoniae than either intratracheal immunization or intramuscular immunization with live bacteria in the absence of exogenous riboflavin. Subsequent studies in which the vaccine inoculating dose, concentration of exogenous riboflavin, and serotype of the vaccine strain were varied demonstrated that immunization with live avirulent riboflavin auxotrophs could elicit significant protection against experimental challenge with both homologous and heterologous virulent serotypes of A. pleuropneumoniae.

Polymerase chain reaction amplification of pseudorabies virus DNA from acutely and latently infected cells.

A characteristic of alphaherpesviruses, including pseudorabies virus (PRV), is that the acute phase of the disease is followed by lifelong latency. Latently infected animals are asymptomatic but can transmit reactivated virus. Corticosteroid administration, tissue explanation, blot- and in situ hybridizations have been used to demonstrate the presence of latent PRV infections. The use of blot hybridization as a convenient method for defining the incidence of PRV infections in swine herds has been hampered by the detection limit of this method. The objective of this study was to increase this sensitivity of blot hybridization by polymerase chain reaction (PCR) amplification of target sequences. Two sets of 20-mer primers were synthesized and used to amplify gX and gII glycoprotein gene sequences in two different strains of PRV. The specificity of the amplification was verified by Southern blot hybridization and restriction endonuclease analysis of the amplified fragments. Amplification of target sequences by PRC increased their detection limit by a factor of at least 10(5). Porcine ganglion samples, in which latency had been demonstrated by in vitro explanation, were analyzed by PCR together with positive and negative controls. Duplicate slot blot analyses of a portion of the amplified products were used to demonstrate latency in seven of eight samples. It was concluded that blot hybridization of PCR amplified DNA appears to be both a sensitive and convenient method for the detection of PRV induced latency.

Cross-protection experiments in pigs vaccinated with Actinobacillus pleuropneumoniae subtypes 1A and 1B.

Cross-protection experiments were conducted to determine whether antigenic differences located within the lipopolysaccharides (LPS) of Actinobacillus pleuropneumoniae subtypes 1A and 1B were important with respect to the efficacy of whole cell, formalin-inactivated bacterins. Based on clinical signs, lung lesions scores and mortality rates, pigs immunized with A. pleuropneumoniae subtype 1A were partially protected against severe challenge with both subtypes 1A and 1B. In contrast, 1B vaccinated pigs were not protected against severe challenge with subtype 1A but were partially protected against 1B challenge. Cross-reactive serum antibody levels were measured with an ELISA using outer membranes of subtype 1A or 1B as the coating antigen. Serum antibodies were detected against both subtypes within 2 weeks after the first immunization. Antibody levels increased with time and were generally higher against the homologous subtype coating antigen. We conclude that antigenic variation within a capsular serotype, due to antigenic variation within LPS, can result in the failure of whole cell bacterins to provide protection against challenge with the same capsular serotype. This lack of cross-protection within a capsular serotype provides partial explanation for vaccination failures observed under field conditions.