Mycoplasma Hyopneumoniae Infection In Pigs Research Articles

Development of ELISA Using Recombinant Proteins for the Diagnosis of Mycoplasma hyopneumoniae Infection.

In order to develop a more sensitive and reliable method for detection of serum antibodies against Mycoplasma hyopneumoniae infection in pigs, six recombinant proteins of M. hyopneumoniae (P102, P95, P46, P97 like, Lppt, and hypothetical P987) were used for the standardization of an indirect enzyme-linked immunosorbent assay (ELISA). The proteins were evaluated against 50 sera of the specific pathogen-free and 50 sera of pigs with lesions suggestive of infection. The sensitivity was 88%, 86%, 78%, 74%, 66%, and 60% for the proteins P102, P95, P46, P97 like, Lppt, and hypothetical protein P987, respectively. Moreover, the proteins were used to establish the seroprevalence in two different commercial herds (254 sera pigs from farm considered free of M. hyopneumoniae and 246 from farm with clinical signs of enzootic pneumonia and positive serology for M. hyopneumoniae) and the positive rate was 65.2% for P95, 54.6% for P102, 40.2% for P46, 37.2% for P97 like, 17.4% for the hypothetical P987, and 14% for Lppt protein. In addition, the ELISA with six recombinant proteins was compared to commercial HerdCheck kit using 118 random pig sera samples and the results showed that ELISA with recombinant proteins were more sensitive than the commercial test. These data show that the recombinant proteins P95 and P102 are potential targets to be used in diagnostic tests to detect antibodies against M. hyopneumoniae. Although more studies are necessary, this study provides insights that these recombinant proteins can be useful in epidemiological investigations and as potential biomarkers in differentiating infected animals from those vaccinated.

Relationships among Fecal, Air, Oral, and Tracheal Microbial Communities in Pigs in a Respiratory Infection Disease Model.

The association of the lower respiratory tract microbiome in pigs with that of other tissues and environment is still unclear. This study aimed to describe the microbiome of tracheal and oral fluids, air, and feces in the late stage of Mycoplasma hyopneumoniae infection in pigs, and assess the association between the tracheal microbiome and those from air, feces, and oral fluids. Tracheal fluids (n = 73), feces (n = 71), oropharyngeal fluids (n = 8), and air (n = 12) were collected in seeder pigs (inoculated with M. hyopneumoniae) and contact pigs (113 days post exposure to seeder pigs). After DNA extraction, the V4 region from 16S rRNA gene was sequenced and reads were processed using Divisive Amplicon Denoising Algorithm (DADA2). Clostridium and Streptococcus were among the top five genera identified in all sample types. Mycoplasma hyopneumoniae in tracheal fluids was associated with a reduction of diversity and increment of M. hyorhinis, Glaesserella parasuis, and Pasteurella multocida in tracheal fluids, as well as a reduction of Ruminiclostridium, Barnesiella, and Lactobacillus in feces. Air contributed in a greater proportion to bacteria in the trachea compared with feces and oral fluids. In conclusion, evidence suggests the existence of complex interactions between bacterial communities from distant and distinct niches.

Serum metabolite markers of early Mycoplasma hyopneumoniae infection in pigs

Mycoplasma hyopneumoniae, the primary pathogenic bacterium causing enzootic pneumonia, significantly affects worldwide swine production. The infection is usually persistent and bacterial identification and isolation of M. hyopneumoniae in clinical samples are challenging due to the fastidious requirements for its growth. Hence, new practical surveillance tools that improve or complement existing diagnostics on M. hyopneumoniae are desirable, especially in early infection. The objective of this study was to identify potential metabolite markers of early M. hyopneumoniae infection in pigs through metabolomics analysis. Samples obtained from pigs in a previous M. hyopneumoniae experimental infection were used in this study. Briefly, two pigs served as mock inoculated controls and ten pigs were intra-tracheally inoculated with M. hyopneumoniae. Sera, laryngeal swabs (LS), and tracheo-bronchial lavage fluid (TBLF) were collected from all pigs at 0, 2, 5, 9, 14, 21 and 28 days post-inoculation (dpi). Bronchial swabs (BS) were collected post-mortem at 28 dpi. Mycoplasma hyopneumoniae infection was confirmed by PCR in LS, TBLF and BS. Serum metabolites were profiled using high-resolution liquid chromatography–mass spectrometry (LC–MS) analysis. Metabolite markers were identified by structural analysis following multivariate analysis of LC–MS data. The results showed that M. hyopneumoniae infection time-dependently altered the serum levels of selective amino acids and fatty acids. α-Aminobutyric acid and long-chain fatty acids were markedly increased at 14 and 21 dpi in inoculated pigs (p < 0.05). These results indicated that M. hyopneumoniae infection caused systemic changes in host metabolism, warranting further studies to determine underlying biochemical and physiological mechanisms responsible for the observed changes.

