Comparison of a modified Edward-type medium and a modified SP4-type medium for primary isolation of Mycoplasma gallisepticum (MG) from chickens vaccinated with the F strain of MG

The efficacy of two media, an Edward-type medium (EPJ) and a modified SP4-type medium (SP4-PS), were compared for primary isolation of Mycoplasma gallisepticum (MG) from commercial layer chickens (n = 58) vaccinated with the live F strain of MG. Three groups of chickens that differed in the interval after vaccinal exposure to the F strain (32, 41, and 102 weeks) were studied at necropsy. Mycoplasma isolation was attempted from the trachea, sinus, and cloaca using lavage and swab techniques but was successful only from the trachea and sinus. MG was isolated from 39 (8.4%) of 463 culture attempts from 58 tracheal inocula and 58 sinus inocula. Isolation of MG was successful more frequently using EPJ medium than SP4-PS medium, and isolation occurred more often from the sinus than from the trachea. Of the 58 chickens studied, 19 (33%) were shown by culture to be infected with MG. Isolation was successful only from 32- and 41-week post-vaccination exposure groups. However, all chickens studied were serologically positive for MG antibody by rapid-plate agglutination and hemagglutination-inhibition assays.     

A historical account of the diagnosis and characterization of strains of Mycoplasma gallisepticum of low virulence

Numerous chicken flocks were studied beginning in 1970 because of questionable results on their serologic tests for Mycoplasma gallisepticum (MG). Typically a low number of hens in the flocks were positive reactors to the rapid serum plate test and rarely had hemagglutination-inhibition (HI) titers over 1:80. Usually no clinical signs were observed. Isolates of MG eventually were cultured from most of the flocks that exhibited that type of marginal serologic pattern. In the laboratory, the MG isolates were frequently less virulent and less pathogenic than the typical field isolates recovered in previous years. Most isolates produced airsacculitis of varying severity when broilers were exposed to the MG cultures as aerosols following exposure to infectious bronchitis virus. They became positive on the rapid serum plate test and developed moderate to high HI titers. Egg-transmission appeared to be the most likely means of transmission, even though the infected progeny rarely showed clinical signs of disease.     

Breeder turkey hens seropositive and culture-negative for Mycoplasma synoviae.

Four flocks of clinically normal turkey breeder hens were shown to have suspect and positive Mycoplasma synoviae (MS) hemagglutination-inhibition (HI), enzyme-linked immunosorbent assay, and, in some cases, serum plate agglutination serology in the absence of MS isolation. In all cases, HI serology for Mycoplasma gallisepticum (MG) and M. meleagridis was negative. Acholeplasma laidlawii was isolated from some hens in each of these MS-seropositive culture-negative flocks. Immunoblotting was used to help determine if this positive MS serology was a result of cross-reactive antibodies to A. laidlawii or to some other Mycoplasma species. When sera from two of the flocks were reacted with MS antigen in immunoblotting, a strong and characteristic MS immunoblot profile was seen. Immunoblotting gave no evidence of a strong antibody response to A. laidlawii, M. iowae, or MG. This suggests the presence (or earlier presence) of MS in these flocks that is difficult to isolate by routine methods. Furthermore, this work shows that immunoblotting can be an important tool in the diagnosis of poultry diseases.     

Prevalence of antibody to chicken anaemia virus (CAV) in Swedish chicken breeding flocks correlated to outbreaks of blue wing disease (BWD) in their progeny

A serological survey for antibody to Chicken Anaemia Virus (CAV) was performed on broiler breeders as well as layer breeding birds in Sweden at the end of their rearing period. Grandparents (GP) of both types leaving quarantine were in 21 out of 26 cases free from antibody to CAV, but often became infected soon thereafter. A total of 10 outbreaks of blue wing disease (BWD) in 3 series were recorded in the broiler and layer parent generation, all of which were progeny of 3 late seroconverting GP-flocks. All but one of 22 layer parent flocks had been infected and had seroconverted during the rearing period. Subsequently BWD was not recorded from commercial layers. Broiler parent flocks were more protected from CAV infection during rearing. Eighteen out of 94 broiler parent flocks had not developed antibody to CAV before coming into lay. Outbreaks of BWD were reported in progeny flocks from all these broiler breeders, with the exception of those that had been vaccinated. Good hygienic routines along with isolation of the birds delayed the seroconversion to CAV in broiler breeders and vaccination of these breeders protected their progeny from outbreaks of BWD. Broiler flocks in houses where BWD had occurred recently had always antibodies to CAV at slaughter. It was possible to eradicate the infection from the house and prevent the infection between flocks by proper cleaning and disinfection of the broiler houses.     

