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.     

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.     

A statistical model to optimize enzyme-linked immunosorbent assay parameters for detection of Mycoplasma gallisepticum and M. synoviae antibodies in egg yolk

An enzyme-linked immunosorbent assay (ELISA) was developed to quantify Mycoplasma gallisepticum (MG) and M. synoviae (MS) antibodies in egg-yolk extract. Various parameters of ELISA were evaluated and optimized. A statistical model was developed to study the relationship between ELISA absorbance (A) and antigen concentration, antibody concentration, and time of reading of the test. These factors explained 62% of the variability in A for the MG antigen and 74% of the variability in A for the MS antigen. The optimum concentration of MG antigen was 4 micrograms protein/ml, and that for MS antigen was 3 micrograms protein/ml. A similar model was developed to study the relation between A and conjugate concentration, antibody concentration, and time of reading of the test. The optimum concentration of the conjugate was found to be 1:4000. The within-run and between-run coefficients of variation for MG were 8% and 9%, respectively, and those for MS were 9% and 7%, respectively, indicating a high degree of reproducibility of these tests. There was a high correlation between ELISA and hemagglutination-inhibition test results.     

Comparison of egg yolk and serum for the detection of Mycoplasma gallisepticum and M. synoviae antibodies by enzyme-linked immunosorbent assay

Egg yolk was evaluated in the enzyme-linked immunosorbent assay (ELISA) as an alternative source of antibodies for detection of Mycoplasma gallisepticum (MG) and M. synoviae (MS) infections in chickens. There was no statistically significant difference (P greater than 0.05) between the ELISA geometric mean titers (GMTs) of saline-diluted egg yolk and chloroform-extracted egg yolk, and both preparations had a high correlation coefficient (0.87 for MG; 0.97 for MS). The saline-diluted and chloroform-extracted yolk had a relative sensitivity of 90% and specificity of 98% in the MG ELISA; in MS ELISA they were 100% and 96%, respectively. Hemagglutination-inhibition (HI) results with chloroform-extracted samples were satisfactory, but those with saline-diluted samples were not. Neither preparation was satisfactory for use in the rapid plate agglutination (RPA) test. A 1-ml sample of yolk was compared with the whole-yolk method. The chloroform-extracted whole yolk yielded a significantly higher (P less than 0.05) GMT in the MG ELISA; however, there was no statistically significant difference (P greater than 0.05) between GMTs yielded by the two procedures in the MS ELISA. The correlation coefficients for the two sampling methods were 0.73 for MG ELISA and 0.63 for MS ELISA. ELISA detected no statistically significant difference (P greater than 0.05) between GMTs of serum and chloroform-extracted yolk from individual birds. Results with the HI test were comparable to those with ELISA on the same samples. The RPA test yielded comparable results on the serum samples. No statistically significant differences (P greater than 0.05) were observed in HI or ELISA antibody levels between egg-yolk samples and sera on random samples collected from nine flocks that were MG- and MS-free or were infected with MG, MS, or both; however, egg-yolk samples tended to have slightly higher titers than sera in both tests. The optimum screening dilution of chloroform-extracted yolk for detecting MG and MS antibodies by ELISA was 1:800.     

Elimination of mycoplasmal plate agglutination cross-reactions in sera from chickens inoculated with infectious bursal disease viruses

Sera from chickens inoculated with various challenge infectious bursal disease viruses or infectious bursal disease vaccines were found to cross-react in the Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) serum plate agglutination (SPA) tests. Two-fold dilutions of these cross-reacting sera with phosphate-buffered saline before retesting eliminated all non-specific agglutination in the MG and MS SPA tests. Cross-reactions were observed in the SPA test using sera from chickens inoculated with either MG or MS. Dilutions of these sera 1:2 had little effect on the number of these cross-reactions. At 1:4 serum dilutions, however, the number of cross-reactions between MG and MS was reduced. At 1:8 dilution of test sera, cross-reactions between MG and MS were further reduced. Some reduction in specific MG and MS SPA reactions, however, also occurred at the 1:8 dilution of sera with some of the plate antigen.     

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.     

