Decrease of Mycoplasma gallisepticum seroprevalence and introduction of new genotypes in Dutch commercial poultry during the years 2001-2018.

Almost two decades ago, in addition to a compulsory M. gallisepticum (Mg) monitoring programme of breeding stock based on European Union regulations, the Dutch poultry industry added national regulations to further reduce the Mg prevalence in Dutch commercial poultry. Currently, all commercial chicken and turkey flocks except broilers are monitored for Mg. All breeding flocks on a farm where one or more flocks tested Mg positive are culled. Mg positive layer pullets are channelled and layer pullets placed on Mg positive multi-age farms are vaccinated. The monitoring data obtained were analysed covering a period of 17 years. Moreover, 31 Dutch Mg isolates from the same period were analysed by multilocus sequence typing (MLST) and compared to available PubMLST data. The results show that in breeding stock the seroprevalence decreased from 1.6% to 0.0%, in commercial layers from 6.3% to 1.9%, and in meat turkeys from 17.6% to 2.4%. The MLST results showed the presence of closely related and identical sequence types (STs) within the different Dutch poultry types. Similar STs were found in Northern and Southern Europe only. The results show a fast decline in the Mg prevalence since 2001, although in layers the Mg prevalence has stabilized and suggests backyard poultry might pose a risk for commercial poultry. The need for Mg control across poultry sectors and in trade was confirmed by the similarity in STs found in different types of poultry and regions. These results from the Dutch poultry industry can be extrapolated to Mg control in general.

Relative contribution of vertical, within-farm and between-farm transmission of Mycoplasma synoviae in layer pullet flocks.

In this study, the relative contribution of vertical transmission, within-farm transmission and between-farm transmission of Mycoplasma synoviae in layer pullet flocks was quantified using logistic regression analysis. Data from 311 Dutch pullet flocks, of which 172 (55%) were positive for M. synoviae, were included in the study. Also the M. synoviae status of the parent stock of these flocks was included. The M. synoviae status was determined with the M. synoviae rapid plate agglutination test. Data analysis showed that vertical transmission was the most important transmission route for M. synoviae in layers as is demonstrated by an odds ratio of 5.8 (P = 0.000). A positive association with M. synoviae infections was found for layer pullet flocks on a multi-house farm where at least one other flock was M. synoviae-positive compared to single-house farms (odds ratio 3.1, P = 0.022), while a negative association was found when no other M. synoviae-positive flocks were present (odds ratio = 0.2, P = 0.003). No association was found between M. synoviae status of pullet flocks and poultry farm density. Odds ratios were 0.54 (P = 0.288) and 0.34 (P = 0.073), respectively, for medium and highest poultry farm density compared to lowest poultry farm density. This is the first time that the relative contribution of horizontal and vertical transmission of M. synoviae has been quantified. These results can be extrapolated to M. synoviae control in general, and emphasize the importance of M. synoviae control in parent stock and practical channelling.

Development, validation and field evaluation of a quantitative real-time PCR able to differentiate between field Mycoplasma synoviae and the MS-H-live vaccine strain.

A quantitative PCR (qPCR) able to differentiate between field Mycoplasma synoviae and MS-H vaccine strain was developed, validated and evaluated. It was developed using nucleotide differences in the obg gene. Analytical specificity and sensitivity assessed using DNA from 194 M. synoviae field samples, three different batches of MS-H vaccine and from 43 samples representing four other avian Mycoplasma species proved to be 100%. The detection limit for field M. synoviae and MS-H vaccine strain was 102-3 and 102 colony-forming units PCR equivalents/g trachea mucus, respectively. The qPCR was able to detect both, field M. synoviae and MS-H vaccine strain in ratios of 1:100 determined both using spiked and field samples. One hundred and twenty samples from M. synoviae-infected non-vaccinated birds, 110 samples from M. synoviae-vaccinated birds from a bird experiment and 224 samples from M. synoviae negative (serology and PCR) birds were used to determine the relative sensitivity and specificity using a previously described M. synoviae PCR as reference. The relative sensitivity and specificity for field M. synoviae were 95.0% and 99.6%, respectively, and 94.6% and 100% for the MS-H-live vaccine, respectively. Field validation and confirmation by multi locus sequence typing revealed that the qPCR correctly distinguished between MS-H and field M. synoviae. Evaluation of the differentiating M. synoviae qPCR in three commercial flocks suggested transmission of MS-H-live vaccine from vaccinated to non-vaccinated flocks at the same farm. Furthermore, it showed evidence for the colonization with field M. synoviae in MS-H-vaccinated flocks.

