M. Gallisepticum Research Articles

Detection of Mycoplasma Synoviae and Other Pathogens in Laying Hens with Respiratory Signs in the Rearing and Production Phases

The present study aimed to investigate the occurrence of Mycoplasma synoviae (MS), M. gallisepticum (MG), Ornitobacterium rhinotracheale (OR), Avibacterium paragallinarum (AP), Pasteurella multocida (PM) and Infectious Bronchitis Virus (IBV) in laying hens with respiratory clinical signs in two phases of production. 140 tracheal swabs and 140 blood samples were collected from laying hens in the rearing and production phases, the chickens belonged to six farms (A-F) located in the state of São Paulo, Brazil. The samples were analyzed by PCR for MG, MS, OR, AP, PM and IBV and by ELISA for MG and MS. The highest frequencies observed by PCR were for MS at farms B and C with 95 and 100% positivity, followed by MG at farms D and E with 35% and 65%, IBV with 35% at farm F and ORT with 15% at farm A. All flocks were positive for MG and MS in serology. Although MG and IBV have been detected, this can be explained by the vaccination protocols, since live attenuated vaccines are widely used for immunization against these pathogens. It was also possible to detect OR and AP thorugh PCR in some flocks. The occurrence of several etiological agents that cause respiratory diseases in laying hens was confirmed by PCR and serology, with MS being the most prevalent and being present in all farms studied.

Evaluation of the potential influence of the disinfection cycle on the efficacy of strain F Mycoplasma gallisepticum vaccine administered by in ovo injection to layer hatching eggs , ,

Summary In ovo administration of a live-attenuated F-strain Mycoplasma gallisepticum vaccine (FMG) has proven to be a promising alternative vaccination route for layer chickens to combat M. gallisepticum (MG) infections. Its practical application within current in ovo vaccination procedures, employing a disinfection step after each injection, has been uncertain. The objective of this experiment was to determine the ability of FMG to survive the in ovo disinfection step to elicit a posthatch immune response. In 2 trials, layer eggs were in ovo-vaccinated with or without the disinfection step between injections. Utilizing an Embrex Inovoject M machine, treatments included noninjected eggs, diluent-injected eggs with or without a disinfection step, and eggs vaccinated with an FMG vaccine with or without a disinfection step. Ninety hatched chicks per treatment in both trials were raised to 6 wk of age and swabbed in the choanal cleft for FMG detection via PCR and were bled for IgM (serum plate agglutination) and IgG (ELISA) testing for detection of antibody production against MG. The percentage of birds in which FMG was detected at 6 wk of age were similar among both FMG vaccination treatments in both trials (overall 89.4% without and 91.6% with disinfectant step). Birds vaccinated with FMG also developed antibody responses regardless of employment of the disinfection step. These results indicate that FMG survives the disinfection step employed by commercial machines and can be readily applied to current commercial procedures of in ovo vaccination with no detriment to vaccine efficacy.

A picture of Mycoplasma gallisepticum and Mycoplasma synoviae in poultry in Egypt: Phenotypic and genotypic characterization

ObjectivesThe study reveals a picture for M. gallisepticum (MG) and M. synoviae (MS) in poultry industry of Egypt. MethodsA total of 628 samples were collected from different species of diseased and apparently healthy poultryspeciesfrom 15 Egyptian governorates.The isolates identified based on mgc2and 16 s rRNAspecific primers for MG and MS respectively. Sequencing for two isolates usingmgc2gene. ResultsTwo hundred and five samples were microscopic fried egg colony.Using digitonin test, 160 samples were identified asgenusMycoplasma.Outofthe160 isolates, 86wereidentified by PCRas MG,while 39 were identified as MS. Sequencing reveals thatoneisolateMGC2-EGY1 was related totheEgyptian strainMG-EIS6-T-10, while, the other isolate MGC2-EGY2 was related totheIndian strain MG-MGS-9B. ConclusionThe study suggested that strictly applied programmes for the prevention and control of avian mycoplasmosis and confirmed the use of PCR on a large scale to help disease eradication programmes to limit economic losses in poultry farms.

