Quadrivalent neuraminidase RNA particle vaccine protects pigs against homologous and heterologous strains of swine influenza virus infection.

Influenza A virus in swine (IAV-S) continues to cause significant negative impact to both sows and growing pigs. The viral hemagglutinin (HA) and neuraminidase (NA) genes continue to evolve with HA diversifying at a faster rate than NA. Depending on country, whole inactivated virus (WIV) commercial and autogenous vaccines, as well as veterinary prescription vaccines targeting HA, are currently available. The use of these vaccines is focused on reducing virus and clinical signs in sows and to provide HA-specific maternally derived antibodies (MDA) to their suckling pigs. The deficiency in this strategy is that HA-MDA does not persist long enough to protect pigs through their growing phase from infection, and HA-MDA can interfere with effective pig immunization. This study evaluated the immunogenicity and efficacy of an adjuvanted, quadrivalent RNA Particle vaccine (Sequivity NA), currently licensed as Sequivity® IAV-S NA. This vaccine was formulated based on four NA antigens representing the major NA clades of IAV subtypes H1N1, H1N2 and H3N2 circulating in swine herds in the United States. In a series of trials, pigs were vaccinated twice, at three days and three weeks of age (WOA), followed by challenge with either homologous or heterologous IAV strains at 8 or 15 WOA. The Sequivity NA vaccine induced robust serum NA inhibition (NI) antibody and protected against IAV-S strains with homologous and heterologous NA to that of the vaccine. The magnitude and duration of nasal shedding was reduced in vaccinated-pigs challenged with either homologous or heterologous virus within the same NA clade. This NA-based RNA Particle vaccine avoids the known impact of HA-MDA on pig vaccination and provides a new tool to successfully reduce IAV-induced disease in the pig population.

Bivalent hemagglutinin and neuraminidase influenza replicon particle vaccines protect pigs against influenza a virus without causing vaccine associated enhanced respiratory disease.

Alphavirus-derived RNA replicon particle (RP) vaccines represent the next generation of swine influenza A virus (IAV) vaccines, as they were shown to be safe, effective, and offer advantages over traditional vaccine platforms. IAV is a significant respiratory pathogen of swine and there is a critical need to improve current commercial swine IAV vaccine platforms. Adjuvanted whole inactivated virus (WIV) IAV swine vaccines provide limited heterologous protection and may lead to vaccine-associated enhanced respiratory disease (VAERD). This study investigated the ability of RP IAV hemagglutinin (HA) vaccines to avoid VAERD and evaluated experimental multivalent HA and neuraminidase (NA) RP vaccines. RP vaccines were formulated with HA or NA heterologous or homologous to the challenge virus in monovalent HA or HA and NA bivalent combinations (HA/NA bivalent). Pigs were vaccinated with an HA RP, HA/NA bivalent RP, or heterologous HA WIV, followed by IAV challenge and necropsy 5 days post infection. RP vaccines provided homologous protection from challenge and induced robust peripheral and local antibody responses. The RP vaccine did not induce VAERD after challenge with a virus containing the heterologous HA, in contrast to the traditional WIV vaccine. The HA monovalent and HA/NA bivalent RP vaccines showed superior protection compared to traditional WIV. Additionally, the RP platform allows greater flexibility to adjust HA and NA content to reflect circulating IAV in swine antigenic diversity.

Detection of porcine parainfluenza virus type-1 antibody in swine serum using whole-virus ELISA, indirect fluorescence antibody and virus neutralizing assays.