Quantification of the horizontal transmission of Mycoplasma synoviae in non-vaccinated and MS-H-vaccinated layers.

The number of newly infected birds attributable to one infectious bird per day (= transmission rate ß) was assessed in non-vaccinated and MS-H-vaccinated experimental specified pathogen-free White Leghorns after Mycoplasma synoviae challenge. Furthermore, the effect of vaccination on the shedding of the challenge strain was determined. The following groups were made: a negative control group (n = 5), a vaccinated (MS-H vaccine by eye drop (>105.7 colour changing units/bird)) non-challenged group (n = 5), two non-vaccinated challenged groups (n = 18 each) and two vaccinated challenged groups (n = 18 each). In the challenged groups, six seeder birds were intratracheally inoculated with 105.4 colony forming units (CFUs)/bird. Trachea swabs were taken at day (D)2, D3, D4, D5, D7, D9, D11, D14, D17, D21, D25, D28, D32, D35, D42 and D46 after contact with seeders and analyzed with a quantitative PCR able to detect the vaccine and field strain separately. The transmission rate and shedding were estimated using the susceptible exposed infectious transmission model and a linear mixed model, respectively. The mean shedding of the challenge strain was 106.4 CFU equivalents M. synoviae/g trachea mucus in vaccinates shedding MS-H, while in the birds not shedding the vaccine (non-vaccinates and vaccinates not shedding MS-H) it was 106.9 CFU equivalents M. synoviae/g trachea mucus. In vaccinates shedding MS-H, ß was 0.0012 (95% C.I.: 0.00048 - 0.0024), while in birds not shedding vaccine (non-vaccinates and vaccinates not shedding MS-H) a significantly higher ß of 0.022 (95% C.I.: 0.015 - 0.031) was found.

Development and evaluation of a multi-locus sequence typing scheme for Mycoplasma synoviae.

Reproducible molecular Mycoplasma synoviae typing techniques with sufficient discriminatory power may help to expand knowledge on its epidemiology and contribute to the improvement of control and eradication programmes of this mycoplasma species. The present study describes the development and validation of a novel multi-locus sequence typing (MLST) scheme for M. synoviae. Thirteen M. synoviae isolates originating from different poultry categories, farms and lesions, were subjected to whole genome sequencing. Their sequences were compared to that of M. synoviae reference strain MS53. A high number of single nucleotide polymorphisms (SNPs) indicating considerable genetic diversity were identified. SNPs were present in over 40 putative target genes for MLST of which five target genes were selected (nanA, uvrA, lepA, ruvB and ugpA) for the MLST scheme. This scheme was evaluated analysing 209 M. synoviae samples from different countries, categories of poultry, farms and lesions. Eleven clonal clusters and 76 different sequence types (STs) were obtained. Clustering occurred following geographical origin, supporting the hypothesis of regional population evolution. M. synoviae samples obtained from epidemiologically linked outbreaks often harboured the same ST. In contrast, multiple M. synoviae lineages were found in samples originating from swollen joints or oviducts from hens that produce eggs with eggshell apex abnormalities indicating that further research is needed to identify the genetic factors of M. synoviae that may explain its variations in tissue tropism and disease inducing potential. Furthermore, MLST proved to have a higher discriminatory power compared to variable lipoprotein and haemagglutinin A typing, which generated 50 different genotypes on the same database.

