Transfer of Mycoplasma hyopneumoniae-specific cell mediated immunity to neonatal piglets.

Mycoplasma hyopneumoniae is the primary agent of enzootic pneumonia in pigs. Although cell mediated immunity (CMI) may play a role in protection against M. hyopneumoniae, its transfer from sows to their offspring is poorly characterized. Therefore, maternally-derived CMI was studied in piglets from vaccinated and non-vaccinated sows. The potential influence of cross-fostering before colostrum ingestion on the transfer of CMI from dam to piglets was also investigated. Six M. hyopneumoniae vaccinated sows from an endemically infected herd and 47 of their piglets, of which 24 piglets were cross-fostered, were included, as well as three non-vaccinated control sows from an M. hyopneumoniae-free herd and 24 of their piglets. Vaccinated sows received a commercial bacterin intramuscularly at 6 and 3 weeks prior to farrowing. The TNF-α, IFN-γ and IL-17A production by different T-cell subsets in blood of sows, colostrum and blood of piglets was assessed using a recall assay. In blood of sows cytokine producing T-cells were increased upon M. hyopneumoniae vaccination. Similarly, M. hyopneumoniae-specific T-cells were detected in blood of 2-day-old piglets born from these vaccinated sows. In contrast, no M. hyopneumoniae-specific cytokine producing T-cells were found in blood of piglets from control sows. No difference was found in M. hyopneumoniae-specific CMI between cross-fostered and non-cross-fostered piglets. In conclusion, different M. hyopneumoniae-specific T-cell subsets are transferred from the sow to the offspring. Further studies are required to investigate the role of these transferred cells on immune responses in piglets and their potential protective effect against M. hyopneumoniae infections.

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 variability: Current trends and proposed terminology for genomic classification.

Mycoplasma hyopneumoniae (M. hyopneumoniae) is the aetiologic agent of enzootic pneumonia in swine, a prevalent chronic respiratory disease worldwide. Mycoplasma hyopneumoniae is a small, self-replicating microorganism that possesses several characteristics allowing for limited biosynthetic abilities, resulting in the fastidious, host-specific growth and unique pathogenic properties of this microorganism. Variation across several isolates of M. hyopneumoniae has been described at antigenic, proteomic, transcriptomic, pathogenic and genomic levels. The microorganism possesses a minimal number of genes that regulate the transcription process. Post-translational modifications (PTM) occur frequently in a wide range of functional proteins. The PTM by which M. hyopneumoniae regulates its surface topography could play key roles in cell adhesion, evasion and/or modulation of the host immune system. The clinical outcome of M. hyopneumoniae infections is determined by different factors, such as housing conditions, management practices, co-infections and also by virulence differences among M. hyopneumoniae isolates. Factors contributing to adherence and colonization as well as the capacity to modulate inflammatory and immune responses might be crucial. Different components of the cell membrane (i.e. proteins, glycoproteins and lipoproteins) may serve as adhesins and/or be toxic for the respiratory tract cells. Mechanisms leading to virulence are complex and more research is needed to identify markers for virulence. The utilization of typing methods and complete or partial-gene sequencing for M. hyopneumoniae characterization has increased in diagnostic laboratories as control and elimination strategies for this microorganism are attempted worldwide. A commonly employed molecular typing method for M. hyopneumoniae is Multiple-Locus Variable number tandem repeat Analysis (MLVA). The agreement of a shared terminology and classification for the various techniques, specifically MLVA, has not been described, which makes inferences across the literature unsuitable. Therefore, molecular trends for M. hyopneumoniae have been outlined and a common terminology and classification based on Variable Number Tandem Repeats (VNTR) types has been proposed.

Impact of diversity of Mycoplasma hyopneumoniae strains on lung lesions in slaughter pigs.

