Progression of Mycoplasma hyosynoviae infection in three pig herds. Development of tonsillar carrier state, arthritis and antibodies in serum and synovial fluid in pigs from birth to slaughter

In this investigation, natural infection with Mycoplasma hyosynoviae was followed in groups of individual pigs in three different herds with regard to occurrence of tonsillar carrier state, clinical arthritis and development of antibodies in serum and in synovial fluid. Antibodies were detected by a polyclonal enzyme-linked immunosorbent assay (ELISA) developed for experimental use. The infection with M. hyosynoviae progressed very differently in the three herds investigated. In one herd, the infection was apparently limited to adult pigs. In a second herd, all pigs became tonsillar carriers of M. hyosynoviae, but no mycoplasma-related arthritis nor any serological response was demonstrated within the growing-finishing period. In the third herd investigated, tonsillar infection was detected in all pigs, clinical cases of M. hyosynoviae arthritis followed and a moderate serological response was observed in some, but not all, pigs. In all three herds, M. hyosynoviae infection was carried in the tonsils of the adult pigs, but it was only occasionally transmitted from sows to piglets. Maternal antibodies were transferred to the piglets and persisted for approximately 8-12 weeks. After weaning, some pigs became infected before 20 weeks of age, while others did not. In the majority of cases, the tonsillar infection was established from 11 weeks of age or older. A latent tonsillar infection was present for a period of several weeks within the group of investigated pigs before cases of generalized infection and arthritis were seen. In some cases, generalization of M. hyosynoviae infection in the blood and in joints was observed in spite of the detection of an active serological response a few weeks earlier. The present work suggests that generalization of the infection and development of arthritis may depend on age, immunity, virulence factors and/or infection pressure; in some herds maybe combined with certain triggering mechanisms such as stress and lowered general resistance.     

Use of a novel serum ELISA method and the tonsil-carrier state for evaluation of Mycoplasma hyosynoviae distributions in pig herds with or without clinical arthritis

A novel indirect ELISA test using deoxycholate extracted antigens coated onto a hydrophobic polystyrene surface for measurement of serum antibodies specific for Mycoplasma hyosynoviae was developed. Sensitivity and specificity of the test were found to be superior to previous ELISAs as tested on porcine serum following experimental Mycoplasma infections as well as by analysis of samples from one Danish herd known to be free of M. hyosynoviae and samples from two Norwegian herds without clinical suspicion of M. hyosynoviae infections since their establishment. The epidemiology of M. hyosynoviae infection was then investigated in Danish pig herds with evidence of clinical M. hyosynoviae arthritis (MhA herds, n = 4) and in herds with M. hyosynoviae-carriers among slaughter pigs, but with limited clinical lameness (MhC herds, n = 4). M. hyosynoviae bacteriaemia and tonsil-carrier state were determined by culture of cross-sectional samples of whole-blood (n = 238) and tonsil scrapings (n = 322), respectively. Levels of serum antibodies (n = 396) were measured by the novel indirect ELISA test. There was no significant difference in the ELISA results between the MhA and the MhC herds. Pigs that were tonsil-carriers had a significantly higher response in the ELISA test (P < 0.001) than non-carriers. Slaughter pigs showed higher ELISA values (P < 0.001) and they were more prone to be tonsil-carriers (P < 0.001). The most critical period for spread of the infection seems to be the nursery period (4-12 weeks). The results indicate that M. hyosynoviae infection progresses similarly in herds irrespective of the presence of clinical arthritis. Thus, clinical arthritis due to M. hyosynoviae is probably triggered by other host or herd factors than low levels of serum antibodies or by differences in virulence factors between M. hyosynoviae strains.     

In vitro activity of tiamulin against porcine mycoplasmas

The activity of tiamulin against Mycoplasma hyopneumoniae, M flocculare, M hyorhinis and M hyosynoviae grown in liquid medium was assessed in vitro. With the first three of these mycoplasmas, the activity of tylosin and oxytetracycline was observed in parallel. Tiamulin was more active against M hyopneumoniae and M flocculare, but there was less disparity between the three antibiotics with the strain of M hyorhinis tested. Tiamulin was notably more active against M hyosynoviae than against M hyopneumoniae. It was more difficult to suppress M hyopneumoniae than the other mycoplasmas with tiamulin. This persistence of M hyopneumoniae was more striking when M hyopneumoniae and M hyosynoviae were tested in parallel.     