BACKGROUND: Porcine parainfluenza virus 1 (PPIV-1) is a respiratory virus in the family Paramyxoviridae and genus Respirovirus. It is closely related to bovine parainfluenza virus 3, human parainfluenza virus 1, and Sendai virus. Recent reports suggest PPIV-1 is widespread in swine herds in the United States and abroad. However, seroprevalence studies and the ability to evaluate cross neutralization between heterologous strains is not possible without validated antibody assays. This study describes the development of an indirect fluorescence antibody (IFA) assay, a whole virus enzyme-linked immunosorbent assay (wv-ELISA) and a serum virus neutralization (SVN) assay for the detection of PPIV-1 antibodies using 521 serum samples collected from three longitudinal studies and two different challenge strains in swine. RESULTS: The area under the curve (AUC) of the wv-ELISA (95% CI, 0.93-0.98) was significantly higher (p = 0.03) compared to the IFA (95% CI, 0.90-0.96). However, no significant difference was observed between the IFA and wv-ELISA when compared to the SVN (95% CI, 0.92-0.97). All three assays demonstrated relatively uniform results at a 99% true negative rate, with only 11 disagreements observed between the IFA, wv-ELISA and SVN. CONCLUSIONS: All three serology assays detected PPIV-1 antibody in swine serum of known status that was collected from experimental studies. The SVN detected seroconversion earlier compared to the IFA and the wv-ELISA. Both the wv-ELISA and the SVN had similar diagnostic performance, while the IFA was not as sensitive as the wv-ELISA. All three assays are considered valid for routine diagnostic use. These assays will be important for future studies to screen seronegative swine for research, determine PPIV-1 seroprevalence, and to evaluate vaccine efficacy against PPIV-1 under experimental and field conditions.

Two clinical isolates of Mycoplasma hyosynoviae showed differing pattern of lameness and pathogen detection in experimentally challenged pigs.

Mycoplasma (M.) hyosynoviae is known to colonize and cause disease in growing-finishing pigs. In this study, two clinical isolates of M. hyosynoviae were compared by inoculating cesarean-derived colostrum-deprived and specific-pathogen-free growing pigs. After intranasal or intravenous inoculation, the proportion and distribution pattern of clinical cases was compared in addition to the severity of lameness. Tonsils were found to be the primary site of colonization, while bacteremia was rarely detected prior to the observation of clinical signs. Regardless of the clinical isolate, route of inoculation, or volume of inocula, histopathological alterations and tissue invasion were detected in multiple joints, indicating an apparent lack of specific joint tropism. Acute disease was primarily observed 7 to 10 days post-inoculation. The variability in the severity of synovial microscopic lesions and pathogen detection in joint cavities suggests that the duration of joint infection may influence the diagnostic accuracy. In summary, these findings demonstrate that diagnosis of M. hyosynoviae-associated arthritis can be influenced by the clinical isolate, and provides a study platform to investigate the colonization and virulence potential of field isolates. This approach can be particularly relevant to auxiliate in surveillance and testing of therapeutic and/or vaccine candidates.

Ring test evaluation of the detection of influenza A virus in swine oral fluids by real-time reverse-transcription polymerase chain reaction and virus isolation.

The probability of detecting influenza A virus (IAV) in oral fluid (OF) specimens was calculated for each of 13 assays based on real-time reverse-transcription polymerase chain reaction (rRT-PCR) and 7 assays based on virus isolation (VI). The OF specimens were inoculated with H1N1 or H3N2 IAV and serially diluted 10-fold (10(-1) to 10(-8)). Eight participating laboratories received 180 randomized OF samples (10 replicates × 8 dilutions × 2 IAV subtypes plus 20 IAV-negative samples) and performed the rRT-PCR and VI procedure(s) of their choice. Analysis of the results with a mixed-effect logistic-regression model identified dilution and assay as variables significant (P < 0.0001) for IAV detection in OF by rRT-PCR or VI. Virus subtype was not significant for IAV detection by either rRT-PCR (P = 0.457) or VI (P = 0.101). For rRT-PCR the cycle threshold (Ct) values increased consistently with dilution but varied widely. Therefore, it was not possible to predict VI success on the basis of Ct values. The success of VI was inversely related to the dilution of the sample; the assay was generally unsuccessful at lower virus concentrations. Successful swine health monitoring and disease surveillance require assays with consistent performance, but significant differences in reproducibility were observed among the assays evaluated.

