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.     

Use of egg yolk to determine antibody levels in chickens inoculated with a hemagglutinating duck adenovirus (adenovirus 127-like)

Chickens were experimentally infected with a duck adenovirus that has been shown to be serologically indistinguishable from Adenovirus 127. Sera and eggs were collected at intervals after exposure for antibody determination by the hemagglutination-inhibition (HI) test, the enzyme-linked immunosorbent assay (ELISA), and the immunodiffusion (ID) test. Egg yolks were processed for use in the serological tests by (a) dilution in phosphate-buffered saline (PBS), (b) extraction of the water-soluble fraction with chloroform, or (c) freezing and thawing PBS-diluted yolks and testing the supernatant fluid. HI antibody titers from serum and extracted yolk were similar except during the initial 2 weeks, when yolk antibody levels were low or absent. Chloroform-extracted yolks were suitable material for the HI, ELISA, and ID tests. Heat inactivation of the chloroform-extracted yolk had no effect on titers.     

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.     

Experimental infection of turkeys with Mycoplasma gallisepticum of low virulence, transmissibility, and immunogenicity.

Three-week-old turkeys were inoculated intranasally with approximately 10(6) colony-forming units (CFU) of putative variant Mycoplasma gallisepticum (MG) strains M876, M35, or the virulent S6 reference strain. Uninoculated turkeys in each group served as contact sentinels. The hemagglutination-inhibition (HI) test and enzyme-linked immunosorbent assay (ELISA) were used to determine serologic responses. MG was isolated from 100% and 92% of S6- and M876-inoculated turkeys, respectively, on day 7 PI. However, culture-positive rates among M876-inoculated turkeys declined more rapidly, transmission to contact sentinels took longer and occurred at lower rates, and serologic responses measured by HI and ELISA were lower than in S6-infected turkeys. Testing sera from inoculated turkeys for antibodies to MG in homologous and heterologous ELISA systems indicated that strain M876 was significantly (P less than 0.05) less immunogenic than S6 (days 62 and 95 PI), and that the homologous ELISA was more sensitive (P less than 0.005). MG strain M35 failed to infect turkeys in three attempts, even though the inocula used were viable on culture media.     

Laboratory diagnosis of psittacine beak and feather disease by haemagglutination and haemagglutination inhibition

SUMMARY Simple and sensitive haemagglutination and haemagglutination Inhibition assays were developed for psittacine beak and feather disease (PBFD) virus and serum antibody, respectively. The assays were used in the examination of samples from 73 birds clinically affected with PBFD. High antigen titres (log₂ 9 to log₂ 12) were detected In feathers, faeces and cloacal contents of PBFD-affected birds. Antigen was not detected In either faecal or feather samples from 20 normal galahs (Eolophus roselcapillus) and 9 normal sulphur crested cockatoos (Cacatua galerita). After kaolin treatment and haemadsorption of serum, haemagglutination inhibition (HI) antibody titres could not be detected in serum from 42 PBFD-affected birds, whereas serum HI titres from 64 normal psittacine birds ranged from less than log₂ 1 to log₂ 8. Serum and yolk HI antibody responses of 6 PBFD virus-inoculated layer hens were measured. Pre-inoculation chicken sera contained high concentrations of non-specific haemagglutination inhibitors (not detected in chloroform-extracted yolk), which were removed by kaolin treatment and haemadsorption.     

Evaluation of the specificity and sensitivity of two commercial enzyme-linked immunosorbent assay kits, the serum plate agglutination test, and the hemagglutination-inhibition test for antibodies formed in response to Mycoplasma gallisepticum

Two commercial enzyme-linked immunosorbent assay (ELISA) kits, seven serum plate agglutination (SPA) antigens, and the hemagglutination-inhibition (HI) test for antibodies to Mycoplasma gallisepticum (MG) were compared for sensitivity and specificity using known MG-positive and MG-negative sera from leghorn chickens. All SPA antigens proved to be highly sensitive when testing MG-positive sera. Laboratory-prepared SPA antigens yielded fewer positive reactions when testing MG-negative sera than commercial SPA antigens. Both MG ELISA kits showed high rates of positive reactions when testing sera from birds given commercial M. synoviae bacterin, fowl coryza (Haemophilus paragallinarum) bacterin, inactivated infectious bursal disease virus vaccine, and to a lesser extent fowl cholera (Pasteurella multocida) bacterin. Immunization with Frey's medium with 12% swine serum-in-oil or Staphylococcus aureus-in-oil resulted in sera which yielded numerous positive ELISA reactions. During the first 1 to 3 weeks, antibodies induced by experimental infection with MG were better detected by the SPA test than by the ELISAs and the HI test, thus confirming the SPA test's importance in Mycoplasma diagnostic serology. The HI test can serve to confirm positive SPA results.     