Mycoplasma gallisepticum (HS strain) surface lipoprotein pMGA interacts with host apolipoprotein A-I during infection in chicken.

The adhesin protein from Mycoplasma gallisepticum (HS strain), namely pMGA1.2, is required for M. gallisepticum (MG) infection in chicken. However, the host factor(s) that interact with pMGA1.2 is not known. In this study, we prepared the membrane fraction of trachea epithelial cells from chicken embryos. Using an improved virus overlay protein blot assay (VOPBA) and glutathione S-transferase (GST) pull-down assay, we found that pMGA1.2 specifically bound to a ∼30kDa host protein. This host protein was further identified by mass spectrometry as chicken apolipoprotein A-I (ApoA-I). We expressed and purified the recombinant ApoA-I protein in Escherichia coli and confirmed that it bound to the purified pMGA1.2 protein in vitro. Transiently expressed pMGA1.2 and ApoA-I were colocalized in HeLa cells. Finally, we designed small interfering RNA (siRNA) molecules to knock down the expression of either ApoA-I or pMGA1.2, which inhibited the MG-induced cell cycle disruption in cells of chicken embryo fibroblast cell line (DF-1). Similarly, knockdown of ApoA-I inhibited the cilia loss and damage in chicken trachea cells in MG infection. In summary, ApoA-I may be an essential host factor in MG infection through interacting with pMGA1.2.

Eggshell apex abnormalities in a free-range hen farm with mycoplasma synoviae and infectious bronchitis virus in Rio de Janeiro state, Brazil

A farm with 3,000 free-range hens between 24 and 65 weeks of age was investigated. These hens were separated in small flocks of 400 to 700 birds, presenting 10 to 23% egg production reduction. Twenty serum samples were collected during the period of drop in egg production and three weeks later for the investigation of Mycoplasma synoviae (MS), M. gallisepticum (MG) and Infectious Bronchitis Virus (IBV) antibodies using ELISA. At the time of the second collection, egg production had resumed to normal levels; however, with 10.23% of the eggs showed eggshell abnormalities limited to the apex. Eggshell strength was significantly different between normal and those with eggshell apex abnormalities, but not other egg-quality parameters. ELISA tests showed that MS and IBV titers increased during the evaluated period. MS infection was confirmed by culture and by PCR of tracheal swabs. All samples were negative for MG by ELISA and PCR. Further studies with larger samples to ensure the occurrence of this disease in industrial layer flocks in Brazil are under way.

A comparative survey on performance of M gallisepticum-Free and M gallisepticum-infected layer flocks in Tabriz area of Iran

The aim of this study was to determine the effect of M gallisepticum (MG) on performance of commercial layer flocks in Tabriz, Iran. Eight Hy-line W36-strain commercial layer flocks, 4 MG-Free and 4 MG infected-flocks, from two breeding companies were reared in the same condition and compared for production factors during rearing phase and production period. In rearing period, livability and body weight at the age of 17 weeks in MG-infected group were significantly lower than MG-Free group (P<0.05). These factors in both groups were lower than the standard group. Feed intake in rearing period did not differ between the two groups. At production period (18–80 weeks), livability, hen housed egg production and peak production rates in MG-infected group were markedly less than MG-Free group. Overall, the result showed that MG-infected group had lower economic performance.

Mycoplasmas in wild turkeys living in association with domestic fowl.

One hundred and nineteen Merriam's wild turkeys (Meleagris gallopavo merriami) and 31 domestic chickens coexisting on a ranch in west-central Colorado (USA) were surveyed for mycoplasmosis by serologic and cultural methods. Although no clinical signs were apparent in any wild turkeys tested, 51 (43%) had positive rapid plate agglutination (RPA) reactions for M. gallisepticum (MG) and/or M. synoviae (MS); 37% of 56 adults and 48% of 63 subadults were classified as positive reactors to MG and/or MS. No turkeys tested in 1992 (n = 61) and 17 (29%) of 58 turkeys tested in 1993 were RPA-positive for M. meleagridis (MM). Hemagglutination inhibition (HI) test results were negative for MG, MS and MM as were most enzyme-linked immunosorbent assay (ELISA) test reactions (MG = 99%, MS = 93%, MM = 87%). Immunoblotting showed mild to moderate reactivity to MG proteins in 49% of 41 samples tested. Most chickens were strongly positive for MS by RPA (81%), HI (58%) and ELISA (87%); 48% also were positive for MG by RPA but all were MG-negative by HI and ELISA. No pathogenic mycoplasmas were isolated from either group of birds. Mycoplasma gallopavonis was commonly identified from the wild turkeys, and M. gallinaceum was isolated from both the chickens and wild turkeys. In a transmission study conducted in 1994, disease-free domestic turkeys failed to seroconvert when co-housed with wild turkeys from this population that were RPA-positive for MG. Collectively, the results of this study were inconclusive regarding the status of pathogenic mycoplasmas within this wild turkey population.