An abattoir survey of pneumonia and pleuritis in slaughter weight swine from 9 selected herds. II. Enzootic pneumonia of pigs: microbiological findings and their relationship to pathomorphology

Lungs from 191 slaughter pigs with gross lesions indicative of enzootic pneumonia of pigs (EPP) and 80 grossly normal lungs, all originating from 9 different herds, were subjected to microbiological and pathological examinations. The microbiological studies included both bacterial and mycoplasmal culture and also testing for Mycoplasma hyopneumoniae antigen in tissue by indirect immunofluorescent technique. M. hyopneumoniae, Pasteurella multocida and Mycoplasma hyorhinis were detected in 83%, 43% and 37% of the pneumonic lungs, respectively. Mycoplasma flocculare was the most frequently isolated organism in the non-pneumonic lungs. The greatest amounts of macroscopic pneumonia (25.2%) were recorded in lungs with all the three agents M. hyopneumoniae, P. multocida and M. hyorhinis present. The amounts of pneumonia in lungs with M. hyopneumoniae alone and in concurrence with P. multocida, were 9.3% and 15.6%, respectively. M. hyorhinis was also, in this study, associated with higher frequency of diffuse pleuritis. These findings indicate that M. hyorhinis might be involved in the pathogenesis of pneumonia in slaughter pigs. Ninety-six per cent of the isolates of P. multocida from pneumonic lungs could be characterized as type A. In the herds which had the most severe pneumonia problems, toxin production was detected in 83% of the P. multocida strains while only 28% were toxigenic in herds with subclinical to moderate pneumonia problems.     

Mycoplasma hyosynoviae in joints with arthritis in abattoir baconers.

The occurrence of Mycoplasma hyosynoviae in synovial fluid of baconers with chronic arthritis was studied at an abattoir. Cultural examination of synovial fluid samples from diseased tarsal joints of 50 animals from 42 herds yielded M. hyosynoviae in 10 cases from 8 herds. Streptococci were found in 6 cases from 6 other herds. M. hyosynoviae antigen was found in 1 of 47 of the samples, and antibody to the mycoplasma was found in 14 of 40 of the samples by ELISA test. The presence of M. hyosynoviae in a joint was usually accompanied by the corresponding antibody. In joints with streptococcal infection antibody to M. hyosynoviae could not be found.     

Specificity of oligonucleotide probes complementary to evolutionarily variable regions of 16S rRNA from Mycoplasma hyopneumoniae and Mycoplasma hyorhinis.

Mycoplasma is the common name for the smallest free-living microorganisms, the Mollicutes. Mycoplasma hyopneumoniae is of great importance in veterinary medicine, causing enzootic pneumonia in pigs. M hyorhinis can cause polyserositis and may cause pneumonia in piglets. Oligonucleotides complementary to variable regions of 16S rRNA from these mycoplasmas were designed and used as probes for detection and identification of these mycoplasmas. The probe complementary to 16S rRNA of M hyorhinis gave a very weak cross-hybridisation with M hyosynoviae in filter hybridisation experiments, but not with any of the other porcine mycoplasmas tested. Three oligonucleotide probes complementary to M hyopneumoniae 16S rRNA were tested. One of the probes (Mhp6/30) was found to be specific to M hyopneumoniae, but the other two gave cross-hybridisation with M flocculare. Using the Mhp6/30 probe in direct filter hybridisation experiments, it proved possible to detect M hyopneumoniae in lung biopsies from experimentally infected pigs.     

Determination of sensitivity of Mycoplasma hyosynoviae to tylosin and selected antibacterial drugs by a microtiter technique.

