Development of an indirect ELISA for detection of anti-Mycoplasma hyopneumoniae IgG in naturally infected pathogen-induced convalescent sera.

BACKGROUND: Immunization of pigs with an inactivated Mycoplasma hyopneumoniae vaccine (bacterin) generates hyperimmune serum that contains high concentrations of anti-M. hyopneumoniae IgG. Commercially available IgG-ELISA kits cannot distinguish between anti-M. hyopneumoniae IgG in inactivated bacterin-induced hyperimmune sera and convalescent sera resulting from natural M. hyopneumoniae infection. Establishment of an ELISA to detect anti-M. hyopneumoniae IgG in convalescent sera will facilitate the evaluation of the M. hyopneumoniae status of pig farms. RESULTS: In this study, we expressed and purified recombinant Mhp366-N protein, which contains an epitope recognized by M. hyopneumoniae convalescent sera but not hyperimmune sera, for use as a coating antigen. For the M. hyopneumoniae convalescent serum IgG-ELISA, the optimal antigen concentration, blocking buffer, blocking time, dilution of serum, incubation time with serum, secondary antibody dilution, secondary antibody incubation time and colorimetric reaction time were 0.25 µg/mL, 2.5 % skim milk, 1 h, 1:500, 0.5 h, 1:10,000, 1 h and 15 min, respectively. Validation of the M. hyopneumoniae convalescent serum IgG-ELISA showed a cut-off value of 0.323, the intra-assay CV ranged from 3.27 to 7.26 %, the inter-assay CV ranged from 3.46 to 5.93 %, and the assay was able to differentiate convalescent sera from antibodies to 7 other porcine respiratory pathogens. The convalescent serum IgG-ELISA detected no anti-M. hyopneumoniae IgG in hyperimmune serum samples while a commercial IgG-ELISA identified 95/145 of these sera as positive. The accuracy of the M. hyopneumoniae convalescent serum IgG-ELISA was comparable to the sIgA-ELISA but better than the commercial IgG-ELISA. CONCLUSIONS: The convalescent serum IgG-ELISA is a reproducible, sensitive, and specific indirect ELISA to detect anti-M. hyopneumoniae IgG in naturally infected pathogen-induced convalescent sera. This ELISA could be used to carry out large scale surveillance of M. hyopneumoniae infection in pig farms regardless of vaccination status.

Development of an indirect ELISA for detecting humoral immunodominant proteins of Mycoplasma hyopneumoniae which can discriminate between inactivated bacterin-induced hyperimmune sera and convalescent sera.

BACKGROUND: Mycoplasma hyopneumoniae (M. hyopneumoniae) is the primary pathogen of porcine enzootic pneumonia, which has been associated with economic losses due to reduced daily weight gain and feed efficiency. Although it has a small genome and no more than 1000 genes, M. hyopneumoniae can be cultured in cell free media. However, some proteins were not expressed or were only expressed in negligible amounts under culture conditions. Nevertheless, some of these proteins can be expressed at a high level and induce a strong and rapid immune response after M. hyopneumoniae infection. The unexpressed or less expressed proteins may play critical roles in pathogenesis and/or immune response. In order to find the differentially expressed proteins of M. hyopneumoniae between culture condition and infected animals, we established an indirect ELISA for the detection of humoral immunodominant proteins which can discriminate between inactivated bacterin-induced hyperimmune sera and convalescent sera by using Mhp366 protein which did not react with sera from bacterin-immunized pigs, but revealed a strong immunoreaction with porcine convalescent sera. RESULTS: The checkerboard titration method was done by using porcine convalescent sera as positive sera and inactivated bacterin-induced hyperimmune sera as negative sera. The bacterial lysates of fusion proteins and free GST protein without dilution were the optimal coating antigens. The optimal blocking buffer was PBS with 10% FBS and 2.5% skimmed milk. In the checkboard ELISAs, when the sera were diluted at 1:500 and the HRP-labeled rabbit anti-pig IgG were diluted at 1:20000, most positive result was obtained for the assay. CONCLUSIONS: This established indirect ELISA can be used as a tool for the detection of humoral immunodominant proteins of M. hyopneumoniae which can discriminate between inactivated bacterin-induced hyperimmune sera and convalescent sera.

