Erin L. Strait

Real-Time PCR Assays To Address Genetic Diversity among Strains of Mycoplasma hyopneumoniae

ABSTRACT Mycoplasma hyopneumoniae is an important cause of pneumonia in pigs around the world, but confirming its presence in (or absence from) pigs can be difficult. Culture for diagnosis is impractical, and seroconversion is often delayed after natural infection, limiting the use of serology. Numerous PCR assays for the detection of M. hyopneumoniae have been developed, targeting several different genes. Recently, genetic diversity among strains of M. hyopneumoniae was demonstrated. The effect of this diversity on the accuracy and sensitivity of the M. hyopneumoniae PCR assays could result in false-negative results in current PCR tests. In this study, a panel of isolates of M. hyopneumoniae, M. flocculare, M. hyorhinis, and M. hyosynoviae were tested with a number of M. hyopneumoniae-specific PCR assays. Some M. hyopneumoniae PCR assays tested did not detect all isolates of M. hyopneumoniae. To increase the efficiency of PCR testing, two new real-time PCR assays that are specific and capable of detecting all of the M. hyopneumoniae isolates used in this study were developed.

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.

Swine influenza matrix 2 (M2) protein contributes to protection against infection with different H1 swine influenza virus (SIV) isolates.

A swine influenza virus (SIV) vaccine-challenge pig model was used to study the potential of a conserved matrix 2 (M2) protein vaccine alone or in combination with an inactivated H1N1-vaccine to protect against H1N1 and H1N2 viruses. The H1N1-vaccine and heterologous H1N2-challenge virus model has previously been shown to prolong fever and increase SIV-associated pneumonic lesions. The M2 vaccine in combination with the H1N1-vaccine reduced the H1N2 induced fever but not virus shedding. The M2 vaccine alone reduced respiratory signs and pneumonic lesions to levels similar to the negative control pigs following H1N2 infection. This study found that the M2 protein has potential as a vaccine for SIV-associated disease prevention. However, development of an immune response towards the major envelope HA protein was required to reduce SIV shedding.

Species characterization and minimum inhibitory concentration patterns of Brachyspira species isolates from swine with clinical disease

Typhlocolitis and dysentery due to Brachyspira hyodysenteriae infection represent an economically important disease syndrome in growing pigs. Largely disappearing from U.S. swine herds in the late 1990s and early 2000s, Brachyspira-associated disease and bacterial isolation from swine with clinical disease has increased in the last several years, and non–B. hyodysenteriae isolates are commonly identified. Antimicrobial resistance has been demonstrated in Brachyspira spp. isolates from Europe and Asia, and may be the reason for the resurgence in U.S. herds. Seventy-nine clinical isolates identified at the Iowa State University Veterinary Diagnostic Lab were tested with multiple polymerase chain reaction assays to establish species identity, and evaluated for minimum inhibitory concentrations (MICs) using an agar dilution method against lincomycin, gentamicin, valnemulin, tiamulin, salinomycin, and carbadox. Only 38.0% of isolates could be confirmed as the known pathogens B. hyodysenteriae (30.4%) or Brachyspira pilosicoli (7.6%). Twenty of the 79 isolates (25.3%) were identified as Brachyspira murdochii, and 13.9% could not be identified to species. The MIC values were consistently high against lincomycin and moderately high against gentamicin. The remaining antimicrobials had MICs that were at the low end of the test ranges. Brachyspira murdochii and Brachyspira spp. had significantly greater MIC values against several of these drugs than other Brachyspira spp. examined. The increased incidence of these less definitively characterized Brachyspira species with increased MIC values to commonly prescribed antimicrobials may, at least in part, explain the increased prevalence and severity of this disease complex in recent years. Further research is necessary to understand these changes.

Mycoplasma Bovis Real-Time Polymerase Chain Reaction Assay Validation and Diagnostic Performance

Mycoplasma bovis is an important bacterial pathogen in cattle, producing a variety of clinical diseases. The organism, which requires specialized culture conditions and extended incubation times to isolate and identify, is frequently associated with concurrent infection with other pathogens which can potentially be more easily identified. Real-time polymerase chain reaction (real-time PCR) is a valuable diagnostic technique that can rapidly identify infectious agents in clinical specimens. A real-time PCR assay was designed based on the uvrC gene to identify M. bovis in diagnostic samples. Using culture as the gold standard test, the assay performed well in a variety of diagnostic matrices. Initial validation testing was conducted on 122 milk samples (sensitivity: 88.9% [95% confidence interval (CI): 68.4-100%], specificity: 100%); 154 lung tissues (sensitivity: 89.0% [95% CI: 83.1-94.9%], specificity: 97.8% [95% CI: 93.5-100%]); 70 joint tissue/fluid specimens (sensitivity: 92.3% [95% CI: 82.1-100%], specificity: 95.5% [95% CI: 89.3-100%]); and 26 nasal swabs (sensitivity: 75.0% [95% CI: 45.0-100%], specificity: 83.3% [95% CI: 66.1-100%]). Low numbers of other sample matrices showed good agreement between results of culture and PCR. A review of clinical cases from 2009 revealed that, in general, PCR was used much more frequently than culture and provided useful diagnostic information in conjunction with clinical signs, signalment, and gross and histopathologic lesions. Diagnostic performance of the real-time PCR assay developed as a testing method indicates that it is a rapid, accurate assay that is adaptable to a variety of PCR platforms and can provide reliable results on an array of clinical samples.