The importance of diversity of Mycoplasma hyopneumoniae (M. hyopneumoniae) strains is not yet fully known. This study investigated the genetic diversity of M. hyopneumoniae strains in ten pig herds, and assessed associations between the presence of different strains of M. hyopneumoniae and lung lesions at slaughter. Within each herd, three batches of slaughter pigs were investigated. At slaughter, from each batch, 20 post mortem bronchoalveolar lavage fluid samples were collected for multiple locus variable-number tandem repeat analysis (MLVA), and lung lesions (Mycoplasma-like lesions, fissures) were examined. Multivariable analyses including potential risk factors for respiratory disease were performed to assess associations between the number of different strains per batch (three categories: one strain, two-six strains, ≥seven strains), and the lung lesions as outcome variables. In total, 135 different M. hyopneumoniae strains were found. The mean (min.-max.) number of different strains per batch were 7 (1-13). Batches with two-six strains or more than six strains had more severe Mycoplasma-like lesions (P = 0.064 and P = 0.012, respectively), a higher prevalence of pneumonia [odds ratio (OR): 1.30, P = 0.33 and OR: 2.08, P = 0.012, respectively], and fissures (OR = 1.35, P = 0.094 and OR = 1.70, P = 0.007, respectively) compared to batches with only one strain. In conclusion, many different M. hyopneumoniae strains were found, and batches of slaughter pigs with different M. hyopneumoniae strains had a higher prevalence and severity of Mycoplasma-like lung lesions at slaughter, implying that reducing the number of different strains may lead to less lung lesions at slaughter and better respiratory health of the pigs.

Mycoplasma hyorhinis is a potential pathogen of porcine respiratory disease complex that aggravates pneumonia caused by porcine reproductive and respiratory syndrome virus.

The porcine respiratory disease complex (PRDC) caused by numerous bacterial and viral agents has a great impact on pig industry worldwide. Although Mycoplasma hyorhinis (Mhr) has been frequently isolated from lung lesions from pigs with PRDC, the pathological importance of Mhr may have been underestimated. In this study, 383 serum samples obtained from seven herds with a history of PRDC were tested for specific antibodies to Mhr, Mycoplasma hyopneumoniae (Mhp), and porcine reproductive and respiratory syndrome virus (PRRSV). Seropositive rates of PRRSV were significantly correlated with those of Mhr (correlation coefficient, 0.862; P-value, 0.013), but not with those of Mhp (correlation coefficient, -0.555; P-value, 0.196). In vivo experiments demonstrated that pigs co-infected with Mhr and PRRSV induced more severe lung lesions than pigs infected with Mhr or PRRSV alone. These findings suggest that Mhr is closely associated with pneumonia caused by PRRSV and provide important information on Mhr pathogenesis within PRDC. Therefore, effective PRDC control strategies should also consider the potential impact of Mhr in the pathogenesis of PRDC.

Porcine respiratory disease complex: Interaction of vaccination and porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, and Mycoplasma hyopneumoniae.

Porcine respiratory disease is a multifactorial and complex disease caused by a combination of infectious pathogens, environmental stressors, differences in production systems, and various management practices; hence the name porcine respiratory disease complex (PRDC) is used. Porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), and Mycoplasma hyopneumoniae are considered to be the most important pathogens that cause PRDC. Although interactions among the three major respiratory pathogens are well documented, it is also necessary to understand the interaction between vaccines and the three major respiratory pathogens. PRRSV and M. hyopneumoniae are well known to potentiate PCV2-associated lesions; however, PRRSV and mycoplasmal vaccines can both enhance PCV2 viraemia regardless of the effects of the actual PRRSV or M. hyopneumoniae infection. On the other hand, M. hyopneumoniae potentiates the severity of pneumonia induced by PRRSV, and vaccination against M. hyopneumoniae alone is also able to decrease PRRSV viraemia and PRRSV-induced lung lesions in dually infected pigs. This review focuses on (1) interactions between PCV2, PRRSV, and M. hyopneumoniae; and (2) interactions between vaccines and the three major respiratory pathogens.

Full-length genome sequence analysis of a Hungarian porcine reproductive and respiratory syndrome virus isolated from a pig with severe respiratory disease.

Here, we report the isolation of a type 1 porcine reproductive and respiratory syndrome virus (PRRSV) strain from a clinical outbreak of severe respiratory problems and high fever. Next-generation sequencing was used to determine the complete genome sequence of the isolate (9625/2012). The virus belongs to a new branch within subtype 1, clade D, and shows the highest similarity to PRRSV Olot/1991 and to the Amervac vaccine strain. Mutation analysis of 9625/2012 revealed no evidence of recombination but did show a high proportion of amino acid substitutions in the putative neutralizing epitopes, suggesting an important role of selective immune pressure in the evolution of PRRSV 9625/2012.