Dynamics of Mycoplasma hyopneumoniae seroconversion and infection in pigs in the three main production systems.

In this study, we investigated the dynamics of Mycoplasma hyopneumoniae infections in 66 pig farms, with different production systems (one-, two-, and three-site systems), and considered different risk factors. Serological assay was used to detect serum antibodies against M. hyopneumoniae and real time polymerase chain reaction (RT-PCR) was performed to detect M. hyopneumoniae DNA in tracheobronchial swabs. Results demonstrated that M. hyopneumoniae infection status was predominantly influenced by the age of the animals and the type of production system. Infection rates were higher in older animals and the prevalence was higher in the one- and two-site systems than in the three-site systems. Dynamics of infection by RT-PCR showed that earlier M. hyopneumoniae infection on one-site farms occurs earlier, while on two- and three-site farms occurs later but spreads faster, suggesting that contact between animals of different age favors the transmission.

Exposure of extensively farmed wild boars (Sus scrofa scrofa) to selected pig pathogens in Greece.

BACKGROUND: Increased density and distribution of wild boar populations are likely to promote interactions and transmission of certain pathogens, not only among wild boar but also from wild boar to livestock or humans and vice versa. OBJECTIVE: The purpose of this study was to determine seroprevalence against seven selected pathogens in wild boar living in four different areas in Greece. ANIMALS AND METHODS: In total, 359 serum samples were collected from extensively farmed wild boar (Sus scrofa scrofa) originating from four distinct geographical areas throughout Greece from April 2012 to August 2013. Samples were tested for antibodies to Actinobacillus pleuropneumoniae, African swine fever virus (ASFV), Aujeszky's disease virus (ADV), classical swine fever virus (CSFV), Erysipelothrix rhusiopathiae, Mycoplasma hyopneumoniae and porcine reproductive and respiratory syndrome virus (PRRSV). Prevalence was compared among the four regions using Fisher's exact test. RESULTS: Low overall seropositivities of 2.4% and 5.6% were detected for E. rhusiopathiae and PRRSV, respectively, higher ones for ADV (32.0%) and the highest (72.5% and 90.5%) for M. hyopneumoniae and A. pleuropneumoniae, respectively. All sera tested were found negative for antibodies directed against CSFV and ASFV. CONCLUSIONS: This is the first report of exposure of wild boars to selected pig pathogens in Greece. These results are indicative of the circulation of these pathogens in Greece with the exception of CSFV and ASFV and suggestive of the potential role of wild boars on their maintenance and transmission to their domestic counterparts and vice versa.

Development of colloidal gold-based immunochromatographic assay for rapid detection of Mycoplasma suis in porcine plasma.

A one-step immunochromatographic assay using gold nanoparticles coated with polyclonal antibody (pAb) against Mycoplasma suis (M. suis) was developed in this study for the detection of M. suis in porcine plasma. The colloidal gold was prepared by the reduction of gold salt with sodium citrate coupled with pAb against M. suis. The pAb was produced by immunizing the BALB/c mice with recombinant MSG1 (rMSG1) protein from M. suis expressed in Escherichia coli. The optimal concentrations of the capture antibody and the coating antibody were 12 µg/ml and 1.5 mg/ml, respectively, and that of the blocking buffer was 1% bovine serum albumin. The lower detection limit of the immunochromatographic assay test was 100 ng/ml with visual detection under optimal conditions of analysis. Classical swine fever virus, porcine reproductive and respiratory syndrome virus, swine pneumonia mycoplasma, swine toxoplasma, and porcine parvovirus were used to evaluate the specificity of the immunochromatographic strips. No cross-reaction of the antibodies with other related swine pathogens was observed. This qualitative test based on the visual evaluation of the results did not require any equipment. The assay time for M. suis detection was less than 10 min, suitable for rapid detection at the grassroots level. The one-step colloidal gold immunochromatographic strips that we developed had high specificity and sensitivity. Therefore, this method would be feasible, convenient, rapid, and effective for detecting M. suis in porcine plasma.