Clinical impact of deoxynivalenol, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol on the severity of an experimental Mycoplasma hyopneumoniae infection in pigs

BackgroundThe mycotoxin deoxynivalenol (DON) is highly prevalent in cereals in moderate climates and therefore pigs are often exposed to a DON-contaminated diet. Pigs are highly susceptible to DON and intake of DON-contaminated feed may lead to an altered immune response and may influence the pathogenesis of specific bacterial diseases. Therefore, the maximum guidance level in feed is lowest in this species and has been set at 900 μg/kg feed by the European Commission. This study aimed to determine the effect of in-feed administration of a moderately high DON concentration (1514 μg/kg) on the severity of an experimental Mycoplasma hyopneumoniae (M. hyopneumoniae) infection in weaned piglets. Fifty M. hyopneumoniae-free piglets were assigned at 30 days of age [study day (D)0] to four different groups: 1) negative control group (NCG; n = 5), 2) DON-contaminated group (DON; n = 15), 3) DON-contaminated and M. hyopneumoniae-inoculated group (DONMHYO; n = 15), 4) M. hyopneumoniae-inoculated group (MHYO; n = 15). The piglets were fed the experimental diets ad libitum for five weeks and were monitored during this period and euthanized at day 35 [27 days post infection (DPI)] or 36 (28 DPI). The main parameters under investigation were macroscopic lung lesions (MLL) at euthanasia, respiratory disease score (RDS) from day 8 until day 35, histopathologic lesions and log copies of M. hyopneumoniae DNA detected by qPCR, determined at the day of euthanasia.ResultsNo significant difference was obtained for MLL at euthanasia, RDS (8–35), histopathologic lung lesions and log copies of M. hyopneumoniae DNA in the DONMHYO and MHYO group and consequently, no enhancement of the severity of the M. hyopneumoniae infection could be detected in the DONMHYO compared to the MHYO group.ConclusionsUnder present conditions, the findings imply that feed contaminated with DON (1514 μg/kg) provided to weaned pigs for five weeks did not increase the severity of an experimental M. hyopneumoniae infection. Further research is needed to investigate the impact of DON on M. hyopneumoniae infections in a multi-mycotoxin and multi-pathogen environment.

Use of serological and mucosal immune responses to Mycoplasma hyopneumoniae antigens P97R1, P46 and P36 in the diagnosis of infection

Currently available ELISAs used to diagnose Mycoplasma hyopneumoniae infection in pigs have high specificity but low sensitivity. To develop more sensitive assays, the kinetics of specific serum IgG and respiratory mucosal sIgA responses against three M. hyopneumoniae antigens, namely, P97R1 (an adhesin protein), P46 (a membrane protein), and P36 (a cytosolic protein), were characterised over 133 days following experimental infection.Immunoglobulin G against the three proteins remained at high concentrations from 28 to 133 days post-infection (dpi), although IgG against P97R1 was detected earlier and was more reactive than the other two antigens under assessment. Mucosal sIgA appeared earlier than serum IgG but did not persist as long; sIgA concentrations against P97R1 were the highest. Seroconversion was detected 2 weeks earlier with the P97R1-based ELISA than with a commercially available ELISA. On analysis of serum samples from five pig farms that did not use a M. hyopneumoniae vaccine, the P97R1-based IgG ELISA demonstrated a 73.6% coincidence rate with the commercial kit. Moreover, this more specific P97R1-based ELISA detected more positive samples than the commercial kit (52.8% vs. 39.2%).It was concluded that the systemic immune response to M. hyopneumoniae infection in pigs was delayed in onset but persistent whereas the mucosal response developed more rapidly but was less sustained. The P97R1 antigen was identified as a suitable serological marker for diagnosing M. hyopneumoniae infection in pigs, particularly early stage infection.