Mycoplasma synoviae in a Released Pen-Raised Wild Turkey

Mycoplasma synoviae (MS) was isolated from the sinus of an adult female "wild-type" turkey found feeding with backyard chickens at a private residence in Randolph County, N.C. Clinical signs included sinusitis, dyspnea, emaciation, diarrhea, and nasal discharge. The bird was seropositive for MS and M. gallisepticum (MG) on the rapid plate agglutination test and had titers of 1:160 for MS and 1:20 for MG on the hemagglutination-inhibition test. Isolations of MS and M. gallopavonis were confirmed by the fluorescent antibody test. This case represents the first and only report of MS in a free-ranging "wild-type" turkey in the eastern United States. Behavioral and other evidence suggests that the bird was a released pen-raised turkey.

Recombinant DNA Probes for Mycoplasma synoviae

A genomic library was prepared from Mycoplasma synoviae (MS) strain WVU 1853 cloned in plasmid vector pUC8 and transformed in Escherichia coli host JM83. In dot blot assays, four transformed E. coli clones hybridized with 32P-labeled chromosomal DNA of MS but not with 32P-labeled chromosomal DNA of M. gallisepticum (MG) strain S6. In Southern hybridization, each of the CsCl-purified recombinant plasmid clones was shown to contain two MS DNA fragments between 1.0 to 2.3 kbp in length. 32P-Labeled probes prepared from each of the four recombinant plasmids hybridized in dot blot assays with MS strain WVU 1853 and nine MS field isolates but not with MG strains S6, K810, F2F10, four MG field isolates, and 15 other species of avian mycoplasmas.

A study of ELISA systems incorporating pooled viral andMycoplasmaantigen preparations for antibody screening of chicken sera

Enzyme linked immunosorbent assays (ELISAs) incorporating up to three different antigens for screening of avian sera for antibodies to several viruses or mycoplasma are described. A triple antigen test comprising Newcastle disease virus (NDV), infectious laryngotracheitis (ILT) virus and avian influenza (AI) antigens for screening sera normally negative for antibodies to these viruses, was shown to be as sensitive as the corresponding single antigen ELISA in detecting seroconversion in experimentally inoculated birds and was also as sensitive as the haemagglutination-inhibition (HI) test for NDV and AI, and the serum neutralisation test for ILT virus. Sensitivity was also demonstrated by comparison of end-points in serially diluted NDV, ILT or AI positive sera. A Mycoplasma synoviae ELISA was shown to be as sensitive as HI test for detection of MS and M. gallisepticum (MG) antibodies in experimentally inoculated birds and in field sera, and this antigen combined with NDV detected antibodies to MG, MS and NDV with sensitivity equivalent to the HI test in each case. The advantages of using pooled ELISA preparation for screening large numbers of sera which are normally negative for the pathogens concerned are discussed.

Use of an Avidin-Biotin Enhanced Dot-Immunobinding Assay to Detect Antibodies for Avian Mycoplasma in Sera from Iowa Market Turkeys

A dot-immunobinding assay, amplified with avidin and biotin (DAB assay), was used to detect serum antibodies to Mycoplasma iowae in immunized turkeys. The DAB assay was used to test serum samples from 122 commercial market turkey flocks obtained from four Iowa processing plants. The samples were pooled and tested for the presence of antibodies to four species of Mycoplasma spp. considered to be important pathogens for turkeys: M. gallisepticum (MG), M. iowae (MI), M. meleagridis (MM), and M. synoviae (MS). The occurrence of antibodies against these mycoplasmas, as determined by the DAB assay, were 5.7% for MG, 18.0% for MI, 77.9% for MM, and 9.8% for MS.