A microtiter technique was used for determination of the sensitivity of Mycoplasma hyosynoviae to antibiotics and other drugs. Use of a biphasic agar-broth medium in microtiter plates allowed direct visualization of growth. Results were more reproducible with this system than when broth alone was used and evaluation based on color change was required. Attempts to adapt the test for use with Mycoplasma hyorhinis were not successful. Minimal inhibitory concentrations of 12 drugs and drug combinations for 12 strains of M. hyosynoviae are presented. Drugs with the lowest minimal inhibitory concentrations were tylosin (0.37 mcg/ml)and lincomycin (0.88 mcg/ml), both of which have been used for treatment and control of M. hyosynoviae arthritis. Comparison of the minimal inhibitory concentrations of tylosin for 43 isolates of M. hyosynoviae obtained in 1959 and 1960 and from 1966 through 1971 indicated the possibilty of decreasing sensitivity to the drug although differences between recent isolates and earlier ones were not statistically significant.     

THE ISOLATION OF MYCOPLASMA HYOSYNOVIAE AND EXPOSURE OF PIGS TO EXPERIMENTAL INFECTION

Arginine-utilising mycoplasmas isolated from the pneumonic lungs of 4 pigs were identified as M. hyosynoviae by their growth characteristics, biochemical activity and serological similarity to each other and to 2 type strains of M. hyosynoviae in growth inhibition tests. A purified culture of one M. hyosynoviae isolate was inoculated intravenously into 3 pigs which were killed 9 days later. Although arthritis was not observed clinically, M. hyosynoviae was isolated from 8 of 9 joints cultured, the tonsils and retropharyngeal lymph nodes of all 3 pigs and the lungs of 2 pigs. No histopathological changes were observed in synovial membranes of the joints cultured or lungs but severe depletion of lymphocytes in the cortex of lymphoid follicles in lymph nodes and tonsils was observed.     

Mycoplasms of the swine--A review]

The mycoplasmas constitute a group of microorganisms placed between bacteria and virus. The name, Mycoplasma, is derived from the mycelial morphology of the organisms. The minimal reproductive unit, the elementary body, measures 0.2-0.5 mum. Unlike bacteria, mycoplasmas are not confined by a rigid cell wall, but just by a thin membrane. For their cultivation, though common bacteriological technique is adequate, especially enriched media are required. Antibiotics, as a rule penicillins, are added to the medium for inhibition of bacteria. Up to the present, 5 porcine species of mycoplasma are known: Mycoplasma suipneumoniae, Mycoplasma hyorhinis, Mycoplasma hyosynoviae, Mycoplasma flocculare, and Acholeplasma granularum. The 4 species first mentioned are very common among swine in Denmark. A. granularum has not been demonstrated so far. Occasionally, other species of mycoplasma are found in swine. M. suipneumoniae is by far the most important porcine mycoplasma, being to-day regarded as the primary etiologic agent in porcine enzootic pneumonia. A pure mycoplasma infection usually results in only weak clinical signs of pneumonia, but the disease may be aggravated by secondary factors as bacteria, parasites, and bad housing conditions. Enzootic pneumonia is usually prevalent only in fattening units, where it tends to persist indefinitely. The mycoplasma infection is practically incurable. Control of the disease is attempted by the SPF-program launched by the Danish Meat Research Institute, Roskilde. In this connexion the high sensitivity of mycoplasmas to physico-chemical influence is of advantage, because it results in a low rate of survival of the organisms outside the host. A further advantage is afforded by the fast that M. suipneumoniae is a definitely swine-specific organism. The rest of the porcine mycoplasmas are of far lesser importance. Yet, M. hyorhinis may produce a sero-fibrinous inflammation of serous cavities and joints in pigs less than 10 weeks old, and M. hyosynoviae may produce arthritis in fattening pigs.     

Preparation of Mycoplasma hyopneumoniae antigen for the enzyme-linked immunosorbent assay.

Methods of preparation of Mycoplasma hyopneumoniae antigens for the enzyme-linked immunosorbent assay to detect specific antibody, and properties of the antigens, are described. The reactivity and specificity of antigen prepared by Sephacryl S-300 column chromatography after treatment of M. hyopneumoniae cells with Tween 20 (S-300 antigen) were superior to those of antigen prepared by Sephadex G-25 column chromatography after treatment with Tween 20, or to lipid antigen. There were no differences among strains MI-3, J and VPP11 of M. hyopneumoniae. The S-300 antigen did not show cross-reactivity against porcine hyperimmune sera produced by M. hyorhinis, M. hyosynoviae, M. hyopharyngis, M. flocculare and Acholeplasma granularum. Antibody was first detected in sera of pigs inoculated intranasally with M. hyopneumoniae at two to four weeks after inoculation and seven to eight weeks after pigs were contact-exposed to the same mycoplasma.     