Herd-level risk factors for the seropositivity to Mycoplasma hyopneumoniae and the occurrence of enzootic pneumonia among fattening pigs in areas of endemic infection and high pig density.

The aim of the present study was to identify potential risk factors for the occurrence of enzootic pneumonia (EP) in herds situated in a region of high pig density, where a majority of herds is endemically infected with Mycoplasma hyopneumoniae. Between 2006 and 2010, overall 100 herds were enrolled in a case-control study. Data were collected through personal interview with the farmers, clinical examination of pigs and their environments, and serological testing for M. hyopneumoniae, swine influenza virus and porcine reproductive and respiratory syndrome virus. There were 40 case herds (coughing index high, seroprevalence high) with a mean coughing index of 4.3 and a seroprevalence of 86.6%. There were two control groups. Control group I consisted of 25 herds (coughing index low, seroprevalence low) with mean values of 0.7 and 11.2%, and 35 herds were allocated to control group II (coughing index low, seroprevalence high) where the mean coughing index was 0.9 and seroprevalence 86.3%. Case herds and control II herds had an increased age of piglets at weaning compared to control I herds. Any contact between fattening pigs of different age during restocking of compartments increased the risk for the occurrence of EP in a herd. Finally, farms that use living animals for the exposure to gilts during the acclimatization and farms that had increased number of weaned piglets per sow and year were less likely to test positive for M. hyopneumoniae and less likely to develop clinical symptoms of EP in fattening pigs.

Evaluation of serovar-independent ELISA antigens of Actinobacillus pleuropneumoniae in pigs, following experimental challenge with A. pleuropneumoniae, Mycoplasma hyopneumoniae and Pasteurella multocida.

OBJECTIVE: To compare the sensitivity and specificity of six serological enzyme-linked immunosorbent assays (ELISAs) based on serovar-independent antigens of Actinobacillus pleuropneumoniae (App) and investigate cross-reactivity in disease-free pigs challenged with Mycoplasma hyopneumoniae and Pasteurella multocida. DESIGN: Five experimental pig trials using direct challenge with App serovars 1, 7 or 15 or direct challenge with M. hyopneumoniae and/or various dose rates of P. multocida. PROCEDURE: A 39-kDa outer membrane protein antigen and five recombinant antigens from the apxIVA gene of App were evaluated. The latter were derived from the ApxIVA N-terminus (ApxIVA-N, ApxIVA-NP, ApxIVA-NPS) or C-terminus (ApxIVA-C, ApxIVA-CP). Pigs were sampled after challenge and clinical and necropsy findings evaluated. RESULTS: The 39-kDa ELISA had high sensitivity but lacked specificity, with significantly increased cross-reactivity following P. multocida challenge. ELISAs based on ApxIVA N-terminus antigens were significantly more sensitive than C-terminus antigens for the detection of App-induced disease. Although ApxIVA-N and ApxIVA-NP ELISAs had increased reactivity following P. multocida challenge, they retained high specificity for App-induced disease (90-93%). Affinity purified ApxIVA-NP antigen had marginally better specificity than ApxIVA-N, without reduced sensitivity. Mycoplasma hyopneumoniae did not affect serological cross-reactivity. In disease-free pigs, the specificity of the ApxIVA-NPS ELISA may be adversely affected by nasal carriage of apparently low-virulence App strains. CONCLUSIONS: ApxIVA-N-based ELISAs can be used for evaluating App status in commercial herds, but some appear limited by high carriage rates of low-virulence App. The 39-kDa antigen is only of merit in exclusion of App disease by negative serology.