Variable lipoprotein haemagglutinin (vlhA) gene sequence typing of mainly Dutch Mycoplasma synoviae isolates: comparison with vlhA sequences from Genbank and with amplified fragment length polymorphism analysis.

Molecular typing techniques with sufficient discriminatory power are required to better understand the transmission of Mycoplasma synoviae, a poultry pathogen with increasing clinical and economic relevance. A promising molecular technique is polymerase chain reaction and subsequent sequencing based on the conserved 5' region of the M. synoviae variable lipoprotein and haemagglutinin (vlhA) gene. This technique was used for genotyping 27 mainly Dutch M. synoviae isolates from different organs of various categories of poultry housed on different farms and collected during a period of 10 years. The obtained vlhA sequences were compared with those of M. synoviae strains from Genbank and data obtained by amplified fragment length polymorphism (AFLP). Grouping based on 100% similarity revealed nine genotypes. Some isolates had identical vlhA gene sequences although they originated from different geographical areas, different years and organs. AFLP analysis results largely confirmed the results obtained by vlhA sequence typing. Our findings raise concern regarding the discriminatory power of these techniques for its use in molecular epidemiology of Dutch M. synoviae isolates and for the differentiation between M. synoviae vaccine strains and field isolates, and indicate that molecular typing based on additional markers should be considered.

The effect of the air sampling method on the recovery of Mycoplasma gallisepticum from experimentally produced aerosols.

BACKGROUND: A reliable air sampling method is a prerequisite to calculate the inhaled aerosol dose by animals exposed to the aerosol as precise as possible.[Comp]: Set abstract according to the journal style.[/Comp] OBJECTIVE: To examine if aerosol collection in a fluid medium (buffered peptone water (BPW) in the impinger) improves detection of viable mycoplasmas. Also the effect of adding Mycoplasma Experience (ME) broth and/or BPW to the aerosol fluid on aerosol titres was assessed. METHODS: Aerosols containing a Mycoplasma gallisepticum field or vaccine strain were simultaneously sampled with gelatin filters and by impinger immediately after ending aerosolization and 25 min later. RESULTS: Sampling of M. gallisepticum aerosols using the impinger did not yield higher aerosol titres compared to sampling with gelatin filters. Initial loss during generation of the field strain aerosol and the half-life time of viable mycoplasmas in the aerosol were 1.1-2.4 log10 and <4-15 min, respectively. The vaccine strain was more vulnerable compared to its field counterpart. In spite of higher aerosolized doses of the vaccine strain (10(8.0) to 10(8.1) versus 10(7.5) cfu per m(3) of air of the field strain), mycoplasmas were not recovered from the aerosols neither by gelatin filter nor by impinger. Therefore, half-life times could not be calculated. Addition of BPW to the aerosol fluid did not clearly improve the recovery of the field strain from the aerosol, while addition of ME broth and BPW did. CONCLUSION: Gelatin filters likely due to their relative high moisture content (10-14% wt/wt) are at least as useful as the impinger for the recovery of M. gallisepticum from aerosols, provided exsiccation of the filters is prevented.

Validation of a previously developed quantitative polymerase chain reaction for the detection and quantification of Mycoplasma synoviae in chicken joint specimens.