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.     

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.     

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.     

Serological profiles of commercial broiler breeders and their progeny. 2. Newcastle disease virus

Newcastle disease virus (NDV) hemagglutination-inhibition (HI) titers were determined for serum samples from eight commercial broiler breeder flocks and their progeny. The chickens sampled had been vaccinated and reared by different producers in different regions of the United States. Breeder flocks had the highest number of NDV-positive HI titers (greater than or equal to 1:10). Eighty percent or more of the samples from six of eight breeder flocks were positive; the geometric mean titers (GMTs) for those six breeder flocks ranged from 19 to 92. Only 3 of 8 broiler flocks had an increased frequency of positive titers and higher GMTs after vaccination. The frequency of positive titers was greater than 80% in only 2 of 8 of the oldest broiler flocks. The number of NDV-negative titers (less than 1:10) increased with age in most broiler flocks, even though all had been vaccinated once or more with live NDV vaccines.     

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.     

Comparison of egg-yolk and serum antibody titers to four avian viruses by enzyme-linked immunosorbent assay using paired field samples

Eggs and blood were collected from 11 hens in each of nine broiler-breeder flocks in Quebec. Serum and egg-yolk extracts were assayed for antibody titers to infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), Newcastle disease virus (NDV), and reovirus (RV) by a commercial enzyme-linked immunosorbent assay (ELISA) kit. Comparison was made between egg-yolk and serum antibody titers by a regression analysis. A high correlation was observed between serum and yolk antibody titers to all the viruses tested (r = 0.9 for IBDV, 0.84 for IBV, 0.84 for NDV, and 0.91 for RV). Antibody monitoring of commercial breeder flocks using egg yolk instead of serum with commercial ELISA plates is thus feasible and is recommended.     

用HI和ELISA方法检测SPF鸡血清和蛋黄中ND抗体的比较

分别用血凝抑制试验（HI）和酶联免疫吸附试验（ELISA）检测42周和48周SPF鸡血清、以及同期SPF鸡蛋蛋黄中新城疫（ND）抗体。结果表明,两种方法检测的120份血清中均无ND抗体,呈阴性;120枚SPF鸡蛋蛋黄检测,ELISA方法检测结果全为阴性;HI方法检测结果25份阳性,差异较为明显。     

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.     

Use of egg yolk in serological tests (ELISA and HI) to detect antibody to Newcastle disease, infectious bronchitis, and Mycoplasma gallisepticum

Serum and yolks from commercial flocks and from hens exposed to Newcastle disease virus (NDV), infectious bronchitis virus (IBV), and Mycoplasma gallisepticum (MG) were tested for immunoglobulin G antibody by the enzyme-linked immunosorbent assay (ELISA) and the hemagglutination-inhibition (HI) test. Yolks prepared by chloroform extraction and low-speed centrifugation performed well in the serological tests used and were a suitable alternative to serum for antibody determination by the ELISA for NDV, IBV, and MG and by HI test for NDV.     

检测鸡慢性呼吸道病抗体ELISA方法的建立

用败血支原体（ＭＧ）Ａ５９６９株制备ＥＬＩＳＡ抗原,与抗鸡ＩＧ单抗ＩＢ７酶结合物建立了检测鸡血清抗体水平的间接ＥＬＩＳＡ方法,交叉试验、阻断试验、重复性试验等表明该方法重复性好、特异性强、灵敏度高。确立了将鸡血清６４倍稀释监测ＥＬＩＳＡ效价（ＥＴ）的回收方程ｙ＝１．３８３＋０．２２４ｘ,可用于定量测定,血凝抑制试验（ＨＩ）与ＥＬＩＳＡ比较试验表明,ＥＬＩＳＡ法比ＨＩ试验敏感性高４倍以上。     

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.     

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.     