Mycoplasma hyorhinis in Taiwan: diagnosis and isolation of swine pneumonia pathogen

This study attempted to determine whether one multiplex polymerase chain reaction (PCR) is an effective adjunct method for diagnosing Mycoplasma hyopneumoniae and Mycoplasma hyorhinis infection, and whether M. hyorhinis should be considered as an enzootic pneumonia or porcine respiratory disease complex pathogen in Taiwan. To our knowledge, this study is the first to isolate and identify M. hyorhinis as a porcine pathogen in Taiwan. A novel isolation method and a multiplex PCR test were applied to detect and isolate M. hyorhinis. The correlation of M. hyorhinis with swine pneumonia was also examined using a challenge test. Based on weight, 18 pigs were assigned to three groups and housed throughout the study in a specific-pathogen-free (SPF) facility and provided with aseptic feed and water. Groups 1 (n=6) and 2 (n=6) were challenged with 5mL M. hyorhinis culture via tracheal intubation on day 1. The M. hyorhinis strains ATIT-1, -3, and-7 were used to infect group 1 and the strain ATCC 27717 was used for group 2. Culture medium was replaced by phosphate-buffered saline in group 3 (n=6). All pigs were slaughtered on day 28, and their lungs were removed for examination of lesions. Of the six pigs in group 1 challenged with wild-type strains, two had typical mycoplasma pneumonia lesions. No gross lung lesions were observed in groups 2 and 3. Although further examination is necessary to confirm that wild-type strains can cause pneumonia, it appears that M. hyopneumoniae is no longer the only mycoplasma pathogen implicated in the diagnosis of swine enzootic pneumonia (SEP).     

Identification of Mycoplasma hyopneumoniae in formalin-fixed porcine lung, using an indirect immunoperoxidase method

An indirect immunoperoxidase staining technique was evaluated for detection of Mycoplasma hyopneumoniae in formalin-fixed, paraffin-embedded porcine lung. Lungs from swine with induced (n = 4) or naturally occurring M hyopneumoniae infection (n = 31) were examined grossly, by light and immunofluorescent microscopy, and by an indirect immunoperoxidase test, using antibody raised in swine against M hyopneumoniae as the primary antibody. Organisms stained by the indirect immunoperoxidase method were identified in tissue sections as pleomorphic brown-staining structures corresponding to those observed with immunofluorescence. Mycoplasma hyosynoviae, M hyorhinis, and Acholeplasma laidlawii did not stain with the indirect immunoperoxidase method, using antibody raised against M hyopneumoniae.     

Genomic diversity among Danish field strains of Mycoplasma hyosynoviae assessed by amplified fragment length polymorphism analysis

Genomic diversity among strains of Mycoplasma hyosynoviae isolated in Denmark was assessed by using amplified fragment length polymorphism (AFLP) analysis. Ninety-six strains, obtained from different specimens and geographical locations during 30 years and the type strain of M. hyosynoviae S16(T) were concurrently examined for variance in BglII-MfeI and EcoRI-Csp6I-A AFLP markers. A total of 56 different genomic fingerprints having an overall similarity between 77 and 96% were detected. No correlation between AFLP variability and period of isolation or anatomical site of isolation could be demonstrated. An examination of the clonal appearance of M. hyosynoviae isolates recovered from seven herds affected with arthritis revealed presence of several genotypically distinct variants of the organism in a single herd, indicating that different strains may simultaneously be involved in an outbreak of the disease.     

Western blot immunoassay as a confirmatory test for the presence of anti-Mycoplasma hyopneumoniae antibodies in swine serum.