Longitudinal assessment of two Mycoplasma hyopneumoniae enzyme-linked immunosorbent assays in challenged and contact-exposed pigs.

The objective of the current study was to compare the sensitivity of 2 Mycoplasma hyopneumoniae enzyme-linked immunosorbent assays (ELISAs) in experimentally challenged and contact-exposed pigs in a long-term longitudinal assessment. On day 0 of the assessment, twelve 2-month-old M. hyopneumoniae-negative pigs were inoculated with M. hyopneumoniae strain 232 (group A). Twelve negative pigs were placed alongside the inoculated pigs, allowing direct contact exposure (group B). A third group of 12 pigs was allocated into 2 independent pens; no direct contact was allowed (group C). A longitudinal serologic profile was performed; samples were collected on days 0, 28, 35, 42, 49, 63, 91, 119, 154, and 170 of the study. Serum samples were tested using a blocking ELISA and an indirect ELISA. Results of the study demonstrated higher sensitivity of the blocking ELISA during early infection (clinical signs). Both ELISAs were 100% sensitive in challenged and naturally infected groups at several testing points during late infection (63, 91, 119, 154, and 170 days of the study) and showed a long antibody detection period. Both tests worked equally well during the chronic phase of infection but the blocking ELISA was more sensitive during acute stages of infection.

Exploratory study on the influence of climatological parameters on Mycoplasma hyopneumoniae infection dynamics.

The objective of the present work was to elucidate the potential relationship between Mycoplasma hyopneumoniae infection and seroconversion dynamics and climatological conditions in four groups of pigs from the same farm born in different seasons of the year. Nasal swabs and blood samples were taken from 184 pigs at 1, 3, 6, 9, 12, 15, 18, 22 and 25 (slaughter age) weeks of age. Outside climatologic parameters, including temperature (°C), relative humidity (%), precipitation (l/m(2)) and wind speed (m/s) were recorded weekly from January 2003 to June 2004. Percentage of nPCR detection of M. hyopneumoniae in nasal swabs was associated significantly with the weekly precipitation rate [P = 0.0018, OR = 1.31 (IC = 1.11-1.55)]; the higher the precipitation rate, the higher the probability of being M. hyopneumoniae nPCR-positive. On the other hand, the percentage of seropositive pigs had a significant association with mean weekly temperature rate [P = 0.0012, OR = 0.89 [IC = 0.84-0.95]); the lower the temperature, the higher the probability of being M. hyopneumoniae seropositive. Animals born in autumn (when higher precipitations rates were recorded), entering finishing units in winter (when lower temperatures were recorded), and reaching slaughter in spring, had the highest probability of being infected by M. hyopneumoniae and the highest probability of being M. hyopneumoniae seropositive.

One dose of a porcine circovirus 2 subunit vaccine induces humoral and cell-mediated immunity and protects against porcine circovirus-associated disease under field conditions.

This study investigated the efficacy of a one-dose porcine circovirus 2 (PCV2) subunit vaccine based on the PCV2 Cap protein expressed in a baculovirus system on two different farms at which a history of porcine circovirus-associated disease (PCVD) was present. Morbidity, mortality, average daily weight gain, carcass weight, PCV2 load in serum and vaccine immunogenicity were assessed. Serology to porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae was performed. A double-blind, randomised, and controlled field trial was performed distributing 818 piglets between two treatment groups. At inclusion (weaning at 21 ± 3 days of age), 408 animals (group B) received a 2-mL intramuscular dose of Porcilis PCV(®) (vaccinated group). Controls (group A, 410 pigs) received 2 mL of the adjuvant Diluvac Forte(®) intramuscularly. Weights were recorded at inclusion and at 12 and 26 weeks of age, and the average daily weight gain (ADWG) was calculated. The carcass weights of the pigs from farm 2 were recorded at slaughter (274 days old). All dead animals (died or culled) underwent autopsy to classify them as PMWS-affected or not. At each farm, blood samples were taken from 22 pigs/group for serologic studies. A beneficial effect was found after vaccination with a single dose of a PCV2 Cap vaccine against PCVD. The vaccination reduced the mortality rate and morbidity, reduced PCV2 viremia and viral load, improved productive performances (e.g. ADWG: +70 g/day between 12 and 26 weeks of age when viremia and the specific disease occurred) as well as carcass weight at slaughter age (+4.5 kg). These effects were associated with virologic and clinical protection from the immunogenicity of the vaccine measured as activation of both a humoral and a cellular immune response.