Detection of serum antibodies against Mycoplasma gallisepticum and Mycoplasma synoviae by a dot-immunobinding technique

Both Mycoplasma gallisepticum (MG) and M. synoviae (MS) antigens prepared for the routine haemagglutination inhibition (HI) test were diluted and absorbed to the separate pieces of durapore membrane for the measurement of dot-immunobinding (DIB) titers of test sera. Besides, durapore strips bearing both antigens were employed for a DIB test with chicken sera definitely diluted 100-fold. Shortening of reaction time of chicken sera with antigens as well as with the secondary serum markedly eliminated non-specific DIB reactions exhibited at low dilutions although the same condition was not so effective on the elimination of non-specific reactions among rabbit hyperimmune sera. Rapid and specific development of DIB antibody which continued at high titer up to 1:640 for 10 weeks postinoculation was proved in the sera of SPF chickens inoculated with MG or MS, while DIB titers of sera from uninoculated chickens remained 1:20 or lower. Non-specific reactions, which occurred in the routine serum plate agglutination test with a part of sera from the inoculated chickens, were not exhibited in the DIB as well as in the HI test with the same sera. Results of the DIB test with serum samples from 287 conventionally reared chickens definitely diluted 100-fold coincided with the results of HI test at a level of 90% with MG and 89% with MS antigen. This technique seems to be useful for a rapid, simple and specific diagnosis of avian mycoplasmosis.     

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.     

An avidin-biotin enhanced dot-immunobinding assay for the detection of Mycoplasma gallisepticum and M. synoviae serum antibodies in chickens

A dot-immunobinding assay was enhanced by the incorporation of avidin and biotin reagents into the test system (DAB assay). This assay was used to detect serum antibodies to Mycoplasma gallisepticum (MG) and M. synoviae (MS) from chickens. Serum samples were tested by rapid serum plate (RSP), hemagglutination-inhibition (HI), and DAB assay methods. These results were compared. The DAB assay was at least 20 times more sensitive in detecting antibodies for MS and at least 75 times more sensitive in detecting antibodies for MG than the HI test. The DAB assay was as specific as the HI test. The DAB assay was also more sensitive and specific than the RSP test. Some cross-reactions occurred when low dilutions of high-titer sera were used in the DAB assay. Parameters for determining negative, suspicious, and positive samples were established. The DAB assay for MG and MS may have several applications, including use as a screening test and a confirmatory test.     

Detection of Mycoplasma gallisepticum by direct immunofluorescence using a species-specific monoclonal antibody

A monoclonal antibody against Mycoplasma gallisepticum (MG) (strain S6) was prepared in mice and identified as isotype IgG1 by standard procedures. Although it did react at high titers (1:100,000) in the enzyme-linked immunosorbent assay (the original method for its identification), it failed to react in the agglutination, hemagglutination-inhibition, and growth-inhibition tests. When conjugated to fluorescein isothiocyanate, the monoclonal antibody reacted with the homologous and eight "atypical" strains of MG but not with M. meleagridis or M. synoviae in the direct fluorescent-antibody test. This reagent may be useful for detecting field infections involving atypical strains of MG.     

Monitoring Mycoplasma gallisepticum and Mycoplasma synoviae infection in breeder chickens after treatment with enrofloxacin.

Three experimental strains of breeder chickens were accidentally exposed to Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS), presumably from a newly introduced group of leghorn-type pullets. The experimental strains subsequently became infected and were diagnosed positive for MG and MS by the serum plate agglutination (SPA) test and confirmed by the hemagglutination inhibition (HI) test and the polymerase chain reaction (PCR) of tracheal swabs. Treatment with 10 mg/kg enrofloxacin via drinking water for 14 days was elected. Before and after initiation of treatment, MG and MS were monitored for changes by SPA, HI, PCR, and culture, with sampling intervals ranging from 1 wk to 7 wk. MG and MS SPA, HI, PCR, and culture were performed at each sampling period, with the exception of weeks 1.0 and 6.5. Week 1.0 included SPA and His for MG and MS. Week 6.5 included PCR and culture for MG and MS. The MG and MS SPA results were positive throughout the 29-wk trial period. MG HI titers declined until the last sampling, whereas the MS HI titers did not decline significantly. PCR for MG yielded only one positive result, which occurred before treatment. MS PCR remained positive throughout the trial period. MG was never isolated from any sample; however, one MS organism was isolated during treatment. The treatment regimen was effective for MG on the basis of PCR results. Treatment with enrofloxacin did not eliminate SPA reactions during the 29-wk trial period. MG HI titers remained in the suspicious range throughout the remainder of the trial period. Four weeks after the treatment ended, MG HIs were reduced by approximately 40%, with MS HIs remaining high throughout the 29-wk period. PCR appeared to be a sensitive and specific test on the basis of correlation with HIs. On the basis of the isolation of MS during treatment and continued subsequent PCR positive reactions, the treatment for MS with enrofloxacin was not as efficacious as for MG.     