A Western blot immunoassay (WBI) was developed as a confirmatory test for 2 commercial Mycoplasma hyopneumoniae (Mhyo) ELISAs. The WBI detected at least 5 antigen bands (150, 130, 74, 70, and 30 kDa) from Mhyo whole membrane proteins that were not present in the antigens prepared from M. hyorhinis and M. hyosynoviae. Among discrepant sera from vaccinated pigs (n = 17) and field samples (n = 91) assayed by WBI: 1) 2 of the ELISA-positive samples reacted with all 5 antigens bands; 2) all blocking ELISA-positive samples (n = 53) bound 150-, 130-, 74-, and 70-kD antigen bands; and 3) all indirect ELISA-positive samples (n = 55) bound 150-, 130-, and 30-kD antigens. We conclude that the WBI targeting the top 4 antigen bands is a useful confirmatory test for samples initially screened using the commercial Mhyo ELISAs.     

Associations between pathogens in healthy pigs and pigs with pneumonia

The aim of this study was to evaluate the associations between different pathogens in the development of pneumonia and bronchopneumonia in pigs. Samples of bronchoalveolar lavage fluid from 100 pigs showing no clinical signs and 239 pigs with clinical signs of respiratory disease were examined for Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, US-type porcine respiratory and reproductive syndrome virus (PRRSV), EU-type PRRSV, porcine circovirus type 2 (pcv-2), influenza virus type A, alpha-haemolytic Streptococcus species, beta-haemolytic Streptococcus species, Pasteurella multocida, Bordetella bronchiseptica, Haemophilus parasuis and Actinobacillus pleuropneumoniae. These potential pathogens were detected more frequently in the pigs with respiratory problems than in the pigs with no clinical signs. pcv-2 and alpha-haemolytic streptococci were the pathogens most frequently detected; A pleuropneumoniae was isolated in only two cases. There were more often associations between the organisms in the pigs with clinical signs than in the healthy pigs. In particular, alpha-haemolytic streptococci and M hyopneumoniae were both associated with the presence of M hyorhinis, EU-type PRRSV, P multocida and B bronchiseptica, and alpha-haemolytic streptococci also occurred more often in pigs that were already infected with other pathogens. P multocida and B bronchiseptica were both significantly associated with M hyopneumoniae, alpha-haemolytic streptococci, EU-type PRRSV and US-type PRRSV.     

Absence of strictly age-related resistance to Mycoplasma hyosynoviae infection in 6-week-old pigs

Mycoplasma hyosynoviae has never been reported to cause arthritis in pigs younger than 10 weeks of age. In order to investigate whether a strict age-related resistance exists, four 6-week-old pigs and four 13-week-old pigs, all immunologically na?ve with respect to M. hyosynoviae, were inoculated intranasally with the agent and autopsied at day 11 or 13 after infection. One uninoculated pig per age group was included as a negative control. Just as the 13-week-old pigs, the 6-week-old piglets were susceptible to blood, joint and tonsillar infection with M. hyosynoviae and developed clinical arthritis following inoculation with the agent. Thus, we found no evidence of an age-related resistance to the infection.     

Comparison of Mycoplasma hyopneumoniae strains by serologic methods

Six field strains of Mycoplasma hyopneumoniae isolated from pneumonic lungs of pigs, reference strains 11 and J of M hyopneumoniae, Ms 42 strain of Mycoplasma flocculare, and BTS 7 strain of Mycoplasma hyorhinis were compared serologically, using hyperimmune antisera produced in rabbits. All strains of M hyopneumoniae were closely related as determined with the disk growth-inhibition test; however, differences in zone sizes indicated that some antigenic heterogeneity existed. Cross-reactions were not detected between M hyopneumoniae, M flocculare, and M hyorhinis with the growth-inhibition test. The metabolic-inhibition test was more useful for detection of intraspecies antigenic difference than was the growth-inhibition test, since antigenic diversity was clearly detected among M hyopneumoniae strains. Slight cross-reactions were observed between M hyopneumoniae and M flocculare. Using 2-dimensional immunoelectrophoresis, antigenic differences were observed among M hyopneumoniae strains, although many common components also were detected in electropherograms. Mycoplasma flocculare possessed a close antigenic relationship to M hyopneumoniae, as determined by two-dimensional immunoelectrophoresis, whereas both organisms were less related to M hyorhinis. Evidence obtained in this study indicated that strains of mycoplasmas tentatively identified as M hyopneumoniae were similar antigenically, but evidence was obtained also of some diversity in antigenic structure among these strains.     