Reduced lung lesions in pigs challenged 25 weeks after the administration of a single dose of Mycoplasma hyopneumoniae vaccine at approximately 1 week of age.

Two independent studies assessed the duration of immunity of an inactivated adjuvanted Mycoplasma hyopneumoniae vaccine against mycoplasmal pneumonia in seronegative (study A, n=52) and seropositive (study B, n=52) pigs. The pigs were allocated randomly to treatment and were then injected with a single dose of either the vaccine or a placebo at approximately 1 week of age. Twenty-five weeks after treatment administration, the pigs were challenged with a virulent strain (LI 36, Strain 232) of M. hyopneumoniae and the extent of lung lesions consistent with mycoplasmal pneumonia was assessed 4 weeks later. In study A, the geometric mean lung lesion score (expressed as least squares mean percentages of lung lesions) was significantly (P=0.0001) lower in vaccinated (0.3%, n=20) than in control pigs (5.9%, n=24) seronegative to M. hyopneumoniae at enrolment; similarly, in study B, the extent of lung lesions was significantly reduced (P=0.0385) in seropositive vaccinated pigs (2.0%, n=22) compared to controls (4.5%, n=26). At the end of the investigation period, 4 weeks after challenge, mean antibody sample-to-positive (S/P) ratios were significantly higher both in seronegative (P=0.0012) and seropositive (P=0.0001) vaccinated pigs (mean values=0.77 and 0.81, respectively) than in controls (mean values=0.51 and 0.38, respectively).

Current perspectives on the diagnosis and epidemiology of Mycoplasma hyopneumoniae infection.

Mycoplasma hyopneumoniae is the principal aetiological agent of enzootic pneumonia (EP), a chronic respiratory disease that affects mainly finishing pigs. Although major efforts to control M. hyopneumoniae infection and its detrimental effects have been made, significant economic losses in pig production worldwide due to EP continue. M. hyopneumoniae is typically introduced into pig herds by the purchase of subclinically infected animals or, less frequently, through airborne transmission over short distances. Once in the herd, M. hyopneumoniae may be transmitted by direct contact from infected sows to their offspring or between pen mates. The 'gold standard' technique used to diagnose M. hyopneumoniae infection, bacteriological culture, is laborious and is seldom used routinely. Enzyme-linked immunosorbent assay and polymerase chain reaction detection methods, in addition to post-mortem inspection in the form of abattoir surveillance or field necropsy, are the techniques most frequently used to investigate the potential involvement of M. hyopneumoniae in porcine respiratory disease. Such techniques have been used to monitor the incidence of M. hyopneumoniae infection in herds both clinically and subclinically affected by EP, in vaccinated and non-vaccinated herds and under different production and management conditions. Differences in the clinical course of EP at farm level and in the efficacy of M. hyopneumoniae vaccination suggest that the transmission and virulence characteristics of different field isolates of M. hyopneumoniae may vary. This paper reviews the current state of knowledge of the epidemiology of M. hyopneumoniae infection including its transmission, infection and seroconversion dynamics and also compares the various epidemiological tools used to monitor EP.

Immune response of gnotobiotic piglets against Mycoplasma hyopneumoniae.

