Incomplete autophagy promotes the replication of Mycoplasma hyopneumoniae.

Autophagy is an important cellular homeostatic mechanism for recycling of degradative proteins and damaged organelles. Autophagy has been shown to play an important role in cellular responses to bacteria and bacterial replication. However, the role of autophagy in Mycoplasma hyopneumoniae infection and the pathogenic mechanism is not well characterized. In this study, we showed that M. hyopneumoniae infection significantly increases the number of autophagic vacuoles in host cells. Further, we found significantly enhanced expressions of autophagy marker proteins (LC3-II, ATG5, and Beclin 1) in M. hyopneumoniae-infected cells. Moreover, immunofluorescence analysis showed colocalization of P97 protein with LC3 during M. hyopneumoniae infection. Interestingly, autophagic flux marker, p62, accumulated with the induction of infection. Conversely, the levels of p62 and LC3-II were decreased after treatment with 3-MA, inhibiting the formation of autophagosomes, during infection. In addition, accumulation of autophagosomes promoted the expression of P97 protein and the survival of M. hyopneumoniae in PK-15 cells, as the replication of M. hyopneumoniae was down-regulated by adding 3-MA. Collectively, these findings provide strong evidence that M. hyopneumoniae induces incomplete autophagy, which in turn enhances its reproduction in host cells. These findings provide novel insights into the interaction of M. hyopneumoniae and host.

Pathogenicity & virulence of Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae: is the etiological agent of porcine enzootic pneumonia (EP), a disease that impacts the swine industry worldwide. Pathogen-induced damage, as well as the elicited host-response, contribute to disease. Here, we provide an overview of EP epidemiology, control and prevention, and a more in-depth review of M. hyopneumoniae pathogenicity determinants, highlighting some molecular mechanisms of pathogen-host interactions relevant for pathogenesis. Based on recent functional, immunological, and comparative "omics" results, we discuss the roles of many known or putative M. hyopneumoniae virulence factors, along with host molecules involved in EP. Moreover, the known molecular bases of pathogenicity mechanisms, including M. hyopneumoniae adhesion to host respiratory epithelium, protein secretion, cell damage, host microbicidal response and its modulation, and maintenance of M. hyopneumoniae homeostasis during infection are described. Recent findings regarding M. hyopneumoniae pathogenicity determinants also contribute to the development of novel diagnostic tests, vaccines, and treatments for EP.

Lung consolidation caused by Mycoplasma hyopneumoniae has a negative effect on productive performance and economic revenue in finishing pigs.

This study aimed to measure the impact of productivity and the consequent economic losses related to lung lesions caused by M. hyopneumoniae. Five-hundred 75 days-old pigs were selected and weighed at the beginning and at the end of the finishing phase to assess the average daily gain (ADG). These animals were evaluated at the slaughter, and samples were collected for laboratory analysis to confirm the presence of M. hyopneumoniae DNA. The lungs of each pig were examined and classified into groups based on the extension of macroscopic lung lesions. Four-hundred eighty-six lungs were examined and 68.5% (n = 333) had macroscopic lung lesions. All pigs with lesions were positive for M. hyopneumoniae in qPCR. Linear mixed regression models (proc Glimmix) were performed on SAS to estimate the effect of macroscopic lung lesion scores on the ADG of finishing pigs. All pairwise comparisons among lesion score groups were performed using p < 0.05. For each increase of one percent in the lesion area, there was a decrease of 1.8 g in the daily weight gain. All the groups had a numerically lower ADG when compared to Group 1 (no lesions). The economic analysis was performed by simulation on Excel to estimate and compare the financial performance of the different lung lesion score groups. The negative correlation found between the group with no lung lesions and the group with more than 15.1% of lesions, showed a statistical difference in ADG, which could mean an opportunity to gain up to $ 6.55 per pig at slaughter. The presence of lesions causes the animals to decrease their productive potential, causing financial loss and generating impacts on the production system.

Mycoplasma hyopneumoniae evades phagocytic uptake by porcine alveolar macrophages in vitro.