La probabilité de détecter le virus de l'influenza A (VIA) dans des échantillons de fluide oral (FO) a été calculée pour chacune des 13 épreuves basées sur une réaction d'amplification en chaine en temps réel utilisant la polymérase réverse (rRT-PCR) et 7 épreuves basées sur l'isolement viral (IV). Les échantillons de FO ont été inoculés avec du VIA H1N1 ou H3N2 et dilués en série par facteur de 10 (10−1 à 10−8). Huit laboratoires participants ont reçu 180 échantillons randomisés de FO (10 réplicats × 8 dilutions × 2 sous-types de VIA plus 20 échantillons témoins négatifs sans VIA) et ont réalisé la méthode de rRT-PCR et d'IV de leur choix. L'analyse des résultats à l'aide d'un modèle de régression logistique pour les effets mélangés a identifié la dilution et l'épreuve comme étant des variables significatives (P < 0,0001) pour la détection de VIA dans du FO par rRT-PCR ou IV. Le sous-type de virus n'était pas significatif pour la détection de VIA soit par rRT-PCR (P = 0,457) ou par IV (P = 0,101). Pour les épreuves rRT-PCR les valeurs seuils de cycle (Ct) augmentaient de manière constante avec la dilution mais variaient énormément. Ainsi, il n'était pas possible de prédire le succès de l'IV sur la base des valeurs de Ct. Le succès de l'IV était inversement relié à la dilution de l'échantillon; l'épreuve était généralement négative aux faibles concentrations de virus. Pour avoir du succès dans la surveillance des maladies et de la santé des porcs il est nécessaire d'avoir des épreuves avec des performances constantes, mais des différences significatives dans la reproductibilité ont été observées parmi les épreuves évaluées.(Traduit par Docteur Serge Messier).

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.

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.

Identification of Mycoplasma suis antigens and development of a multiplex microbead immunoassay.

The aims of the current study were to identify Mycoplasma suis antigens and develop a multiplex microbead immunoassay (MIA). A M. suis-expression library was screened for immunogens using sera from infected pigs. Based on bioinformatics, putative antigens were identified within positive inserts; gene fragments were expressed and purified as polyhistidine fusion proteins, and immunoreactivity was confirmed by Western blot. Selected antigens were used to develop a MIA. Sera from noninfected and infected pigs were used to set the median fluorescent intensity (MFI) cutoffs and as positive controls, respectively. Assay specificity was tested using sera from pigs seropositive for other pathogens (2 different pigs seropositive for each pathogen). Samples from 51 field pigs and 2 pigs during the course of acute (pig 1) and chronic (pig 2) infections were tested using MIA, indirect hemagglutination assay (IHA), and quantitative polymerase chain reaction (qPCR). Sixteen reactive plaques (52 genes) were detected. A heat-shock protein (GrpE), a nicotinamide adenine dinucleotide-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPN), and 4 proteins from paralogous gene families (PGFs) were identified as antigens by Western blot. While GrpE, GAPN, and 1 PGF protein were strong antigens, the others were not suitable as MIA targets. A MIA using GrpE, GAPN, and the strongly reactive PGF protein was developed. Cross-reactivity with sera from pigs infected with Mycoplasma hyopneumoniae, Porcine circovirus-2, Porcine parvovirus, Porcine reproductive and respiratory syndrome virus, and Porcine respiratory coronavirus with this MIA was not observed. Pig 2 was consistently positive by MIA and qPCR, whereas pig 1, initially negative, seroconverted before becoming qPCR positive. Only 2 samples (from pig 1) were IHA positive. Five (9.8%) field samples were qPCR positive and 40 (78.43%) were positive for all 3 MIA antigens; however, all were IHA negative. In summary, the MIA is specific and more sensitive than qPCR and IHA, providing simultaneous evaluation of antibody response to M. suis antigens.

Detection of Salmonella enteritidis in pooled poultry environmental samples using a serotype-specific real-time-polymerase chain reaction assay.