Experimental infection of chickens and turkeys with Mycoplasma gallisepticum reference strain S6 and North Carolina field isolate RAPD type B

During an epidemic of mycoplasmosis in chicken and turkey flocks in North Carolina between 1999 and 2001, isolates of Mycoplasma gallisepticum (MG) from affected flocks were characterized by random amplification of polymorphic DNA (RAPD), and eight distinct RAPD types were identified. MG RAPD type B accounted for more than 90% of the isolates and was associated with moderate-to-severe clinical signs and mortality. The virulence of MG RAPD type B for chickens and turkeys was compared with sham-inoculated negative controls and MG S6 (a virulent strain)-inoculated positive controls. Clinical signs occurred in chickens and turkeys inoculated with either MG RAPD type B or MG S6. However, they were not as frequent or severe as those seen in naturally affected flocks, and there was no mortality in the experimental groups. Based on gross and microscopic findings, MG RAPD type B was equal to or more virulent than MG S6. All MG-inoculated birds were culture and PCR positive at 7 and 14 days postinoculation (PI). Among serological tests, the serum plate agglutination test was positive for the majority of chickens and turkeys (58%-100%) infected with either strain of MG at both 7 and 14 days PI. The hemagglutination inhibition test was negative for all birds at 7 days PI and positive for a few chickens (8%-17%) and several turkey sera (40%-60%) at 14 days PI. Only a single serum was positive by enzyme-linked immunosorbent assay (an MG S6-infected turkey) at 14 days PI.     

Prevalence of Mycoplasma gallisepticum and M. synoviae in commercial layers in southern and central California

The prevalence of Mycoplasma gallisepticum (MG) and M. synoviae (MS) in commercial pullet and layer flocks in Southern and Central California was estimated by testing serum and egg-yolk samples from 360 sample flocks in Southern California and 41 sample flocks in Central California. Data relating to potential risk factors associated with MG and MS infections were collected. The estimated true prevalence rate of MG was 73% in Southern California and 3% in Central California. The estimated true prevalence rate of MS was 91% in Southern California and 32% in Central California. Compared with uninfected flocks, MG-infected flocks in Southern California were significantly older and were medicated less (P less than 0.05). More managements were under a multiple-age system, more flocks had molted, more were vaccinated with F-strain, and more had concurrent infection with MS (P less than 0.05). Only one sample flock in Central California was MG-infected; none were vaccinated with F-strain. In Southern California, MS-infected flocks were older than uninfected flocks, more had molted, more were medicated, and more had concurrent infection with MG (P less than 0.05). In Central California, MS-infected flocks did not differ significantly from uninfected flocks in any factor examined; the lack of statistical significance may be due to small sample size.     

Characterization of a ts-1-like Mycoplasma galisepticum isolate from commercial broiler chickens

Several commercial broiler flocks in northeastern Georgia that were the progeny of the same parent flock (Flock 40) were diagnosed as Mycoplasma gallisepticum (MG) positive by serology, culture, and PCR. Flock 40 had been vaccinated with ts-11 live MG vaccine. Several isolates were obtained from the MG-positive broiler flocks, and these isolates were indistinguishable from the ts-11 vaccine strain by the molecular strain differentiation methods used. A pathogenicity study was performed to compare the virulence of one of the isolates, K6216D, to the ts-11 vaccine strain. K6216D elicited a significantly stronger antibody response and significantly increased colonization of the tracheas and air sacs. K6216D also elicited significantly greater air sac and tracheal lesions than the ts-11 vaccine strain at 10 and 21 days postinoculation (P < or = 0.05). This is the first report of a field case of the apparent reversion to virulence and vertical transmission of the ts-11 vaccine.     