Mycoplasma hyopneumoniae, the agent of porcine enzootic pneumonia (EP), is able to persist in the lung tissue and evade destruction by the host for several weeks. To understand the mechanism of pathogen survival, phagocytic uptake of M. hyopneumoniae by primary porcine alveolar macrophages was investigated. Intracellular location and survival of the pathogen were explored using gentamicin survival assays, flow cytometry and confocal microscopy of M. hyopneumoniae 232 labelled with green fluorescent protein (GFP). Following 1 h and 16 h of co-incubation, few viable M. hyopneumoniae were recovered from inside macrophages. Flow cytometric analysis of macrophages incubated with M. hyopneumoniae expressing GFP indicated that the mycoplasmas became associated with macrophages, but were shown to be extracellular when actin-dependent phagocytosis was blocked with cytochalasin D. Confocal microscopy detected GFP-labelled M. hyopneumoniae inside macrophages and the numbers increased modestly with time of incubation. Neither the addition of porcine serum complement or convalescent serum from EP-recovered pigs was able to enhance engulfment of M. hyopneumoniae. This investigation suggests that M. hyopneumoniae evades significant uptake by porcine alveolar macrophages and this may be a mechanism of immune escape by M. hyopneumoniae in the porcine respiratory tract.

Lesões pulmonares em suínos abatidos no matadouro Público Municipal de Esperança, Paraíba / Pulmonary lesions of slaughtered pigs at the municipal public slaughterhouse of Esperança, Paraíba

O objetivo do presente estudo foi avaliar as lesões macroscópicas e histológicas em pulmões de suínos abatidos no abatedouro público de Esperança, Paraíba. Foram inspecionados pulmões de 180 suínos entre julho e dezembro de 2013. Destes, 34 (18,8%) apresentaram lesões. Na análise anatomopatológica dos fragmentos coletados, 82,3% (28/34) exibiram lesões sugestivas de Pneumonia Enzoótica Suína (PES). Seis (17,7%) amostras apresentaram alterações causadas pela distribuição irregular de sangue. Casos sugestivos de PES crônica foram observados em 57,1% (16/28) dos fragmentos coletados. Em 42,9% (12/28) das amostras foram definidos como sugestivos de PES subaguda. Nenhum pulmão apresentou lesões sugestivas de PES aguda. A pesquisa demonstrou que lesões pulmonares em suínos são frequentemente detectadas no abatedouro de Esperança, Paraíba, sendo a maioria destas lesões sugestivas de PES.

The objective of the present study was to evaluate the macroscopic and histological lesions in the lungs of slaughtered pigs at the public slaughterhouse of Esperança, Paraíba. Lungs from 180 pigs were inspected between July and December 2013. Lesions were observed in 34 (18.8%) lungs. In the anatomopathological analysis of the collected fragments, 82.3% (28/34) presented suggestive lesions of Swine Enzootic Pneumonia (SEP). Six (17.7%) samples presented alterations caused by irregular blood distribution. Suggestive cases of chronic SEP were observed in 57.1% (16/28) of the collected fragments. In 42.9% (12/28) of the samples were defined as suggestive of subacute SEP. No lungs presented lesions suggestive of acute PES. The research showed that lung lesions in pigs are frequently detected in the Esperança, Paraíba slaughterhouse, with the majority of these lesions suggestive for SEP.

Association of swine leukocyte antigen class II haplotypes and immune-related traits in a swine line selected for resistance to mycoplasmal pneumonia.

By selective breeding for five generations, a Landrace line has been recently established to improve resistance to mycoplasmal pneumonia of swine (MPS), daily gain (DG), back fat thickness (BF), and plasma cortisol concentrations (COR). To clarify the involvement of swine leukocyte antigen (SLA) polymorphisms in the selection process, we investigated possible associations of 11 SLA-class II haplotypes with selected traits or immune parameters. Pigs with the low-resolution SLA haplotype Lr-0.23 or Lr-0.13, which increased in frequency with the passage of generations, had less severe pathological lesions of MPS, increased leukocyte phagocytic activity, and higher white blood cell counts. In contrast, Lr-0.12 and Lr-0.2, which decreased in subsequent generations, were weakly associated with more severe pathological lesions of MPS. Therefore, in the studied Landrace line, the Lr-0.23 and Lr-0.13 haplotypes are potentially useful genetic markers for selecting and breeding animals with less severe pathological lesions of MPS.

Experimental infection of SPF pigs with Actinobacillus pleuropneumoniae serotype 9 alone or in association with Mycoplasma hyopneumoniae.