In this study, several cytokine responses were investigated during Mycoplasma hyopneumoniae (Mhp) infection using a gnotobiotic infection model. We found that several inflammatory cytokines (IL-1beta, IL-8, IL-18, and TNF-alpha) and an anti-inflammatory cytokine IL-10 were induced from peripheral blood mononuclear cells (PBMC) of germ-free (GF) piglets stimulated with heat killed Mhp whole antigens, but no IFN-gamma and IL-4 were induced by Mhp. After the intranasal infection of Mhp, IL-1beta, IL-8, IL-18, and IFN-gamma were also detected in the broncho-alveolar lavage fluids (BALF). The antigen-specific IFN-gamma and IL-10 responses after infection of Mhp were gradually suppressed during Mhp infection as well as non-specific immune response to concanavalin A (ConA) and lipopolysacchalide (LPS) at early stage of infection. These results suggested that Mhp infection modulates the immune response of pigs by inducing several cytokines, and causes immuno-suppression of pigs in a gnotobiotic condition.

Effect of sow vaccination against Mycoplasma hyopneumoniae on sow and piglet colonization and seroconversion, and pig lung lesions at slaughter.

The objectives of the present study were to compare Mycoplasma hyopneumoniae (Mh) colonization and serologic status on Mh vaccinated and non-vaccinated sows and to assess the effect of sow vaccination on colonization and serologic status of their piglets at weaning as well as presence of enzootic pneumonia (EP) lung lesions at slaughter. Fifty sows (25 vaccinated and 25 unvaccinated) as well as five of their piglets were included in the study. Blood samples and nasal swabs from sows at 7 weeks pre-farrowing and 1 week post-farrowing and from piglets at 3-4 weeks of age were taken. Nasal swabs and sera were tested by a nested polymerase chain reaction (nPCR) to detect Mh DNA and by an enzyme-linked immunosorbent assay (ELISA) test to detect antibodies to the pathogen, respectively. Finally, at 23 weeks of age, pigs were sent to the slaughter where the extension of EP-compatible gross lesions was assessed. Vaccination with two doses of Mh vaccine resulted in a significantly higher (p<0.05) percentage of seropositive sows than in the non-vaccinated group at 1 week post-farrowing. On the contrary, no statistical significant differences were found in the number of nasal nPCR positive sows among different treatments (p>0.05). At 3-4 weeks of age, a significantly higher percentage (p<0.001) of seropositive piglets came from vaccinated than from non-vaccinated sows. Although the number of Mh infected piglets coming from non-vaccinated sows was higher than the one from vaccinated sows, the difference was not statistically significant (p>0.05). Overall, piglets from vaccinated sows had a significant lower (p<0.05) mean of EP-compatible lung lesions (1.83+/-2.8) than piglets from non-vaccinated sows (3.02+/-3.6). Under the conditions described in this study, sow vaccination did not affect sow or piglet colonization but increased the percentage of seropositive sows and piglets at weaning and reduced significantly the mean EP-compatible lung lesion scoring at slaughter.

Chronological study of Mycoplasma hyopneumoniae infection, seroconversion and associated lung lesions in vaccinated and non-vaccinated pigs.

A field trial was conducted to study Mycoplasma hyopneumoniae (Mh) infection dynamics by nested polymerase chain reaction (nPCR) and serology in pigs of a farm affected by enzootic pneumonia (EP). Moreover, correlation of Mh detection at different respiratory tract sites with presence of EP gross and microscopic lung lesions was assessed. These parameters were studied and compared between vaccinated (two doses at 1 and 3 weeks of age versus one dose at 6 weeks of age) and non-vaccinated pigs. Animals were monitored from birth to slaughter by nPCR from nasal swabs and by serology. From 3 to 22 weeks of age, an average of three pigs per treatment and per batch were necropsied (n = 302). The remaining pigs were sent to the slaughter (n = 103). Nasal, bronchial and tonsillar swabs were taken from the necropsied/slaughtered pigs; gross and microscopic EP-suggestive lung lesions were also assessed. Single and double vaccination resulted in earlier seroconversion and higher percentage of Mh seropositive pigs compared to control group. At slaughter, double vaccinated pigs showed lower percentage of EP-compatible gross lung lesions and lower Mh prevalence at upper respiratory tract sites (nasal cavity and tonsil) than control pigs.