A quantitative polymerase chain reaction (qPCR) was validated for the detection of Mycoplasma synoviae (PCR equivalents of colony-forming units [CFU eq.]) in chicken joint specimens with time and compared with direct mycoplasma culture. Joint specimens were obtained from 70 layer pullets inoculated intravenously with M. synoviae at 6 weeks of age. Ten control birds were injected intra-articularly with Freund's complete adjuvant. Macroscopic joint lesions were observed in 54 infected birds, of which 11 showed positive M. synoviae culture. The specificity of direct mycoplasma culture was high (100%, 95% confidence interval [CI] = 74 to 100), but its sensitivity low (16%, 95% CI = 8 to 26). Most positive results were obtained during the first 2 weeks after onset of joint swelling using synovial fluid. The qPCR was positive in 26 of 28 synovial fluid samples and in 51 of 70 joint swabs. The sterile joint samples obtained from Freund's complete adjuvant-injected birds were negative in the mycoplasma culture. The specificity and sensitivity of the qPCR for synovial fluid samples were 100% (95% CI = 65 to 100) and 93% (95% CI = 77 to 99); for joint swabs they were 100% (95% CI = 74 to 100) and 73% (95% CI = 61 to 83), respectively. Positive qPCR results (10(0.3) (to) (4.6) CFU eq./ml) were found until the end of the experiment (12 weeks post inoculation). At the end of the study, eight out of 16 joint swabs from birds without macroscopic joints lesions were positive in the qPCR (10(2.0) (to) (2.8) CFU eq./ml). Under the conditions of this study, the sensitivity of the qPCR was higher than that of direct mycoplasma culture (P< 0.0001) during the acute, subacute and chronic stages of arthritis.

Discrepancy between minimal inhibitory concentration to enrofloxacin and mutations present in the quinolone-resistance determining regions of Mycoplasma gallisepticum field strains.

Molecular characterization of the quinolone-resistance determining regions (QRDRs) of DNA gyrase and topoisomerase IV in 93 Mycoplasma gallisepticum field strains isolated in different geographic regions revealed discrepancies between minimal inhibitory concentration values and presence of amino acid substitutions within the QRDRs of GyrA and ParC in 9/93 (10%) strains. This may delimitate applicability of a gene-based assay to detect fluoroquinolone resistance in this avian pathogen.

Longitudinal field study on the occurrence of Mycoplasma synoviae in Dutch turkey flocks with lameness and experimental induction of the condition.

Four meat turkey farms with a history of lameness were investigated for the presence of Mycoplasma synoviae by testing one flock per farm for antibodies with the rapid plate agglutination (RPA) test and/or for M. synoviae DNA by polymerase chain reaction (PCR). Flocks were sampled every 2 weeks from 6 weeks of age until slaughter. If PCR results were positive, tracheal swabs were taken for mycoplasma isolation and swollen joints were sampled for general bacteriology, mycoplasma culture and virology. In one flock, all M. synoviae diagnostic tests were negative but reovirus was isolated. In the other flocks, M. synoviae was the only pathogen associated with lameness due to joint disease. M. synoviae RPA tests became positive 6 to 8 weeks later than PCR. An experimental infection was then conducted in male meat turkeys in which a negative control group was sham inoculated at 8 weeks of age, while three other groups were inoculated intravenously with M. synoviae. Turkeys in group LCh were given 10(5) colony-forming units (CFU) of an arthropathic M. synoviae chicken strain at 8 weeks; group LHCh was given a low (10(5) CFU) dose at 8 weeks, followed by a high (10(8) CFU) dose at 12 weeks, of the same chicken strain; and group HTu was inoculated with 10(8) CFU of a M. synoviae turkey joint isolate from the field study. Post-mortem examination, histopathology, serology, bacteriology and mycoplasma culture were performed at 19 weeks of age. A dose effect was found after comparing the LCh and the LHCh groups. No significant difference was observed between the HTu (10(8) CFU/bird) and the LCh (10(5) CFU/bird) group regarding the number of turkeys with arthritis, the number of M. synoviae reisolations and the mean microscopic lesion scores of joints, indicating that the M. synoviae chicken strain has greater arthropathogenic potential and that infection of turkeys in the field with such a strain may possibly have a greater clinical and economic impact.

Induction of eggshell apex abnormalities in broiler breeder hens.