Characterization of experimental Mycoplasma gallisepticum infection in captive house finch flocks

The use of controlled, horizontal-transmission experiments provides detailed information on the spread of disease within fixed social groups, which informs our understanding of disease dynamics both in an empirical and theoretical context. For that reason, we characterized in 2002, horizontal transmission of Mycoplasma gallisepticum (MG) in two flocks of 11 wild-caught house finches housed in outdoor aviaries over a 6-mo period. All birds were initially free of MG by a polymerase chain reaction (PCR)-based test, rapid plate agglutination (RPA), and the scoring of physical signs. We inoculated one flock member bilaterally in the palpebral conjunctiva and reintroduced it into its cage. Index birds developed conjunctivitis within 3 to 5 days but died 13 and 20 days postinfection (PI) possibly because of very severe weather. The proportion of birds with physical signs increased gradually, reached 40% at 6 wk PI, and fluctuated around 40% until 21 wk PI. By the time our experiment ended at 24.5 wk PI, 28% of the birds still exhibited physical signs. Across both flocks, 80% of the birds developed unilateral or bilateral conjunctivitis, and several birds relapsed. The appearance of physical signs in new individuals occurred between 10 and 144 days PI (median 41 days PI). Physical signs lasted 1-172 days (median 42 days). Birds that became infected earlier during the experiment developed more severe conjunctivitis, and there was a tendency for birds that developed bilateral conjunctivitis to develop physical signs earlier. Most birds that developed physical signs of MG were also PCR- and RPA-positive, although we detected a single asymptomatic carrier and a single symptomatic false negative. No birds died as a result of secondary MG infection.     

Evaluation of criteria for the postmortem diagnosis of mycoplasmal pneumonia of swine.

Ten swine from each of five herds believed to be affected with mycoplasmal pneumonia of swine and ten swine from each of five herds believed to be mycoplasmal pneumonia-free were selected for postmortem study. Lungs from the 100 swine were examined; grossly and microscopically for lesions typical of mycoplasmal pneumonia of swine and culturally and by an indirect immunofluorescent procedure for the presence of Mycoplasma hyopneumoniae. Nineteen of the lungs had both gross and microscopic lesions typical of mycoplasmal pneumonia of swine and 13 (68%) of these were infected, i.e. were culturally and/or indirect immunofluorescent positive. Absence of gross lesions did not prove freedom from mycoplasmal pneumonia, 14 of 73 (19%) grossly normal lungs were found to be infected with M. hyopneumoniae. Comparison of the indirect immunofluorescent and cultural examination, as methods of diagnosing mycoplasma pneumonia, revealed that neither procedure alone was reliable in the case of negative results. Ten lungs were indirect immunofluorescent negative and culturally positive and seven were culturally negative and indirect immunofluorescent positive (11 lungs were positive by both procedures). It was concluded that a definitive diagnosis of mycoplasmal pneumonia of swine requires that M. hyopneumoniae be visualized in indirect immunofluorescent stained lung sections or that it be recovered culturally.     

Poor serologic response to upper respiratory infection with Mycoplasma synoviae in turkeys

Mycoplasma synoviae (MS) was isolated from a flock of commercial tom turkeys in which a small percentage of the birds exhibited clinical signs and lesions typical of MS synovitis. However, serologic testing of such flocks revealed poor to inconsistent reactivity by agglutination, enzyme-linked immunosorbent assay (ELISA) or hemagglutination inhibition; isolation of MS from such flocks proved to be very difficult. Turkeys were challenged with one of the isolates (K4463B) either by aerosol or systemically by a combination of intravenous, foot pad, and eyedrop routes. Turkeys challenged by the systemic route responded normally to all serologic tests, whereas those challenged by aerosol either responded very poorly on all serologic tests or were seronegative up to 6 wk postchallenge even though they were positive for MS by tracheal culture. These results suggest that turkeys may harbor an upper respiratory infection with MS while remaining serologically negative.     

Seroepidemiological survey of sheep flocks from Northern Japan for Mycoplasma ovipneumoniae and Mycoplasma agalactiae

Sheep flocks from Hokkaido, Iwate and Aomori, three northern prefectures of Japan, were screened for antibodies to Mycoplasma ovipneumoniae and Mycoplasma agalactiae by ELISA. Sixty four animals out of 246 (26%) were seropositive to M. ovipneumoniae, with positive results obtained from all three prefectures. None of the sera tested were serologically positive to M. agalactiae.     

Economic impact of Mycoplasma gallisepticum and M. synoviae in commercial layer flocks

An egg-production function was constructed, using data collected from 366 commercial layer flocks in California, to predict the impact of Mycoplasma gallisepticum (MG) and M. synoviae (MS) on egg production while controlling for confounding factors. In the first and second cycles, respectively, an MG-infected flock produced 12 and 5 fewer eggs per hen than an uninfected flock. Flocks that became infected with MG after F-strain vaccination produced 6 eggs/hen more than unvaccinated infected flocks in the first cycle, but no significant difference was observed between such groups in the second cycle. No association was found between MS-infection and egg production. Commercial layer producers in Southern California lost an estimated 127 million eggs because of MG in 1984. This lost egg production and associated MG-control-program costs amounted to an estimated financial loss of approximately $7 million. This represented a loss of approximately $6 million in consumer surplus.     