Porcine acute phase protein concentrations in different diseases in field conditions.

Five acute phase proteins (APPs) [C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin (Hp), pig-MAP and albumin] were measured in pigs with naturally occurring infections by porcine reproductive and respiratory syndrome virus (PRRSV), Aujeszky's disease virus (ADV), porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae, and in animals with tail and ear bites, arthritis and other acute inflammatory processes. Healthy specific pathogen-free (SPF) pigs were used as controls. In PRRSV-infected pigs, all APPs with the exception of pig-MAP exhibited significant changes compared with controls. In animals affected with ADV only Hp presented changes of statistical significance, whereas pigs with PCV2 showed marked modifications in all APPs tested. Animals affected with Mycoplasmosis showed concentrations of all positive APPs significantly above levels obtained in SPF pigs, though albumin concentrations did not differ from controls. Finally, all APPs studied showed substantial changes in pigs with acute inflammation. The results indicated that an acute phase response was developed in the different diseases studied, this response being higher in animals with clinical signs and concurrent bacterial processes. Haptoglobin would be the APP that better reflects pathological states; however, to get more complete and valuable information it might be advisable to perform APPs profiles including another APP, such as CRP or SAA.

Comparative efficacy of two single-dose bacterins in the control of Mycoplasma hyopneumoniae in swine raised under commercial conditions in Brazil.

A field study was conducted in Brazil to evaluate the efficacy of single vaccination of pigs with two bacterins to prevent Mycoplasma hyopneumoniae lung lesions. The first (T1) treatment group (174 pigs) was injected with 2 mL of saline solution; group T2 (177 pigs) with 2 mL of bacterin A, and group T3 (174 pigs) with 2 mL of bacterin B. On days-on-test (DOT) 0, 35, 66, 97 and 125, blood samples and tonsil swabs were collected from selected pigs for antibody determination (indirect ELISA) and PCR assay for the presence of M. hyopneumoniae. Pigs were slaughtered on DOT 126-129 and lung lesions were scored blindly. Bacterin A vaccinated pigs had significantly (P < or = 0.05) lower lung lesion scores (0.2%) than bacterin B (0.4%) or saline-treated pigs (1.2%); there was also a significantly lower (P < or = 0.05) number of pigs with lung lesions (27.1%), than bacterin B (38.2%) or saline-treated (55.4%) pigs. The two vaccines had similar (P>0.05) results in terms of mean weight gain, average daily weight gain, feed efficiency, frequency of PCR positives, and there was similar antibody conversion (ELISA). It was concluded that although the productivity parameters and antibody conversions were similar, bacterin A was more effective in preventing and reducing the severity of lung lesions than bacterin B.

[Possibilities and limits of serological diagnosis in pig stocks in the case of Mycoplasma hyopneumoniae infection]. / Möglichkeiten und Grenzen serologischer Diagnostik im Rahmen der Bestandsbetreuung von Schweinen am Beispiel der Mycoplasma- hyopneumoniae-Infektion.