The purpose of this study was to compare in SPF pigs, the pathogenicity of an Actinobacillus pleuropneumoniae serotype 9 strain 21 (isolated from the palatine tonsils of a healthy gilt on a French nucleus pig farm, with no clinical signs or lung lesions but a highly positive reaction to A. pleuropneumoniae serotype 9 antibodies) with a pathogenic A. pleuropneumoniae strain 4915 serotype 9 (isolated in France from an outbreak of porcine pleuropneumonia). The pathogenicity of one Mycoplasma hyopneumoniae strain alone or associated with A. pleuropneumoniae strain 21 was also compared. Eight groups of 7 pigs were infected (at 6 or 10 weeks of age) and a control group was kept non-infected. Results showed that sensitivity to A. pleuropneumoniae was related to the age of the pig (6 weeks vs 10 weeks) whatever the strain. Surviving pigs infected at 6 weeks of age developed severe clinical signs, lung lesions typical of A. pleuropneumoniae and they seroconverted. In contrast, symptoms and lung lesions were almost non-existent in pigs infected with strain 21 at 10 weeks of age, but a seroconversion was observed with very high ELISA titres. These results were in accordance with those observed in the nucleus pig farm. Infection with M. hyopneumoniae alone induced typical mycoplasmal symptoms, pneumonia and seroconversion. Symptoms and lung lesions were the most noticeable in pigs infected with M. hyopneumoniae at 6 weeks of age and with A. pleuropneumoniae 4 weeks later. Our results show that the presence of A. pleuropneumoniae serotype 9 in a pig herd may be clinically unnoticed and that M. hyopneumoniae may potentiate A. pleuropneumoniae infection.

Plasmid-mediated growth hormone-releasing hormone efficacy in reducing disease associated with Mycoplasma hyopneumoniae and porcine reproductive and respiratory syndrome virus infection.

The purpose of this study was to determine the effects of plasmid-mediated growth hormone releasing hormone (GHRH) supplementation on the clinical outcomes of pigs vaccinated against and challenged with either Mycoplasma hyopneumonia (M. hyo) and/or with porcine reproductive and respiratory syndrome (PRRS) virus. Before the first vaccination, pigs received a single i.m. injection of 0.625 mg of a porcine GHRH-expressing plasmid followed by electroporation of the injection site. Pigs were vaccinated at 2-wk intervals, challenged with either M. hyo and/or PRRS virus 2-wk after the second vaccination, and necropsied at 17 and 36 d after challenge. Clinical parameters associated with M. hyo challenge were improved with the GHRH treatment. Average daily gain between challenge and necropsy was improved (P = 0.04). Respiratory scores for M. hyo-challenged pigs tended to be lower in GHRH-treated animals compared to controls, and coughing scores were improved by the treatment (P = 0.01). Macroscopic lesions associated with M. hyo infection pneumonia were fewer in the group that received the GHRH-expressing plasmid. No differences between treatment groups in the macroscopic pneumonia associated with PRRS virus were observed. No differences in serum antibodies to M. hyo or PRRS virus were observed with GHRH treatment. Nevertheless, IgG in the bronchioalveolar lavage was increased by the GHRH treatment in M. hyo-challenged animals (P < 0.03). The results of this study suggest that GHRH supplementation before vaccination may enhance the protection against M. hyo-induced pneumonia and that a single dose of GHRH-expressing plasmid was sufficient to elicit an improved clinical outcome in this disease challenge model.

Decreased protein accretion in pigs with viral and bacterial pneumonia is associated with increased myostatin expression in muscle.

Chronic respiratory infections reduce growth in pigs but protein accretion (PA) during an ongoing multifactorial respiratory infection has not been determined, and the mechanisms underlying growth inhibition are largely unknown. The objectives of this study were to determine whether viral and bacterial pneumonia in young pigs decrease PA, increase serum IL-1beta and IL-6, and increase myostatin (MSTN) mRNA in biceps femoris and triceps muscles. Mycoplasma hyopneumoniae (Mh) or medium was given intratracheally at 4 wk of age, Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) or medium was given intranasally at 6 wk of age, and pigs were killed 7 or 14 d after PRRSV inoculation for body composition analysis. PRRSV but not Mh induced a marked increase (P < 0.01) in IL-1beta, IL-6, and MSTN mRNA and a decrease (P < 0.01) in food intake, daily weight gain, PA, and lipid accretion. PRRSV also reduced (P < 0.01) myofiber area in the biceps femoris. Food intake, weight gain, PA, and weight of biceps femoris and triceps muscles were negatively correlated (r = -0.4 to -0.8, P < 0.05) with serum IL-1beta and IL-6 and with MSTN mRNA in muscle. These results suggest that the magnitude of increases in inflammatory cytokines during a respiratory infection may be predictive of decreases in PA and growth. They further suggest that during infection growth of skeletal muscle is limited in part by myostatin.

Infection of growing swine with porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae--effects on growth, serum metabolites, and insulin-like growth factor-I.