The use of a specific dna probe for detection ofMycoplasma gallisepticumin field outbreaks

A specific DNA probe (pMG4) was used to diagnose M. gallisepticum (MG) infection in field outbreaks in chicken and turkey breeder flocks in Israel. Dot-blot hybridisation of tracheal swab suspensions with the probe enabled positive identification of MG as early as 4 days after sampling, even in flocks at an early stage of infection when no other specific indications of infection were available.

Evaluation and Use of a Micro-Broth Dilution Procedure for Testing Sensitivity of Fermentative Avian Mycoplasmas to Antibiotics

A micro-broth dilution procedure is described for testing the sensitivity of glucose-fermenting avian mycoplasmas to antibiotics. A systematic evaluation was made of potential sources of variation that could affect the reliability of this procedure. Strain WVU 1853 of Mycoplasma synoviae (MS) was used as a standard culture and was tested against the antibiotics erythromycin, spiramycin, streptomycin, tetracycline, tiamulin, and tylosin. Inoculum density, duration of incubation before the test was read, and initial pH of the medium significantly affected test results. Type of serum and concentration of pig serum in the medium had little effect, provided the test was read when the phenol red indicator in the medium of a culture control (without antibiotic) had changed from deep red (pH 7.8) to orange-yellow (pH 7). The presence of bacterial inhibitors in the medium, the solvents used to dissolve some antibiotics, the use of thawed or actively growing cultures, and the number of passages of the culture before testing did not appear to be significant causes of test variation. By controlling important sources of variation, a standardized test was developed that gave reproducible results. The standardized test was used to determine the sensitivity to antibiotics of 49 strains of M. gallisepticum (MG), eight strains of MS, five strains of M. pullorum, seven strains of M. gallinaceum, and one strain of M. columborale. Minimum inhibitory concentrations of the macrolide antibiotics were lower for the majority of MG strains than for the other Mycoplasma species tested. However, some strains of MG showed acquired resistance to the macrolide antibiotics. These strains also showed increased resistance to lincomycin and tiamulin.

Serologic Studies with Mycoplasma synoviae in Experimentally Inoculated Chickens

Three strains of Mycoplasma synoviae (MS) (Olson-WVU 1853, reported in 1956; and Massachusetts 9895 and 5044, respectively isolated in 1957 and 1972) were used for experimental inoculation of chickens to evaluate the various mycoplasma serologic tests. The MS plate agglutination and hemagglutination-inhibition (HI) tests were highly sensitive for detection of infection; reactions persisted for test periods extending to 63 weeks. Correlation between MS HI-tube and micro methods was good. HI titers were highest in sera from birds inoculated with strain 9895. All three strains produced cross-reactions with M. Gallisepticum (MG) plate antigens, which were detectable for 2 to 12 weeks following MS inoculation; strain 5044 produced the lowest percentage of cross-reactions. MG HI titers of 16 were observed occasionally in sera from all three group inoculated with strain 1853. It appears from these limited serologic studies in chickens that there may be antigenic differences among the three MS strains.

Influence of Dust on &lt;i&gt;Mycoplasma gallisepticum&lt;/i&gt; Contact Infection and Body Weight Gain in Chicks, additionally Oil Spray Effect on Dust Control

Seventy-two one-week-old crossbred male chicks were divided into three groups (group D: the litter used was saw dust added by sterilized broiler house dust, C: saw dust only, O: oil-sprayed saw dust; litters were renewed weekly), each of which was composed of two sub-groups, and were killed for examinations at 5-weeks-old. Each sub-group included two chicks inoculated intraairsacularally with M. gallisepticum (MG).The following results were obtained.1. A tendency of D>C>O was observed in the number of recipient chicks which showed lesions at infraorbital sinus, trachea or air sacs and from which MG were isolated, and also a significant linear regression of the number of chicks positive to the whole blood agglutination plate test to dust concentration order was.2. According to analyses of variance of results on lesions at air sacs, isolation of MG and the plate test, there were very significant differences among three groups. It was considered, therefore, that MG contact infection might be enhanced by the existence of dust in the atmosphere.3. The influence of contact infection enhanced by dust on body weight gain was not cleared up, but dust itself was not considered to influence chick performances.4. The oil application reduced the amount of dust, but its effect on dust control decreased with length of time after its spray over litter.