While real-time-polymerase chain reaction (RT PCR) has been used as a rapid test for detection of Salmonella Enteritidis in recent years, little research has been done to assess the feasibility of pooling poultry environmental samples with a Salmonella Enteritidis-specific RT PCR assay. Therefore the objective of this study was to compare RT PCR Salmonella Enteritidis detection in individual and pooled (in groups of two, three, and four) poultry environmental drag swab samples to traditional cultural methods. The drag swabs were collected from poultry facilities previously confirmed positive for Salmonella Enteritidis and were cultured according to National Poultry Improvement Plan guidelines. Initial, Salmonella Enteritidis-specific RT PCR assay threshold cycle cutoff values of < or = 36, < or = 30, and < or = 28 were evaluated in comparison to culture. The average limit of detection of the RT PCR assay was 2.4 x 10(3) colony-forming units (CFUs)/ml, which corresponded to an average threshold cycle value of 36.6. Before enrichment, samples inoculated with concentrations from 10(2) to 10(5) CFUs/ml were detected by RT PCR, while after enrichment, samples inoculated from 10(0) to 10(5) CFUs/ml were detected by RT PCR. Threshold cycle cutoff values were used in the subsequent field trial from which Salmonella Enteritidis was cultured in 7 of 208 environmental samples (3.4%). Individual samples were 99.0%, 100%, and 100% in agreement with the RT PCR at threshold cycle (C(t)) cutoff values of < or = 36, < or = 30, and < or = 28 respectively. The agreement for pooled samples also followed the same trend with highest agreement at C(t) < or = 28 (pool of 2 = 100.0%, pool of 3 = 100.0%, pool of 4 = 100.0%), midrange agreement at C(t) < or = 30 (pool of 2 = 99.0%, pool of 3 = 100.0%, pool of 4 = 100.0%), and lowest agreement at C(t) < or = 36 (pool of 2 = 98.1%, pool of 3 = 97.1%, pool of 4 = 98.1%). In conclusion, regardless of the level of pooling after tetrathionate enrichment, sensitivity was very good, and results would be comparable to what would have been found with individual culture or individual RT PCR at C(t) < or = 36.

Dysgalactia associated with Mycoplasma suis infection in a sow herd.

CASE DESCRIPTION: A sudden onset of extreme dysgalactia in gilts and sows in a 1,000-head farrow-to-wean herd was observed in December 2009. Signs of dysgalactia were identified in sows beginning 1 day after parturition and lasted 4 to 6 days. This resulted in a mean piglet preweaning mortality rate of 18% because of starvation. CLINICAL FINDINGS: Sows were neither off feed nor febrile. Udders were not inflamed or congested. Feed sample analysis did not find ergotamine, mycotoxin contamination, or ration formulation errors. Management practices were acceptable. Piglets attempted to stimulate milk production but none was elicited. Oxytocin (20 U) caused milk ejection but the effect was short-lived. Blood samples from sows with affected litters were positive for Mycoplasma suis (formerly Eperythrozoon suis) by PCR assay, and blood samples from sows with unaffected litters were negative. TREATMENT AND OUTCOME: Chlortetracycline fed to the entire sow herd at 22 mg/kg/d (10 mg/lb/d) for 2 weeks resulted in a near complete absence of dysgalactia in sows farrowing within 5 weeks after the start of treatment. Dysgalactia did occur in sows that received chlortetracycline > 5 weeks prior to farrowing. Currently, gestating sows and gilts receive chlortetracycline in feed at a dosage of 22 mg/kg/d for 2 weeks beginning 3 weeks prior to farrowing. CONCLUSIONS AND CLINICAL RELEVANCE: M suis is spread primarily by blood contact from animal to animal, and diagnosis of infection with this organism can be easily missed by means of standard diagnostic protocols unless PCR assays or specific stains are used. Therefore, its current prevalence and impact are likely to be greatly underestimated.

Comparative virulence of clinical Brachyspira spp. isolates in inoculated pigs.

Classical swine dysentery is associated with the presence of the strongly beta-hemolytic Brachyspira hyodysenteriae. However, multiple Brachyspira spp. can colonize the porcine colon. Since 2008, several Brachyspira spp. not identified as B. hyodysenteriae by genotypic and/or phenotypic methods have been isolated from the feces of pigs with clinical disease typical of swine dysentery. In the current study, 8 clinical isolates, including 5 strongly beta-hemolytic and 3 weakly beta-hemolytic Brachyspira strains, and a reference strain of B. hyodysenteriae (B204) were inoculated into pigs (n = 6 per isolate) to compare pathogenic potential following oral inoculation. Results revealed that strongly beta-hemolytic isolates induced significantly greater typhlocolitis than those that are weakly beta-hemolytic, regardless of the genetic identification of the isolate, and that strongly beta-hemolytic isolates identified as "Brachyspira sp. SASK30446" and Brachyspira intermedia by polymerase chain reaction (PCR) produced lesions similar to those caused by B. hyodysenteriae. The results suggest that phenotypic culture characteristics of Brachyspira spp. may be a more sensitive indicator of potential to induce dysentery-like disease in pigs than molecular identification alone based on currently available PCR assays. Additionally, culture of mucosal scrapings obtained at necropsy was more sensitive than direct PCR on the same samples for detection of Brachyspira spp.