Detection, quantification and genetic variability of Mycoplasma hyopneumoniae from apparently healthy and pneumonic swine / Detecção, quantificação e variabilidade genética de Mycoplasma hyopneumoniae a partir de suínos pneumônicos e aparentemente saudáveis

Molecular differences among Mycoplasma hyopneumoniae strains present in pneumonic lungs of swine have been largely studied. However, no comparative studies concerning the strains present in apparently healthy pigs have been carried out. This study aimed to detect, quantify and perform molecular analysis of M. hyopneumoniae strains in pig lungs with and without pneumonic lesions. The detection of M. hyopneumoniae was performed using multiplex PCR (YAMAGUTI, 2008), real-time PCR (STRAIT et al., 2008) and multiple VNTR amplification (VRANCKX et al., 2011). Molecular characterization of the strains was achieved by analysis of the VNTR copy number in P97R1, P146R3, H2R1 and H4. M. hyopneumoniae was detected in samples from healthy and pneumonic pigs and the amount of M. hyopneumoniae positive samples detected varied with the type of assay. The greater number of positive samples was identified by the multiple VNTR amplification combined with capillary electrophoresis. Using real-time PCR, 4.9*104 M. hyopneumoniae genome copies/mL was detected in apparently healthy lungs. A mean quantity of 3.9*106 M. hyopneumoniae genome copies/mL was detected in pneumonic lungs. The analysis of VNTR copy number demonstrated a high genetic variability of the M. hyopneumoniae strains present in apparently healthy and pneumonic lungs. Strains having 3 VNTR copy number in P97R1, were detected only in pneumonic lungs and strains having 40 and 43 VNTR copy number in P146R3 were detected only in apparently healthy lungs. Despite the genetic variability of M. hyopneumoniae, predominant strains in the swine farms could be identified...

As diferenças moleculares entre as estirpes de Mycoplasma hyopneumoniae presentes em pulmões de suínos com pneumonia tem sido estudadas. Porém, estudos comparativos relativos as estirpes presentes nos suínos aparentemente saudáveis não foram levados a cabo. O objetivo do estudo foi a detecção, quantificação e analise molecular de M. hyopneumoniae nos pulmões suínos com e sem lesões pneumônicas. Para a detecção de M. hyopneumoniae usaramse o PCR Multiplo (YAMAGUTI, 2008), o PCR a Tempo Real (STRAIT et al., 2008) e a amplificação de múltiplo VNTR (VRANCKX et al., 2011). A caracterização molecular das estirpes foi realizada mediante a análise do número de copias de VNTR em P97R1, P146R3, H2R1 e H4. O M. hyopneumoniae foi detectado em amostras de suínos saudáveis e pneumônicos e a quantidade de M. hyopneumoniae nas amostras positivas variou com o tipo de ensaio. O maior número de amostras positivas foi identificado pela amplificação de múltiplas VNTR combinado com a eletroforese de capilares. Usando o PCR a Tempo Real, 4.9*104 copias de genoma/mL de M. hyopneumoniae foram detectadas em pulmões aparentemente saudáveis. Uma quantidade média de 3.9*106 copias de genoma/mL de M. hyopneumoniae foi detectada em pulmões pneumônicos. A análise do número de copias de VNTR demonstrou uma elevada variabilidade...

Occurrence and severity of lung lesions in slaughter pigs vaccinated against Mycoplasma hyopneumoniae with different strategies.

Different vaccination strategies against Mycoplasma hyopneumoniae have been adopted worldwide. Reports from the field indicate varying levels of protection among currently available vaccines. The goal of the present study was to compare the efficacies of three widespread commercial vaccination strategies against M. hyopneumoniae under field conditions. 20 farms were included. 14 farms used different single dose vaccines (vaccine 1 [V1], 8 herds; vaccine 2 [V2], 6 herds); another 6 farms (V3) used a two dose vaccination strategy. Gross lesions of 854 lungs and histopathology from 140 lungs were quantified, and a quantitative PCR was applied to detect M. hyopneumoniae and porcine circovirus 2 (PCV2) DNA in lung tissue (n=140). In addition, porcine reproductive and respiratory disease virus (PRRSV), swine influenza virus (SIV), Actinobacillus pleuropneumoniae, Haemophilus parasuis and Pasteurella multocida were tested by qualitative PCR. 53% of lungs were positive for M. hyopneumoniae. 55.9% of lungs showed macroscopic enzootic pneumonia (EP)-like lesions. Lung lesion scores (P<0.001) and M. hyopneumoniae-loads (P<0.008) differed significantly among the vaccination groups, with the most severe cases and highest amounts occurring in V1. Histological alterations differed (P<0.001) between V1 and V3. Lung lesion scores and histopathological changes were significantly correlated, with prevalence and load of M. hyopneumoniae indicating that the applied diagnostic tools are valuable in confirming the prevalence and severity of M. hyopneumoniae infections. Comparing different vaccination strategies against M. hyopneumoniae indicates varying levels of protection. M. hyopneumoniae is still a major problem despite the widely applied vaccination.