A field evaluation of two vaccines against Mycoplasma hyopneumoniae infection in pigs.

BackgroundA field trial was carried out with two Mycoplasma hyopneumoniae vaccines in order to investigate the benefit of vaccination under field conditions in modern Danish pig production facilities with pigs being positive for M. hyopneumoniae. The M. hyopneumoniae infection of the herd was confirmed through blood samples that were positive for antibodies against M. hyopneumoniae combined with gross lesions of the lungs related to M. hyopneumoniae at slaughter and detection of M. hyopneumoniae by polymerace chain reaction in these lesions.ResultsA total of 2,256 pigs from two herds were randomly divided into three groups. Group 1 received 2 mL ThoroVAX®VET, Group 2 received 1 mL Ingelvac®MycoFLEX, and Group 3 was a non-vaccinated control group. The vaccination was performed by a person who was not involved in the rest of the trial and vaccination status thereby blinded to the evaluators.The prevalence of lung lesions related to M. hyopneumoniae were significantly lower for pigs vaccinated with ThoroVAX®VET but not for pigs vaccinated with Ingelvac®MycoFLEX®, when compared to non-vaccinated pigs. There was no significant effect of vaccination on growth rate, antibiotic consumption or mortality.ConclusionThis trial demonstrated that vaccination with Thoro®VAX VET was effective in reducing the prevalence of lung lesion in pig units infected with M. hyopneumoniae.

Improved vectors for expression library immunization — application to Mycoplasma hyopneumoniae infection in pigs

Expression library immunization (ELI) has previously been used in a number of disease models in mice. Here, we describe the first example of the application of ELI to a large animal model with the immunization of pigs against enzootic pneumonia, a disease caused by Mycoplasma hyopneumoniae. The development of new plasmid vectors and library screening methods facilitated the application of ELI to this disease by allowing random libraries to be screened for clones expressing recombinant proteins. In this way the vast majority of clones in random libraries that are unproductive can be eliminated, meaning that libraries are more likely to give protection and are subsequently easier to further screen and analyze. By using this approach we have used one library screen and two animal trials to progress from an original library of 20,000 clones to a group of just 96 clones.

Mycoplasma hyopneumoniae infection in pigs: Duration of the disease and evaluation of four diagnostic assays

200 SPF pigs were infected by aerosol with Mycoplasma hyopneumoniae and the development of clinical signs, serological and pathological reactions were studied. Mean time to onset of coughing was 13 days. A mean delay of 9 days was observed from onset of coughing until seroconversion against M. hyopneumoniae as measured by ELISA. At an individual level, the sensitivity for this ELISA was estimated to 98–100% and the specificity to 93–100%. Pasteurella multocida was isolated from the majority of the lungs 4 weeks post inoculation with M. hyopneumoniae and the lung lesions in pigs were significantly larger when P. multocida was present as compared to pigs with M. hyopneumoniae alone. An evaluation of cultivation, immunofluorescence, ELISA and polymerase chain reaction for demonstration of M. hyopneumoniae in lungs showed that all four methods have a high sensitivity in the acute stages of pneumonia. In the later stages the sensitivity of cultivation was superior to the other methods. No differences in specificity were observed between the methods. The antigen-ELISA OD values and the immunofluorescence scores revealed a strong positive correlation. Nasal swabs were additionally used for demonstration of M. hyopneumoniae and the polymerase chain reaction was found superior to the other methods.