Comparison of atypical Brachyspira spp. clinical isolates and classic strains in a mouse model of swine dysentery

Multiple Brachyspira spp. can colonize the porcine colon, and the presence of the strongly beta-hemolytic Brachyspira hyodysenteriae is typically associated with clinical swine dysentery. Recently, several Brachyspira spp. have been isolated from the feces of pigs with clinical disease suggestive of swine dysentery, yet these isolates were not identified as B. hyodysenteriae by genotypic or phenotypic methods. This study used a mouse model of swine dysentery to compare the pathogenic potential of seventeen different Brachyspira isolates including eight atypical clinical isolates, six typical clinical isolates, the standard strain of B. hyodysenteriae (B204), and reference strains of Brachyspira intermedia and Brachyspira innocens. Results revealed that strongly beta-hemolytic isolates induced significantly greater cecal inflammation than weakly beta-hemolytic isolates regardless of the genetic identification of the isolate, and that strongly beta-hemolytic isolates identified as Brachyspira sp. SASK30446 and B. intermedia by PCR produced lesions indistinguishable from those caused by B. hyodysenteriae in this model.

Antibody responses of swine following infection with Mycoplasma hyopneumoniae, M. hyorhinis, M. hyosynoviae and M. flocculare

Several mycoplasma species possessing a range of virulence have been described in swine. The most commonly described are Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Mycoplasma hyosynoviae, and Mycoplasma flocculare. They are ubiquitious in many pig producing areas of the world, and except for M. hyopneumoniae, commercial antibody-based assays are lacking for most of these. Antibody cross-reactivity among these four mycoplasma species is not well characterized. Recently, the use of pen-based oral fluids for herd surveillance is of increasing interest. Thus, this study sought to measure pig antibody responses and the level of cross-reactivity in serum and pen-based oral fluids after challenge with four species of swine mycoplasmas. Four groups of four mycoplasma-free growing pigs were separately inoculated with the different mycoplasma species. Pen-based oral fluids and serum samples were collected weekly until necropsy. Species-specific Tween 20 ELISAs were used to measure antibody responses along with four other commercial M. hyopneumoniae ELISAs. Animals from all groups seroconverted to the challenge species of mycoplasma and no evidence of cross-contamination was observed. A delayed antibody response was seen with all but M. hyorhinis-infected pigs. Cross-reactive IgG responses were detected in M. hyopneumoniae- and M. flocculare-infected animals by the M. hyorhinis Tween 20 ELISA, while sera from M. hyosynoviae and M. flocculare-infected pigs were positive in one commercial assay. In pen-based oral fluids, specific anti-M. hyopneumoniae IgA responses were detected earlier after infection than serum IgG responses. In summary, while some antibody-based assays may have the potential for false positives, evidence of this was observed in the current study.

Quantitative real-time polymerase chain reaction for detecting Mycoplasma hyosynoviae and Mycoplasma hyorhinis in pen-based oral, tonsillar, and nasal fluids

Mycoplasma (M.) hyorhinis and M. hyosynoviae are pathogens known to cause disease in pigs post-weaning. Due to their fastidious nature, there is increased need for culture-independent diagnostic platforms to detect these microorganisms. Therefore, this study was performed to develop and optimize quantitative real-time PCR (qPCR) assays to rapidly detect M. hyorhinis and M. hyosynoviae in pen-based oral fluids as well as nasal and tonsillar fluids as proxies for samples used in swine herd surveillance. Two methods of genomic DNA extraction, automated versus manual, were used to compare diagnostic test performance. A wean-to-finish longitudinal study was also carried out to demonstrate the reproducibility of using pen-based oral fluids. Overall, pen-based oral and tonsillar fluids were more likely to be positive for both types of bacteria whereas only M. hyorhinis was detected in nasal fluids. DNA extraction protocols were shown to significantly influence test result. Although the initial detection time somewhat differed, both organisms were repeatedly detected in the longitudinal study. Overall, this study evaluated two qPCR methods for rapid and specific detection of either mycoplasma. Results from the present investigation can serve as a foundation for future studies to determine the prevalence of the two microorganisms, environmental load, and effectiveness of veterinary interventions for infection control.