This study evaluated the influence of concomitant infections with porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae on growth performance, serum metabolite concentrations, and serum insulin-like growth factor-I (IGF-I) in growing pigs. Twenty-two barrows (10 weeks of age) were treated with either an intranasal administration of PRRSV and an intratracheal infusion of M. hyopneumoniae (treatment; n = 8) or a sham inoculation with medium (sham; n = 8), or were not treated (control; n = 6). The sham pigs were matched by body weight and pair-wise fed with treatment pigs. Pigs were weighed on the day of inoculation (day 0) and at 4 weeks postinoculation (day 28). Blood samples were collected prior to inoculation and at weekly intervals for 4 weeks. Pigs in the treatment group exhibited clinical signs consistent with PRRSV infection and M. hyopneumoniae pneumonia. Diagnostic procedures confirmed that treatment pigs were inoculated with PRRSV and M. hyopneumoniae and that sham and control pigs remained free of both pathogens. Average daily gain and feed conversion did not differ among the 3 groups. The IGF-I levels differed (P < 0.05) between control and treatment pigs, even after feed intake returned to similar levels among groups. At day 7, IGF-I concentrations were greater in sham pigs compared with treatment pigs, despite similar feed intake. Sham inoculation and decreased feed intake in sham pigs did not alter serum IGF-I concentrations. Evidently, IGF-I status of pigs affected with disease is influenced by nutritional and nonnutritional factors during the disease process.

Growth performance and whole-body composition of pigs experimentally infected with Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae (Mh) is the primary infectious pathogen responsible for enzootic pneumonia in pigs. Although Mh is thought to impair growth performance, whole-body composition, and fat and protein accretion in pigs with pneumonia have not been reported and the mechanism through which Mh reduces growth is unknown. The objectives of this study were to evaluate the effects of Mh on growth performance, whole-body composition, and protein and fat accretion in nursery pigs and to determine whether Mh infection increases the expression of interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha (TNF-alpha). Sixty-four 2-wk-old Mh-free pigs were used (two trials) in a randomized complete block design. In each trial, two pigs were housed in each of 16 disease-containment chambers. At 4 wk of age, pigs were inoculated intratracheally with 3 mL of Mh broth (P5722-3, 10(7) cfu/mL) or sterile Friis culture medium. Clinical signs of disease and feed intake were monitored daily and body weight was determined weekly for 4 wk. Whole-body composition was determined from pigs killed 0, 14, and 28 d after inoculation, and the comparative slaughter technique was used to estimate protein and fat accretion. At death, gross lung lesions were quantified, and lung tissue was collected to verify the presence or absence of Mh, and to determine cytokine mRNA levels. Control pigs displayed no overt signs of infection and were Mh-negative and free of pulmonary lesions. Pigs inoculated with Mh showed pneumonic coughing (P < 0.005), were Mh-positive, and had pulmonary lesions that affected 4.5% (P < 0.01) and 14.1% (P < 0.001) of total lung surface area at 14 and 28 d, respectively, after inoculation. Ribonuclease protection assays revealed increased IL-1beta (P < 0.04) and TNF-alpha (P < 0.06) mRNA in lung tissue collected from a lesion site compared with tissue collected 10 cm from a lesion site or from control pigs. Interestingly, Mh did not depress weight gain or feed efficiency during any week of the 28-d study (P > 0.10). Moreover, Mh did not affect whole-body fat or protein accretion (P > 0.10). Thus, in spite of inducing disease and expression of inflammatory cytokines, Mh alone did not affect growth performance and whole-body composition of nursery pigs during the 4-wk experiment. The ability of pigs to contend with Mh may have resulted from the absence of other pathogens that generally co-exist with Mh under commercial conditions.

A field trial to evaluate a Mycoplasma hyopneumoniae vaccine: effects on lung lesions and growth rates in swine.

A killed Mycoplasma hyopneumoniae vaccine was evaluated in a single swine herd in which the farrowing barn and weaner rooms were on one Mycoplasma-free farm, while the growing and finishing barn was on a separate farm on which Mycoplasma was present. The study was carried out in a cohort of pigs born in a 12-week period. Pigs born in 6 of the 12 wk were vaccinated and the rest were left as controls. The vaccine was administered twice at approximately 3 and 6 wk of age. Carcass characteristics, lung lesions, and growth rates were recorded on 893, 390, and 220 pigs, respectively. The vaccine reduced the prevalence of pneumonic lesions in slaughter hogs from 69% to 36% (P < 0.001). It also appeared to reduce the prevalence of pleuritis from 20% to 13%, but the difference was only statistically significant at P = 0.07. The vaccine had no effects on carcass characteristics except that carcasses of vaccinated pigs were, on average, 1 kg heavier than those of nonvaccinated pigs, and a smaller percentage of vaccinated pigs were shipped "light" (carcass weight < 70 kg). Two methods were used to estimate the effect of the vaccine on growth rates (as measured by days to 80 kg carcass weight) resulting in estimates of 11 and 2 d reduction attributable to vaccination, respectively. The latter estimate was probably an underestimate for reasons discussed in the paper.