Intranasally inoculated Mycoplasma hyorhinis causes eustachitis in pigs

Specific-pathogen-free pigs were experimentally inoculated with Mycoplasma hyorhinis, Pasteurella multocida, or both bacterial isolates to evaluate the role of these bacteria in the pathogenesis of otitis media. Six pigs were inoculated intranasally with 4.4 X 10(8) colony-forming units (CFU) of M. hyorhinis. Twenty-one days later, three of these six pigs were inoculated intranasally with 5.0 X 10(8) CFU of P. multocida. Three additional pigs were also inoculated intranasally at the time with P. multocida alone. Two pigs served as uninoculated controls. Seven days later, all pigs were euthanatized. Histologically, subacute inflammation was found in 10 auditory tubes of six pigs and two tympanic cavities of two pigs inoculated with M. hyorhinis. Immunohistochemically, M. hyorhinis antigens were detected on the luminal surface of eight of 10 inflamed auditory tubes, and ultrastructural examination confirmed mycoplasmal organisms in two pigs. M. hyorhinis was isolated from the inflamed tympanic cavities of two pigs. None of the pigs inoculated only with P. multocida had otitis, and P. multocida was not isolated from the tympanic cavity. These findings indicate that M. hyorhinis can cause eustachitis but rarely otitis media in specific-pathogen-free pigs.     

Macrolides and lincomycin susceptibility of Mycoplasma hyorhinis and variable mutation of domain II and V in 23S ribosomal RNA

A total of 151 strains of Mycoplasma hyorhinis isolated from porcine lung lesions (weaned pigs, n=71, and finishers, n=80) were investigated for their in vitro susceptibility to 10 antimicrobial agents. Thirty-one strains (28 from weaned pigs and 3 from finishers) showed resistance to 16-membered macrolide antibiotics and lincomycin. The prevalence of the 16-membered macrolide-resistant M. hyorhinis strain in weaned pigs from Japanese herds has approximately quadrupled in the past 10 years. Several of the 31 strains were examined for mutations in the 23S ribosomal RNA (rRNA). All field strains tested showed a transition of A to G at position 2059 of 23S rRNA-rendered Escherichia coli. On the other hand, individual tylosin- and lincomycin-resistant mutants of M. hyorhinis were selected in vitro from the susceptible type strain BTS7 by 3 to 9 serial passages in subinhibitory concentrations of each antibiotic. The 23S rRNA sequences of both tylosin and lincomycin-resistant mutants were compared with that of the radical BTS7 strain. The BTS7 mutant strain selected by tylosin showed the same transition as the field-isolated strains of A2059G. However, the transition selected in lincomycin showed mutations in domains II and V of 23S rRNA, G2597U, C2611U in domain V, and the addition of an adenine at the pentameric adenine loop in domain II. The strain selected by lincomycin showed an additional point mutation of A2062G selected by tylosin.     

Interferon-alpha and haptoglobin in pigs selectively bred for high and low immune response and infected with Mycoplasma hyorhinis.

Pigs selected for high (H) or low (L) combined antibody and cell-mediated immune response were infected with Mycoplasma hyorhinis. Following the infection, arthritis was more severe in the H pigs, while pleuritis and peritonitis were more severe in the L pigs. Since Mycoplasma infections in pigs often cause just mild signs, indicators of the inflammatory response may aid diagnosis of such infections. In addition, data about the genetic influence on inflammatory response indicators are scanty in the pig. The objectives of the study were therefore: firstly, to determine interferon-alpha (IFNalpha) and haptoglobin in M. hyorhinis infected pigs and, secondly, to investigate if the inflammatory response as determined by these indicators was influenced by genetic selection. There was no consistent increase of IFN-alpha in serum following infection. The serum haptoglobin concentration started to increase 3 days post-infection and there was no difference between the two breeding lines. Hence, M. hyorhinis infection in pigs is reflected in increased serum haptoglobin concentration, but no effect of the magnitude of the inflammatory response on this indicator by selection for high or low immune response was observed.