Effect of cutting height and genetics on composition, intake, and digestibility of corn silage by beef heifers

This research was conducted to determine the effect of corn genetics and cutting height on the composition and nutritive characteristics of corn silage. An in situ study involving eight commercially available corn hybrids indicated main effects and interactions (P < 0.01) of hybrid and cutting height on NDF, ADF, and starch content and on in situ DM and NDF degradablility. Four ruminally cannulated Angus heifers (initial BW = 378 +/- 3 kg) were used in a 4 x 4 Latin square digestion experiment with a 2 x 2 factorial treatment arrangement. Main effects and interactions of hybrid (Pioneer Hi-Bred Int., Inc., hybrids 3335 and 3223) and cutting height (LO = 20.3 cm, and HI = 61 cm) were evaluated. Dietary treatment consisted of 40% chopped alfalfa hay and 60% corn silage. Although corn silage hybrids used were of equivalent maturity at harvest (60% milkline), 3335 treatments had 37.8% starch and 34.8% NDF, whereas 3223 treatments had 33.7% starch and 38.6% NDF. The LO treatments averaged 3.1 percentage units greater in NDF and 3.45 percentage units less in starch content than the HI treatments. Intake of DM was greater for heifers fed 3335-HI than 3335-LO; however, DMI was greater by heifers fed 3223-LO than 3223-HI (hybrid x cutting height interaction, P < 0.05). Starch intake was greater (P < 0.05) and NDF intake was less (P < 0.05) by heifers fed HI vs. LO and fed 3335 vs. 3223 dietary treatments. Digestibility of DM, starch, and NDF was greater (P < 0.05) by heifers fed 3223 than 3335 dietary treatments, but digestibility differences were not observed (P > 0.10) between cutting heights. Rate of in situ DM and starch degradability was not affected (P > 0.10) by hybrid or cutting height; however DM degradability was greater (P < 0.05) for HI than LO corn silage substrates at 8, 16, and 24 h of incubation. Rate of NDF degradability tended (P = 0.08) to be greater for 3223 than for 3335, and for LO compared with HI corn silage. Degradability of NDF was greater (P < 0.05) for 3223 compared with 3335 substrates at 24, 36, and 48 h of incubation. These data suggest fiber may not be an accurate measure of corn silage quality. Whereas cutting height affected chemical composition, we observed genetics to have a greater effect on corn silage quality.     

Induction of precocious puberty in heifers I: enhanced secretion of luteinizing hormone

In beef heifers weaned between 3 and 4 mo of age and fed a high-concentrate diet, approximately 50% reach puberty before 300 d of age (precocious puberty). The objectives of this experiment were 1) to determine whether precocious puberty could be induced experimentally by weaning heifers early and feeding a high-concentrate diet, and 2) to determine the dynamics of secretion of LH associated with precocious puberty. Crossbred Angus and Simmental heifer calves were weaned at 73 +/- 3 d of age and 115 +/- 3 kg of BW and fed a high-concentrate (60% corn; HI, n = 9) or control diet (30% corn; CONT, n = 9). Heifers were fed individually, and target BW gains were 1.50 and 0.75 kg/d for the HI and CONT treatments, respectively. Heifers were weighed every 2 wk. Blood samples were collected weekly and assayed for progesterone concentration to determine age at puberty. Serial blood samples were collected at 20-min intervals for 24 h at mean ages of 102, 130, 158, 172, 190, 203, 217, 231, and 259 d and assayed for LH concentration to evaluate the dynamics of secretion of LH. Heifers fed the HI diet exhibited greater BW gain (P < 0.01) than CONT heifers (1.27 +/- 0.05 vs. 0.85 +/- 0.05 kg/d, respectively). As a result, BW in the HI treatment was greater (P < 0.01) than in the CONT treatment by 188 d of age and remained different through the end of the experiment. Precocious puberty occurred in 8 of 9 heifers fed the HI diet and 0 of 9 heifers fed the CONT diet. Age at puberty was reduced in the HI (P < 0.01) compared with the CONT heifers (262 +/- 10 vs. 368 +/- 10 d of age, respectively). Body weight at puberty was also reduced in the HI (P < 0.05) compared with the CONT treatment (327 +/- 17 vs. 403 +/- 23 kg, respectively). Heifers attaining puberty during the experiment continued with subsequent luteal phases as evidenced by cyclic patterns of progesterone concentrations. Frequency of pulses of LH (pulses/24 h) increased with age (P < 0.01) for both treatments. Heifers in the HI treatment exhibited a greater number of pulses of LH (P < 0.01) than those in the CONT treatment by 190 d of age and in all subsequent collection periods (treatment x age, P < 0.05). Mean LH concentrations also increased with age (P < 0.01) for both treatments but did not differ between treatments. In conclusion, precocious puberty induced by early weaning and feeding of a high-concentrate diet is preceded by increasing frequency of pulses of LH.