Detection of M. Synoviae Infection in Chickens and its Differentiation from M. Gallisepticum Infection

Rapid serum agglutination (RSA) and haemagglutination inhibition (HI) tests were used to detect antibodies to Mycoplasma synoviae (MS) in an artificially infected flock of specific pathogen-free (SPF) chickens and sera from commercial flocks. Comparative RSA and HI tests with M. gallisepticum (MG) antigens were also carried out. In both experimental and field sera, weak transitory reactions occurred with the MG-RSA antigen but these were not supported by positive HI titres. Known MS infected birds gave positive MS-RSA reactions for a prolonged period, and these were accompanied by MS-HI titres between 1/5 and 1/160. The specificity of the tests and the relationship of non-specific reactions to rheumatoid arthritis (RA) factors is discussed. L’agglutination rapide du sérum (ARS) et l’inhibition de l’agglutination de l’hémoglobine ont été utilisées pour détecter des anticorps à Mycoplasma synoviae (MS) dans une harde artificiellement infectée de poulets spécifiquement non-pathogéniques (SNP) et des sérums de hardes commerciales. Des tests comparatifs de ARS et IH avec des antigènes de M. gallisepticum (MG) ont également été réalisés. Dans les sérums tant expérimentaux que pathologiques, de faibles réactions transitoires ont eu lieu avec l’antigène de Mg-ARS, mais ceux-ci n’étaient pas supportés par des valeurs d’IH positives. Les poulets qu’on savait être infectés de MS donnèrent des réactions MS-ARS positives pendant une période prolongée, et celles-ci étaient accompagnées de valeurs MS-IH entre 1/5 at 1/160. Cette spécificité du test et le rapport des réactions non-spécifiques aux facteurs de rhumatisme articulaire (RA) sont discutée. Rapide Serumagglutination (RSA) und Inhibition von Haemagglutination dienten zur Entdeckung von Antikörpern gegen Mycoplasma synoviae (MS) bei künstlich infizierten Scharen von spezififisch pathogenfreien (SPF) Hühnern und in Seren von im Handel erworbenen Tieren. Vergleichende RSA und HI Tests mit M.-gallisepticum-Antigenen wurden ausserdem ausgeführt. Mit beiden Seren, den experimentellen und denen der im Handel erworbenen Tiere, traten vorübergehende Reaktionen mit MG-RSA-Antigen auf; sie wurden jedoch nicht durch positive HI-Titer bestätigt. Tiere, deren MS-Infektion bekannt war, gaben positive MS-RSA-Reaktionen für längere Zeit und diese waren von MS-HI-Titern zwischen 1/5 und 1/160 begleitet. Es wird besprochen, in welcher Weise diese Tests spezifisch sind und wie nichtspezifische Reaktionen sich zu rheumatischarthritischen (RA) Fektoren verhalten. Se emplearon la aglutinación rápida del suero (ARS) y la inhibición de la hema-glutinación (IH) para descubrir anticuerpos a Mycoplasma synoviae (MS) en un gallinero de gallinas libres de patógenos específicos (LPE) que se infectó artificialmente y en sueros de gallineros comerciales. También se efectuaron pruebas comparativas de ARS e IH con anticuerpos de M. gallisepticum (MG). Tanto en los sueros experimentales como en los de campo, se dieron reacciones transitorias débiles con el antígeno MG-ARS, pero no se obtuvieron valoraciones positivas de IH. Aves que se sabía estaban infectadas con MS dieron reacciones MS-ARS positivas por un período prolongado, y dichas reacciones fueron acompañadas por valoraciones de MS-IH de entre 1/5 y 1/160. Se discuten la especificidad de las pruebas y la relación entre reacciones no específicas y los factores de la artritis reumatoide (AR).