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.

Development of a real-time polymerase chain reaction assay for detection of Actinobacillus suis in porcine lung.

In the current study, the development and validation of a real-time polymerase chain reaction (PCR) assay using a TaqMan-labeled probe for the detection of Actinobacillus suis from porcine lung samples is described. This real-time PCR amplified a 110-bp region of the 23S ribosomal RNA gene from A. suis but not from other bacteria. First, the assay was validated with 183 bacterial strains representing different species of bacteria. Subsequently, 85 porcine lung specimens that were declared A. suis-positive and -negative by bacterial culture and identification were tested to assess whether it can be performed directly on tissue specimens. The bacterial culture results and real-time PCR results agreed across all the samples tested assigning 100% positive and negative predictive values to the PCR. Further, the detection limit of the assay was 380 colony-forming units (CFU) per ml or approximately 1 CFU per reaction. In conclusion, the TaqMan real-time PCR assay described herein is a highly specific, sensitive, and reproducible test, which can be used to detect A. suis DNA in porcine lung specimens, thus providing a timely diagnosis.

Comparison of RNA extraction and real-time reverse transcription polymerase chain reaction methods for the detection of Porcine reproductive and respiratory syndrome virus in porcine oral fluid specimens.

The objective of the current study was to evaluate various RNA extraction and polymerase chain reaction (PCR) protocols for the detection of Porcine reproductive and respiratory syndrome virus (PRRSV) in porcine oral fluids. Extraction protocols were selected based on ease of use and compatibility with high-throughput, automated systems. The results showed marked differences among extraction protocols, PCR protocols, and combinations thereof in detecting PRRSV in the oral fluid matrix. An important finding was that PCR reactions were partially inhibited by unknown factors in the oral fluid matrix and that inhibition was reduced by use of a higher concentration of PCR enzymes. The results suggest that further optimization of PCR assays for porcine oral fluids is needed and that laboratories should not assume that methods optimized for detection of virus in serum will perform equally with porcine oral fluids.

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 1990 s 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.

Influenza A pandemic (H1N1) 2009 virus infection in domestic cat.

Influenza A pandemic (H1N1) 2009 virus continues to rapidly spread worldwide. In 2009, pandemic (H1N1) 2009 infection in a domestic cat from Iowa was diagnosed by a novel PCR assay that distinguishes between Eurasian and North American pandemic (H1N1) 2009 virus matrix genes. Human-to-cat transmission is presumed.

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.

Real-time PCR assays to address genetic diversity among strains of Mycoplasma hyopneumoniae.

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.

Array-based genomic comparative hybridization analysis of field strains of Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia and a major factor in the porcine respiratory disease complex. A clear understanding of the mechanisms of pathogenesis does not exist, although it is clear that M. hyopneumoniae adheres to porcine ciliated epithelium by action of a protein called P97. Previous studies have shown variation in the gene encoding the P97 cilium adhesin in different strains of M. hyopneumoniae, but the extent of genetic variation among field strains across the genome is not known. Since M. hyopneumoniae is a worldwide problem, it is reasonable to expect that a wide range of genetic variability may exist given all of the different breeds and housing conditions. This variation may impact the overall virulence of a single strain. Using microarray technology, this study examined the potential variation of 14 field strains compared to strain 232, on which the array was based. Genomic DNA was obtained, amplified with TempliPhi, and labeled indirectly with Alexa dyes. After genomic hybridization, the arrays were scanned and data were analyzed using a linear statistical model. The results indicated that genetic variation could be detected in all 14 field strains but across different loci, suggesting that variation occurs throughout the genome. Fifty-nine percent of the variable loci were hypothetical genes. Twenty-two percent of the lipoprotein genes showed variation in at least one field strain. A permutation test identified a location in the M. hyopneumoniae genome where there is spatial clustering of variability between the field strains and strain 232.