Recently, the causal relationship between eggshell apex abnormalities (EAA) and Mycoplasma synoviae was described. This eggshell pathology has only been documented in table egg layers both spontaneously and experimentally infected with M. synoviae, suggesting that meat-type layers are less prone to this condition. In this study the susceptibility of specified pathogen free (SPF) broiler breeder hens to produce eggs with EAA after M. synoviae infection was assessed. Five groups of 12 hens each were made: a negative control group, a group inoculated intratracheally (i.t.) with a M. synoviae EAA strain at 19 weeks of age, a group inoculated i.t. with this strain at 19 and 26 weeks of age, a group inoculated with M. synoviae i.t. at 19 weeks of age and infected 5 days earlier with infectious bronchitis virus D1466 (IBV), and a fifth group similar to the former but inoculated i.t. twice with an M. synoviae EAA strain at 19 and 26 weeks of age. Eggs with EAA were only produced after a single i.t. inoculation with the M. synoviae EAA strain if preceded by an infection with IBV. The production of eggs with EAA started 6 weeks after M. synoviae EAA inoculation and the proportion of eggs with EAA during the experiment was 9/449 (2%), which was much lower than that in SPF layer hens (14-22%). The present results suggest that broiler breeder hens are less susceptible to producing eggs with EAA after an infection with a M. synoviae EAA strain preceded by an IBV infection, compared with table egg layers. Similar to SPF egg layers, the mean daily egg production per hen was significantly reduced by the M. synoviae EAA strain and there was a general negative effect on eggshell strength by this strain, suggesting it could also have a detrimental effect on hatching egg quality.

Effect of a live Mycoplasma synoviae vaccine on the production of eggshell apex abnormalities induced by a M. synoviae infection preceded by an infection with infectious bronchitis virus D1466.

An experimental study was conducted to assess the effect of a live Mycoplasma synoviae vaccine (Vaxsafe MS; Bioproperties Pty Ltd, Ringwood, Victoria, Australia) on M. synoviae-induced eggshell apex abnormalities (EAA). Four experimental groups of specified-pathogen-free white laying hens were made. All groups were inoculated with infectious bronchitis virus D1466 at 18 weeks of age. One group did not receive further treatment (non-vaccinated non-challenged (NVNC)). Two groups were vaccinated at 14 weeks of age against M. synoviae, and one of these groups was also challenged with an EAA-inducing M. synoviae strain 5 days after infectious bronchitis virus challenge (vaccinated non-challenged (VNC) and vaccinated challenged group (VC), respectively). The fourth group was not vaccinated but was challenged with M. synoviae (non-vaccinated challenged (NVC)). Eggs with EAA eggs were produced only in the NVC and VC groups. However, the proportion of eggs with EAA and the mean daily production of eggs with EAA per chicken was significantly lower (P<0.05) in the VC group (88/741 (11.9%) and 0.09+/-0.01 eggs per hen) compared with the NVC group (148/646 (22.9%) and 0.14+/-0.01 eggs per hen). The mean daily egg production per chicken was significantly lower in the NVC group (0.48+/-0.03 eggs) compared with that of the NVNC group (0.60+/-0.03 eggs), but not significantly different from other groups. The eggshell strength of eggs with EAA (22.8 N) was significantly lower (P<0.05) than non-affected eggs from the other groups (33.7 to 39.5 N). Furthermore, the eggshell strength of non-affected eggs in the NVC group was significantly lower (P<0.05) compared with that of non-affected eggs from the flock of origin (33.7 versus 41.2 N), but not different from the other groups. It can be concluded from the present study that vaccination with a live M. synoviae vaccine reduces the occurrence of M. synoviae-induced EAA significantly.

Induction of eggshell apex abnormalities by Mycoplasma synoviae: field and experimental studies.