Interactions of highly and low virulent Mycoplasma hyopneumoniae isolates with the respiratory tract of pigs.

Mycoplasma hyopneumoniae is the etiological agent of swine enzootic pneumonia, a chronic nonfatal disease affecting pigs of all ages. To obtain better insight in the mechanisms responsible for differences in virulence between highly and low virulent M. hyopneumoniae isolates, 23 caesarean-derived, colostrum-deprived piglets were randomly assigned to three groups. Groups 1 and 2 consisted of nine animals each, which were intratracheally inoculated at 1 week of age with a highly or a low virulent isolate of M. hyopneumoniae, respectively. The remaining five animals were inoculated with sterile culture medium. Animals were euthanized at 5, 10, 15 and 28 days post-inoculation (DPI). Animals inoculated with the highly virulent isolate had more neutrophils in BAL fluid at 10, 15 and 28DPI compared to the other groups. At 10 and 15DPI, animals in the highly virulent group had significantly higher concentrations of TNF-alpha in BAL fluid. IL-1beta concentration in this group was higher at 5 and 28DPI compared to the other groups. From 10DPI onwards, significantly higher titres of M. hyopneumoniae were detected in the BAL fluid of animals inoculated with the highly virulent isolate compared to animals inoculated with the low virulent isolate. Additionally, the in vitro generation time of the highly virulent M. hyopneumoniae isolate was significantly shorter than that of the low virulent isolate. The present study indicates that the difference in pathogenicity between the highly and low virulent isolates is associated with a faster in vitro growth, a higher capacity to multiply in the lungs and the induction of a more severe inflammation process by the highly virulent isolate.

Experimental dual infection of pigs with an H1N1 swine influenza virus (A/Sw/Hok/2/81) and Mycoplasma hyopneumoniae.

Dual infection of pigs with swine influenza virus (SIV) and Mycoplasma hyopneumoniae was carried out to compare the clinical and pathological effects of dual infection in caesarian derived and colostrums deprived (CDCD) pigs, with that of a single infection with M. hyopneumoniae. In Experiment 1, 40-day-old CDCD pigs were inoculated only with SIV (A/Sw/Hok/2/81, H1N1). The virus was isolated from nasal swabs for 5-6 days. None of these pigs showed clinical signs of infection throughout the experimental period. These results suggested that this strain can infect pigs but is only slightly pathogenic when it is inoculated singly to a CDCD pig. In Experiment 2, 60-day-old CDCD pigs were inoculated with M. hyopneumoniae and then were inoculated with SIV (A/Sw/Hok/2/81) at 1 week (MHYO-7d-SIV-7d group) or 3 weeks (MHYO-21d-SIV-7d group) after M. hyopneumoniae inoculation. Macroscopically, dark red-to-purple lung lesions were observed in all of pigs at 14 or 28 days post-inoculation. Percentages of dark red-to-purple lung lesions in dual infection groups (MHYO-7d-SIV-7d group: 18.7 +/- 4.2%, MHYO-21d-SIV-7d group: 23.0 +/- 8.0%) were significantly (P < 0.05) increased compared to those of each control group in which pigs were inoculated only with M. hyopneumoniae (MHYO-14d group: 4.7 +/- 2.9%, MHYO-28 group: 3.3 +/- 2.4%). Microscopically, bronchial epithelial lesions (epithelial disruption, degeneration, hyperplasia and formation of microabscess) were frequently observed in dark red-to-purple lung lesions of only the dual infection groups. These results demonstrate that the lung lesion of pigs inoculated with M. hyopneumoniae and SIV is more severe than that of pigs inoculated only with M. hyopneumoniae.