Molecular characterization of Mycoplasma gallisepticum isolates from Jordan

Two groups of Mycoplasma gallisepticum (MG) isolates (n = 24) from Jordan were analyzed by molecular methods and compared with other Middle Eastern isolates, related international isolates, and reference strains. The first group (n = 19) was isolated from July 2004 to January 2005 (isolation period A), and the newer group (n = 5) from June 2007 to April 2008 (isolation period B). The groups of isolates are from chicken flocks from northern Jordan, but are not from the same farms. None of the flocks were vaccinated for MG. Random amplified polymorphic DNA analysis, targeted sequencing of the partial MG cytadhesin 2 (mgc2), and the MG 16S-23S rRNA intergenic spacer region (IGSR) divided the Jordanian isolates into two groups. All of the 19 isolates from time period A, in addition to two isolates from time period B, were indistinguishable from the F strain. Three of five isolates from time period B were characterized as wild types and were indistinguishable from each other. The wild-type field strain was readily distinguished from the F strain. It was 91% and 96.4% similar to the F strain based on Clustal-W alignments of sequences of mgc2 and IGSR, respectively. Sequence similarity of mgc2 gene of the Jordan wild-type strain to isolates from Israel and Egypt ranged from 96.5% to 100%, whereas for IGSR it was 99.4%-100%. We theorize that the F-strain live MG vaccine, commonly used in Jordan prior to 2007, was transmitted to nonvaccinated poultry in the region and was a predominant genotype during time period A.     

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.     

Egg production in relation to the results of a long term serological survey of 73 flocks of fowl

Summary

Seventy‐three flocks of fowl were tested at regular intervals for the presence of precipitins to fowl adenovirus (AV) and infectious bronchitis virus (IBV), haem‐agglutinating inhibiting antibodies to BC14 virus, and of agglutinins to Mycoplasma gallisepticum (M.g.) and Mycoplasma synoviae (M.s.). In all the eight flocks affected with Egg Drop Syndrome (EDS ‘76), egg production problems were associated with increasing numbers of BCI4 virus reactors and AV reactors. In flocks showing production problems other than EDS ‘76 without any apparent cause, the average percentage of AV reactors increased significantly after the rearing period; this was not true of IBV reactors. BC14 reactors were either absent or present only once, in small numbers and with low titres, during the test period. The average percentage of AV reactors did not increase after the rearing period either in normally producing flocks or in flocks with production problems for which other diseases or dietary errors plausibly accounted for these problems. All this suggests a pathogenic role of AV in production problems. One can conclude from the high percentage of reactors in all groups of flocks that sub‐clinical IBV infections are common.

The percentage of IBV reactors during the laying period of flocks with EDS ‘76 was significantly higher than that of normally producing flocks. It is therefore suggested that subclinical IBV infection could be among the factors causing stress, acting as a trigger for EDS ‘76.

All M.g.‐infected flocks showed production problems; M.s. infections could not be related to egg production disturbances.     

Bacterin to control the vertical transmission of Mycoplasma gallisepticum in chickens

A Mycoplasma gallisepticum bacterin was prepared and used in Mycoplasma gallisepticum (MG)-positive primary breeders to control vertical transmission of MG. Two generations were vaccinated, but the third generation was not vaccinated and was monitored serologically. Results showed no evidence of MG at 1 day, 6 weeks, 11 weeks, 16 weeks, or 31 weeks of age. This procedure may offer small breeder organizations and showbird fanciers a way to eliminate MG.     

Maintenance of a captive flock of house finches free of infection by Mycoplasma gallisepticum

Since the beginning of an epidemic of conjunctivitis in wild house finches caused by Mycoplasma gallisepticum (MG), all captive colonies established by capturing free-ranging house finches from the eastern population have also either been infected at the time of capture or developed infection shortly after capture. In an attempt to avoid this infection in captive flocks being maintained for studies of the finches' behavior and ecology, we compared two different flock management strategies and were able to prevent the development of mycoplasmal conjunctivitis with one of the strategies. Single-sex flocks were built by introducing only seronegative wild-caught birds showing no clinical signs of conjunctivitis and covering their outdoor flight cages with netting to prevent interaction with other wild birds although only the female flocks were initially treated with a 6-wk course of tylosin tartrate (0.3 mg/ml). The female flocks never developed conjunctivitis although the disease did develop in the male flocks. Furthermore, serologic assessments of the healthy flock by serum plate agglutination assays for MG indicated that the females remained free of MG infection in the final 7 wk of the study, during which they were unmedicated. We conclude that any low-level MG infection not diagnosed by the initial test for seroconversion was cleared by the prolonged drug treatment.     