Evaluation of sodium dodecyl sulfate-polyacrylamide gel electrophoresis purified proteins of Mycoplasma gallisepticum and M. synoviae as antigens in a dot-enzyme-linked immunosorbent assay

Selected immunogenic proteins of Mycoplasma gallisepticum (MG) strain R and M. synoviae (MS) isolate F10-2AS were purified from sodium dodecyl sulfate-polyacrylamide gels. Purified MG proteins of 65 to 63 (p64) kilodaltons (kDa), and 26 and 24 (p26/24) kDa, and purified MS proteins of 53 (p53) kDa, 41 (p41) kDa, and 22 (p22) kDa were evaluated as potential antigens for an enzyme-linked immunosorbent assay (ELISA). Chicken antisera to MG, MS, or oil-emulsion vaccines were used to evaluate these purified proteins as antigens in a dot-ELISA. MG antigen p64 detected antibodies 3 days after the serum plate agglutination (SPA) test and 7 days before the hemagglutination-inhibition (HI) test. Antigen p64 detected antibodies to 12 MG isolates, and in sera from field outbreaks of MG. No cross-reactions with MS-positive antisera were seen with antigen p64. MG antigen p26/24 did not perform as well as p64. MS antigen p41 detected antibodies 5 days after the SPA test and at least 11 days before the HI test, and in sera from field outbreaks of MS. However, some MG-positive antisera reacted with p41. MS antigens p53 and p22 did not perform well.     

Clinical, cultural, and serologic observations of avian mycoplasmosis in two chicken breeder flocks.

Two chicken breeding flocks from different breeding lines were studied serologically and culturally for Mycoplasma gallisepticum (MG) throughout their growing and laying period. Infection was proven by successful isolation of MG from both breeders and progeny originating from these two flocks. Observations of these flocks which were serologically and culturally negative for Mycoplasma synoviae (MS) further disclosed that: 1) negative plate tests of large numbers of day-old progeny may sometimes be found in flocks known to be infected with MG; 2) it may be very difficult to isolate MG consistently from some infected flocks; 3) overgrowth of M. gallinarum may interfere with successful cultivation of MG; 4) a persistent breeder flock reactor rate of greater than 10-20% but less than 80-100% for a 4-to-12-week period is a strong indication of MG infection despite weak or negative MG hemagglutination-inhibition (HI) test results; and 5) antibodies for all strains of MG may not react equally to the standard USDA MG-HI antigen.     

Nonspecific reactions to Mycoplasma serum plate antigens induced by inactivated poultry disease vaccines

Nonspecific serum plate agglutination reactions to some avian mycoplasma antigens were induced by injecting chickens with several commercial poultry disease vaccines. All of the vaccines were inactivated, and most of them had oil-emulsion adjuvants. The serum plate agglutination reactions appeared within 2 to 3 weeks post-vaccination and generally persisted for several weeks. The plate test reactions were noted with both Mycoplasma gallisepticum (MG) and M. synoviae (MS) antigens, although the degree and duration of the reactions varied with the vaccine involved and the source of MG and MS plate test antigens. Attempts to prevent the nonspecific reactions by heat-inactivation at 56 C for 30 minutes or by addition of equal volumes of solutions of 2-mercaptoethanol, dithiothreitol, or 3 M sodium chloride were ineffective. No hemagglutination-inhibition activity against MG or MS antigens was induced by the vaccines.     

Mycoplasma gallisepticum infection in wild-type turkeys living in close contact with domestic fowl

Mycoplasma gallisepticum was isolated from 2 wild-type turkeys (Meleagris gallopavo) and 1 domestic turkey living in close contact on a farm in Tehama County, California. Sinusitis was detected in 2 of 14 wild-type turkeys and in 1 of 12 feral broad-breasted bronze turkeys, but in none of several chickens on the premises. The entire mixed flock was captured, sinus aspirates were collected from affected birds, and blood samples were obtained from all birds for serologic testing. Blood samples also were obtained from 10 domestic turkeys on adjacent premises from which breeding stock had been borrowed. The M gallisepticum isolated from sinus aspirates was typed and inoculated into susceptible chickens, resulting in airsacculitis. California wild turkeys with and without histories of exposure to domestic fowl and wild turkeys shipped into California from Texas for release were tested for antibodies to M gallisepticum, using the plate agglutination test. Evidence of M gallisepticum infection was not found in wild turkeys at any location other than the original premises.     

Separating Mycoplasma gallisepticum field strains from nonpathogenic avian mycoplasmas

Mycoplasma gallisepticum (MG) has repeatedly emerged as a serious problem in U.S. broiler, layer, and turkey industries. Tracing the source of an outbreak is essential if MG control is to be accomplished. Amplified fragment length polymorphism (AFLP), random amplification of polymorphic DNA (RAPD), and restriction fragment length polymorphism (RFLP) are valuable tools used to study MG epidemiology, allowing diagnosticians to determine the source of MG infections. In some past outbreaks, AFLP, RAPD, and RFLP fingerprinting, which require pure MG cultures, were not successful because of contaminating nonpathogenic mycoplasmas from field samples. The objective of this research was to develop a method to separate rapidly growing nonpathogenic avian mycoplasma species from slower-growing MG field strains. Mixtures of MG and three separate nonpathogenic avian mycoplasmas were inoculated onto chick embryo fibroblasts cells (CEF) allowing MG to penetrate the CEF cells. Later, gentamicin sulphate was added to the culture, eliminating the nonpathogenic mycoplasmas and allowing MG to be isolated in pure culture. Mixtures of Mycoplasma synoviae (MS) and MG could not be separated in this assay. However, removal of nicotinamide adenine dinucleotide and cysteine hydrochloride during serial passage in Frey broth medium successfully eliminated growth of MS.     