A novel eggshell pathology, characterized by an altered shell surface, thinning, increased translucency, and cracks and breaks in the eggshell apex, has become increasingly common in layer flocks of various breeds in The Netherlands. Two field studies found an association between the eggshell apex abnormalities (EAA) and infection with Mycoplasma synoviae. M. synoviae was isolated from the oviduct of birds that produced abnormal eggs, but not from birds in control flocks, although both affected and control birds had agglutinating antibodies against M. synoviae. After a single injection with long-acting oxytetracycline, the production of abnormal eggs ceased, but then reoccurred about 12 days later. A causal relationship between EAA and M. synoviae infection was subsequently demonstrated experimentally. EAA occurred after intratracheal inoculation of birds with M. synoviae, and even more frequently in birds infected with infectious bronchitis virus 5 days before inoculation of M. synoviae. EAA also occurred, although less frequently, in birds inoculated intravenously with M. synoviae and infected with infectious bronchitis virus. EAA did not occur in birds only inoculated intravenously with M. synoviae. M. synoviae was only isolated from the oviducts of birds producing abnormal eggs. The mean daily egg production was reduced in all groups infected with M. synoviae. Examination of the eggshells by scanning electron microscopy revealed that the mammillary layer of the calcified zone was absent and that the inner eggshell membranes were thicker. Isolates of M. synoviae from the oviducts of birds from farms producing abnormal eggs were examined by amplified fragment-length polymorphism analysis and did not appear to be clonal.

In vitro antibiotic susceptibility of Dutch Mycoplasma synoviae field isolates originating from joint lesions and the respiratory tract of commercial poultry.

The in vitro susceptibility of 17 Dutch Mycoplasma synoviae isolates from commercial poultry to enrofloxacin, difloxacin, doxycycline, tylosin and tilmicosin was examined. Three isolates originated from joint lesions and 14 were from the respiratory tract. The type strain M. synoviae WVU 1853 was included as a control strain. Antibiotic susceptibility was tested quantitatively using the broth microdilution test. Based on initial and final minimum inhibitory concentration values, all tested isolates were susceptible to doxycycline, tylosin and tilmicosin. Two isolates from the respiratory tract were resistant to enrofloxacin and showed intermediate resistance to difloxacin.

The effect of a live vaccine on the horizontal transmission of Mycoplasma gallisepticum.

The effect of a live Mycoplasma gallisepticum vaccine on the horizontal transmission of this Mycoplasma species was quantified in an experimental animal transmission model in specific pathogen free White Layers. Two identical trials were performed, each consisting of two experimental groups and one control group. The experimental groups each consisted of 20 birds 21 weeks of age, which were housed following a pair-wise design. One group was vaccinated twice with a commercially available live attenuated M. gallisepticum vaccine, while the other group was not vaccinated. Each pair of the experimental group consisted of a challenged chicken (10(4) colony-forming units intratracheally) and a susceptible in-contact bird. The control group consisted of 10 twice-vaccinated birds housed in pairs and five individually housed non-vaccinated birds. The infection was monitored by serology, culture and quantitative polymerase chain reaction. The vaccine strain and the challenge strain were distinguished by a specific polymerase chain reaction and by random amplified polymorphic DNA analysis. In both experiments, all non-vaccinated challenged chickens and their in-contact 'partners' became infected with M. gallisepticum. In the vaccinated challenged and corresponding in-contact birds, a total of 19 and 13 chickens, respectively, became infected with M. gallisepticum. Analysis of the M. gallisepticum shedding patterns showed a significant effect of vaccination on the shedding levels of the vaccinated in-contact chickens. Moreover, the Cox Proportional Hazard analysis indicated that the rate of M. gallisepticum transmission from challenged to in-contact birds in the vaccinated group was 0.356 times that of the non-vaccinated group. In addition, the overall estimate of R (the average number of secondary cases infected by one typical infectious case) of the vaccinated group (R = 4.3, 95% confidence interval = 1.6 to 49.9) was significantly lower than that of the non-vaccinated group (R = infinity, 95% confidence interval = 9.9 to infinity). However, the overall estimate of R in the vaccinated group still exceeded 1, which indicates that the effect of the vaccination on the horizontal transmission M. gallisepticum is insufficient to stop its spread under these experimental conditions.

The effect of vaccination with a bacterin on the horizontal transmission of Mycoplasma gallisepticum.