When conducting their investigations to diagnose infectious diseases in swine, practitioners are often forced to use reduced numbers of animals in their samples in order to minimize costs for farmers. A cross-sectional study was conducted approximating such field conditions to show the limits of interpretation with reduced sample sizes in case of Enzootic Pneumonia. Compared with other respiratory pathogens, Mycoplasma hyopneumoniae, the etiologic agent of Enzootic Pneumonia spreads very slowly, mainly when animals are in direct contact. Furthermore, the interpretation of serological results is difficult because several weeks must usually pass for serological reactions to become apparent. Serological testing is normally used to confirm a clinical diagnosis by detecting an increase in antibodies against the etiologic agent. Samples are collected at the beginning of disease and four to six weeks later. An increasing number of serological positive animals in a herd is usually interpreted as spread of infection. The ,,true" prevalence we observed in our investigation was used to make a statistical analysis describing the probability of detecting an increasing prevalence from 0.07 to 0.33 with a reduced sample size. We showed that the probability of detecting an increase of two seropositive animals was 44% if 5 samples per group were analysed. When only 3 samples were analysed per group, this probability decreases to 21%. Compromise must be found between epidemiological necessary and financially acceptable sample size; this could be a minimum of 10 samples per age group.

Dynamics and persistence of Mycoplasma hyopneumoniae infection in pigs.

The purpose of this study was to describe the dynamics (shedding and transmission) of Mycoplasma hyopneumoniae infection within a population of swine and to determine the duration of the infection (persistence) through the identification of the agent in bronchial samples. Sixty-three 2-month-old pigs were used in this study. The pigs (n = 28) were experimentally infected by the intratracheal route with M. hyopneumoniae and considered as seeder pigs. The remaining pigs (n = 32) were not inoculated and randomly allocated to 2 different groups: direct contact exposure pigs (n = 12) and indirect contact exposure pigs (n = 20). Blood samples and nasal swabs were collected throughout the study on days 0, 28, 35, 42, 49, 63, 91, and 119 postinfection. To assess the duration of M. hyopneumoniae infection, 9 seeder and 6 contact exposure pigs were slaughtered at days 155 (group 1), 170 (group 2), and 185 (group 3) postinfection. Direct contact pigs showed evidence of infection on day 28 by polymerase chain reaction (PCR) and on day 35 by serology. The indirect contact exposure pigs presented a very delayed and slow seroconversion pattern; they did not present evidence of transmission until 42 d after the infection of seeder pigs. Identification of M. hyopneumoniae in bronchial swabs was confirmed by nested-PCR from days 155 to 185 postinfection. At the last slaughter date, 77.7% and 100% of the seeders and contact exposure pigs, respectively, tested positive. The results of this study reconfirmed direct infection of M. hyopneumoniae and suggest that indirect transmission can occur in a population. Finally, duration of the infection in this study was longer than previously described.

Evaluation of virulence of Mycoplasma hyopneumoniae field isolates.

The course of enzootic pneumonia, caused by Mycoplasma hyopneumoniae, is strongly influenced by management and housing conditions. Other factors, including differences in virulence between M. hyopneumoniae strains, may also be involved. The aim of this study was to evaluate the virulence of six M. hyopneumoniae field isolates and link it to genetic differences as determined by randomly amplified polymorphic DNA (RAPD) analysis. Ninety, conventional M. hyopneumoniae-free piglets were inoculated intratracheally with the field isolates, a virulent reference strain or sterile culture medium. Animals were examined daily for the presence of disease signs and a respiratory disease score (RDS) was assessed per pig. Twenty-eight days post infection, pigs were euthanized, blood sampled and a lung lesion score was given. Lung samples were processed for histopathology, immunofluorescence testing for M. hyopneumoniae and isolation of M. hyopneumoniae. RAPD analysis was performed on all M. hyopneumoniae strains. Significant differences between isolates were found for the RDS, lung lesion score, histopathology, immunofluorescence and serology. Based on the results of the different parameters, isolates were divided into three "virulence" groups: low, moderately and highly virulent strains. Typically, a 5000 bp RAPD fragment was associated with the highly and moderately virulent strains whereas it was absent in low virulent strains. It was concluded that high variation in virulence exists between M. hyopneumoniae strains isolated from different swine herds. Further studies are required to determine whether the 5000 bp fragment obtained in the RAPD analysis can be used as a virulence marker.