Effect of terdecamycin on experimentally induced Mycoplasma hyopneumoniae-infection in pigs.

The effect of terdecamycin, a new antibiotic, was evaluated on experimentally induced Mycoplasma hyopneumoniae infection in pigs. Tylosin and josamycin were used as the positive control drugs. Five to 7-week-old pigs were inoculated intranasally with homogenate of pig lung lesions containing M. hyopneumoniae. In experiments 1 and 2, drugs were given in feed for 42 days, from 7 days before inoculation to 35 days after inoculation, and necropsy was carried out 35 days after inoculation. In experiment 3, drugs were given in feed for 10 days, from 3 days before inoculation to 7 days after inoculation and necropsy was carried out 28 days after inoculation. Drug efficacy was evaluated on the basis of clinical signs, lung lesions, (including percentage of gross lung lesion area), isolation of M. hyopneumoniae and growth performance data. The total number of days on which coughing was observed and the mean percentage of lung lesion area in pigs given feed containing terdecamycin at 50 ppm or above were lower than those in unmedicated control pigs. Average daily weight gain in terdecamycin-treated pigs was higher than that in unmedicated control pigs. Treatment with terdecamycin showed equal or better efficacy as compared with treatment with tylosin or josamycin.

Application of enzyme-linked immunosorbent assay for the surveillance of Mycoplasma hyopneumoniae infection in pigs.

A monoclonal antibody-based blocking enzyme-linked immunosorbent assay (ELISA) was used for serological surveillance of Mycoplasma hyopneumoniae infection in pig herds. A follow-up study was conducted on "herd predictive values" previously reported for this ELISA. Of those herds giving positive results by this ELISA, 42% were subsequently found to be infected, while 100% of herds giving negative results were uninfected. Previous reports recorded positive and negative herd predictive values of 39% and 99.8%, respectively. Among naturally-infected animals, reaction in colostrum was more frequent than in serum, and this difference was most pronounced if the colostrum samples were obtained shortly before or after farrowing. Coughing was found to be the most reliable clinical indicator of infection, but surveillance through clinical herd inspections alone failed to detect 30% of infected herds. The time required for seroconversion following natural exposure to M. hyopneumoniae differed in two herds using different management systems: in one herd antibodies were first detected three weeks post-exposure, while in the other herd antibodies were not detected until five weeks after exposure.

Serological studies in pigs with Mycoplasma hyopneumoniae

Further work is described on the complement-fixation (CF) test for Mycoplasma hyopneumoniae infection in pigs, using unheated pig serum. Young pigs, 7 weeks old, were more susceptible than older pigs to M. hyopneumoniae infection; they developed lung lesions and a positive CF reaction. Three out of eight 14-week-old pigs inoculated with M. hyopneumoniae did not produce a positive M. hyopneumoniae CF reaction. Serum samples from M. hyorhinis infected pigs produced slight reactions in the M. hyopneumoniae CF test. The CF reactions between sera from M. hyorhinis infected pigs and M. hyopneumoniae antigen were greater if lung lesions were present in the pig. The possible nature of these cross-reactions and the specificity of the test is discussed. One hundred and twenty five samples from the laboratory herd gave negative results in the M. hyopneumoniae CF test. Heating the pig serum had the effect of substantially decreasing the CF titres and the addition of 1 50 normal serum from day-old pigs did not fully restore the titres. Formalin had the effect of reducing and often eliminating the CF titres of both heated and unheated serum samples. Using gel diffusion studies M. hyopneumoniae antigen could not be detected in diseased lungs resulting from M. hyopneumoniae infection. A slide agglutination reaction could not be demonstrated using serum samples from M. hyopneumoniae infected pigs. The CF test showed that the strains of M. hyopneumoniae, 11, 6238, M244, EP29 and EP33, were antigenically similar. Cross-reactions occurred in the CF test between M. hyorhinis antigen and M. hyopneumoniae immune rabbit serum. Using gel diffusion studies these cross-reactions were shown to be due to adsorption of medium constituents by organisms when grown in broth. The immune rabbit serum contained antibodies to the medium's constituents.