Pasteurella anatipestifer infections in California turkey flocks: circumstantial evidence of a mosquito vector

Outbreaks of Pasteurella anatipestifer infections in California turkey flocks were investigated and found to have a seasonal distribution, with a peak incidence in fall, coinciding with peak Culex mosquito populations. An experiment was conducted to test the hypothesis that mosquitoes may serve as vectors for P. anatipestifer infections in turkeys. Four 7-week-old turkey poults were exposed for 7 days to mosquitoes captured from turkey barns during a field outbreak of P. anatipestifer serotype 1 infection. One turkey developed serum antibodies to serotype 1, detectable by enzyme-linked immunosorbant assay, and was resistant to an intravenous inoculation of P. anatipestifer serotype 1 at 4 weeks postexposure. Giemsa-stained blood smears from this bird and from three 7-week-old turkeys inoculated intravenously with P. anatipestifer revealed the presence of rod-shaped bacteria in or on the surface of host erythrocytes. No such rod-shaped bodies were found on erythrocytes of an uninoculated control turkey.     

Molecular Detection of Avian Pathogens in Poultry Red Mite (Dermanyssus gallinae) Collected in Chicken Farms

Poultry red mite (PRM, Dermanyssus gallinae) is a blood-sucking ectoparasite as well as a possible vector of several avian pathogens. In this study, to define the role of PRM in the prevalence of avian infectious agents, we used polymerase chain reaction (PCR) to check for the presence of seven pathogens: Avipox virus (APV), Fowl Adenovirus (FAdV), Marek’s disease virus (MDV), Erysipelothrix rhusiopathiae (ER), Salmonella enterica (SE), Mycoplasma synoviae (MS) and Mycoplasma gallisepticum (MG). A total of 159 PRM samples collected between 2004 and 2012 from 142 chicken farms in 38 prefectures in Japan were examined. APV DNA was detected in 22 samples (13.8%), 19 of which were wild-type APV. 16S ribosomal RNA (16S rRNA) of MS was detected in 15 samples (9.4%), and the mgc2 gene of MG was detected in 2 samples (1.3%). Eight of 15 MS 16S rRNA sequences differed from the vaccine sequence, indicating they were wild-type strains, while both of the MG mgc2 gene sequences detected were identical to the vaccine sequences. Of these avian pathogen-positive mite samples, three were positive for both wild-types of APV and MS. On the other hand, the DNAs of ER, SE, FAdV and MDV were not detected in any samples. These findings indicated that PRM can harbor the wild-type pathogens and might play a role as a vector in spreading these diseases in farms.     

The effect of oil-emulsion vaccines on the occurrence of nonspecific plate agglutination reactions for Mycoplasma gallisepticum and M. synoviae

Six groups of ten 18-week-old mycoplasma-free white leghorn pullets were vaccinated with one of the following: Mycoplasma gallisepticum (MG) bacterin. Haemophilus gallinarum bacterin, Pasteurella multocida bacterin, combined infectious bursal disease (IBD)-Newcastle (NDV) chicken-embryo-origin (CEO) vaccine. IBD-NDV tissue-culture-origin (TC) vaccine, or saline emulsified in oil; one group received no vaccine. Plate agglutination tests for M. synoviae (MS) and MG were done for 10 weeks after vaccination using three different test antigens. Pullets vaccinated with H. gallinarum bacterin and IBD-NDV TC vaccine showed the greatest incidence of nonspecific plate agglutination reactions. The incidence of positive plate agglutination reactions varied with test antigens. Five groups of fifty 18-week-old mycoplasma-free heavy-breed pullets were vaccinated with one of the following: saline emulsified in oil, chicken embryo fibroblasts emulsified in oil, allantoic fluid emulsified in oil, chicken embryos emulsified in oil, or MS-contaminated chicken embryos emulsified in oil. Plate agglutination tests for MS and MG were done for 8 weeks after vaccination. Chickens vaccinated with chicken embryo fibroblasts emulsified in oil had the greatest incidence of nonspecific plate agglutination reactions. Pullets vaccinated with MS-contaminated chicken embryo vaccine had only a small increase in MS-positive plate agglutination reactions compared with pullets vaccinated with uncontaminated chicken embryo vaccine.     

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.