Effect of enzootic pneumonia of pigs on growth performance.

Growing pigs were naturally infected with a field strain of Mycoplasma hyopneumoniae to assess the effect of enzootic pneumonia on production. Both the initial ("breakdown") and endemic stages of infection were evaluated. The pigs were reared under environmental and management conditions commonly found on commercial piggeries in South Australia. Growth rate of pigs held in-contact with inoculated pigs was reduced by 12.7% (p less than 0.01) between 50 to 85 kg bodyweight. In the second trial inoculated gilts were used to naturally infect piglets during suckling. Growth rate of infected pigs was reduced by 15.9% (p less than 0.001) between 8 to 85 kg bodyweight, while feed conversion was depressed by 13.8% (p less than 0.05) between 10 to 25 kg bodyweight. At current feed and production costs this reduced performance added approximately $2.80 to the cost of every pig produced. These losses were recorded in groups of pigs in which enzootic pneumonia was present. At slaughter, 40% of lungs contained gross lesions of enzootic pneumonia which were free of significant secondary bacteria. The nature of the infection was established by gross and microscopic pathology and confirmed by the detection of specific complement fixing antibody in infected pigs and the demonstration of M. hyopneumoniae by direct immunofluorescent staining of lung sections.

Pulmonary and hematologic changes in swine with Mycoplasma hyopneumoniae pneumonia.

Studies were made of the physiologic changes in the lung of young growing pigs intratracheally inoculated at 5.5 to 6 weeks of age with Mycoplasma hyopneumoniae. Hematologic changes also were studied. Studies were performed at the 4th, 5th, and 6th weeks after inoculations were done. The inoculated pigs had a marked decrease in body weight gain. There were no significant changes in the heart rate, respiratory rate, and rectal temperature, even though at necropsy the apical, cardiac, and diaphragmatic lobes of the lungs of inoculated pigs had scattered, but well-demarcated, pneumonic lesions. The lungs were edematous and increased in weight, compared with those of control (noninoculated) pigs. There were several changes in respiratory functions in the inoculated animals, including significant increases in esophageal pressure negativity, expiratory flow rate, tidal and minute volumes, and work of breathing and significantly less oxygen consumption. Inspiratory flow rate and airway resistance were markedly increased. The elasticity of infected lungs was reduced, as shown by notable decreases of dynamic lung compliance. According to blood gas analyses, acid-base balance had changed in the inoculated pigs. Arterial blood pH, arterial partial pressures of O2 and CO2, and arterial concentration of HCO3- decreased. To some degree respiratory acidosis indicated by a significant decrease of base excess was evident in the blood of inoculated pigs. As a result, inoculated animals required hyperventilation (compensatory respiratory alkalosis) to maintain the pH of arterial blood near a physiologically normal value. The pigs with mycoplasmal pneumonia showed no drastic hematologic changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Tiamulin feed medication for the maintenance of weight gains in the presence of mycoplasmal pneumonia in swine.

Tiamulin, a semisynthetic antibiotic, was fed to growing pigs and its value in maintaining weight gain and feed efficiency in the face of mycoplasmal pneumonia was determined. Four treatments divided among 64 growing pigs from a conventional pig farm were used to test the effects of tiamulin administered in feed at levels of 10, 20 and 30 ppm. All pigs were artificially infected with Mycoplasma hyopneumoniae. Medication was started on the 14th day after infection (d 0) and continued for 28 d (d 0 to 28). Pigs were continued on test for an additional 14-d period for further observation (d 28 to 42) after the medicated feed was withdrawn. Tiamulin fed at 10, 20 and 30 ppm for 28 d did not cure the mycoplasmal pneumonia. The effect of tiamulin on growth performance was not the same for all periods. During the medication period (d 0 to 28), both daily gain and feed conversion were not significantly affected by dietary tiamulin concentrations. For the post-treatment period (d 28 to 42), tiamulin improved daily gain (P less than .05) and feed conversion (P less than .05) over those of nonmedicated controls, and the responses of both daily gain and feed efficiency increased linearly (P less than .05) as dietary tiamulin levels increased.