Indirect micro-enzyme-linked immunosorbent assay for the detection of antibodies to Mycoplasma synoviae and M. gallisepticum

The sensitivity and specificity of the indirect micro-enzyme-linked immunosorbent assay (ELISA) was compared with that of the rapid serum-plate test (RSPT) and the hemagglutination-inhibition test (HIT) in detecting antibodies to Mycoplasma gallisepticum (MG) and M. synoviae (MS). Membrane antigens of MG strain S6 and MS strain NEL 61800 were used. ELISA was performed with single MS and single MG antigens and a combined MS/MG antigen. The MS-ELISA was as sensitive as the MS-RSPT and more sensitive than and as specific as the MS-HIT in detecting antibodies to MS. The MG-ELISA was less sensitive than the MG-RSPT and slightly less sensitive than the MG-HIT in detecting antibodies to MG in chickens experimentally infected with MG R strain but more sensitive in detecting antibodies in chickens infected with MG F strain. MG-ELISA resulted in fewer cross-reactions than the MG-RSPT but more than the MG-HIT. The combined MG/MS-ELISA was as sensitive as the ELISA with its individual antigen components. No nonspecific reactions were observed with sera from MG/MS-free flocks. The combined MG/MS-ELISA was found to be a practical screening test for antibodies to both MS and MG. Further improvement of the sensitivity and the specificity of the MG antigen is desirable.     

Comparison of culture, PCR, and different serologic tests for detection of Mycoplasma gallisepticum and Mycoplasma synoviae infections

In this study, the technical performance of culture, two commercially available polymerase chain reaction (PCR) tests, rapid plate agglutination (RPA) test, hemagglutination inhibition (HI) test, and eight commercially available enzyme-linked immunosorbent assays (ELISAs) were compared for the detection of avian mycoplasma infections from 3 days postinfection (d.p.i.) through 35 d.p.i. The tests were carried out on samples from specified pathogen-free layers that were infected at 66 wk of age with recent Mycoplasma synoviae (MS) and Mycoplasma gallisepticum (MG) field strains, MS and MG ATCC strains, and Mycoplasma imitans (MIM), respectively. Results showed a high percentage of positive samples in the homologous infected groups and a high percentage of negative samples (100%) in the uninfected and heterologous infected groups during 35 d.p.i. of both culture and PCR tests. For the group infected with the MG 15302 ATCC strain, serology was more sensitive than bacteriology. All MG and MS tests, with the exception of MG ELISA kit D showed a lower percentage of positive samples during 35 d.p.i. for the detection of the MG and MS ATCC strain infection compared with that of the field strains. Also, the number of cross-reactions (false positives) in the serologic tests was lower after infection with an ATCC strain than after an infection with the MG or MS field strain. Contradictory to other studies, the ELISAs and the RPA test using undiluted serum showed a relatively high number of false-positive results. The MG ELISAs (except ELISA kit D) showed more false-positive results (up to 37%) in the MIM-infected group than in the MS-infected groups. This was not unexpected, as MIM and MG have a close antigenic relationship. The results of the serologic tests in this study showed that a certain level of false-positive results can be expected in about any serologic test. Although the level of false-positive results varied between several serologic tests, this study showed that it is not advisable to rely completely on one test (system) only.     

Evaluation of factors associated with infection of commercial layers with Mycoplasma gallisepticum and M. synoviae

Information on factors possibly associated with the risk of infection with Mycoplasma gallisepticum (MG) or M. synoviae (MS) were collected from nearly 400 layer flocks in California. Factors associated with the probability of flock infection with either MG or MS were identified, and their magnitude was quantified by statistical analysis. More frequent administration of several vaccines was associated with decreased probability of both MG and MS infection of flocks. Also identified were housing or hygiene factors and system of management (i.e., multiple-age status) that could reduce the probability of infection of flocks with mycoplasma. The change in probability of MG infection resulting from modifying certain management factors was examined.