The effect of an inactivated vaccine on the horizontal transmission of Mycoplasma gallisepticum was quantified in a transmission model. Twenty non-vaccinated and 20 vaccinated 23-week-old specific pathogen free hens were housed in pairs, while five individually housed hens acted as a negative control group. Each pair consisted of a challenged chicken (10(4) colony forming units intratracheally) and a non-challenged susceptible contact bird. Infection was monitored by serology, quantitative polymerase chain reaction and culture. All non-vaccinated and vaccinated in-contact chickens became infected with M. gallisepticum. The 95% confidence interval of the reproduction ratio, R (a measure of transmission defined as the average number of secondary cases caused by one infectious individual) was 4.48 to infinity in both groups. However, the logarithm of the area under the curve in the vaccinated group was 0.51 lower (P = 0.02) than in the non-vaccinated group, indicating that there was an effect of vaccination on the levels of potential shedding of M. gallisepticum. Nevertheless, the results of this study indicate that the use of an inactivated M. gallisepticum vaccine will not reduce the horizontal transmission of M. gallisepticum between laying hens.

Real-time polymerase chain reaction for the qualitative and quantitative detection of Mycoplasma gallisepticum.

The aim of this study was to evaluate the sensitivity and the detection limit of a real-time polymerase chain reaction (Q-PCR) developed for the qualitative and quantitative detection of Mycoplasma gallisepticum. No cross-reactivity was observed with DNA from other important avian mycoplasmas, including Mycoplasma synoviae and Mycoplasma meleagridis. However, the Q-PCR could not distinguish between M. gallisepticum and Mycoplasma imitans. The Q-PCR had detection limits 10 to 1000 times lower than a conventional commercial PCR method and than culture. The Q-PCR was used quantitatively by incorporating a set of external M. gallisepticum DNA standards, derived from a M. gallisepticum log-phase culture of a known concentration. The number of colony-forming unit equivalents per millilitre in tracheal swabs from experimentally infected birds could be determined from a single sample. The method had good reproducibility and correlated well with standard counting techniques using culture. It can be concluded that the Q-PCR described is suitable for qualitative and quantitative detection of M. gallisepticum in clinical samples.

An experimental model to quantify horizontal transmission of Mycoplasma gallisepticum.

Before interventions to control horizontal transmission of Mycoplasma gallisepticum can be tested, a suitable experimental model should be available. Transmission dynamics in a flock can be quantified by two parameters: the average number of secondary cases infected by one typical infectious case (R0) and the number of new infections that occur due to one infectious animal per unit of time (beta). The transmission dynamics of M. gallisepticum have not been studied experimentally, so the aim of this study was to examine the horizontal transmission of M. gallisepticum. The study was carried out using a pairwise design with three different inoculation doses. Every pair consisted of an inoculated chicken and a susceptible in-contact chicken. Five susceptible individually housed chickens were placed in between pairs in order to measure airborne transmission. Infection was detected by serology, quantitative polymerase chain reaction and culture. The inoculated and in-contact chickens were equally infectious and the pairs could be regarded as independent. The R0 was estimated to be greater than 1 (infinity; 95% confidence interval, 4.5 to infinity), the estimated beta was 0.22 per day and there was no significant difference between the different inoculation doses. It was concluded that the animal model as described in this study meets the conditions for the establishment of transmission dynamics of M. gallisepticum and therefore can be used to establish the quantitative effect of intervention measures on horizontal transmission.

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.

Aerosolization of Mycoplasma synoviae compared with Mycoplasma gallisepticum and Enterococcus faecalis.

In order to study the airborne transmission of an arthropathic strain of Mycoplasma synoviae, preliminary aerosol experiments were performed. They were conducted in duplicate in an empty isolator (1.3 m3) to assess the yield and viability of M. synoviae with time compared with Mycoplasma gallisepticum and Enterococcus faecalis. After aerosol generation air samples were taken with two different devices using gelatine or cellulose nitrate filters. There was no difference between the devices, but cellulose nitrate filters yielded very low bacterial counts. The aerosolized dose per isolator for M. synoviae was 3.4 x 10(10) colony-forming units (cfu), for M. gallisepticum was 2.6 x 10(10) cfu and for E. faecalis was 3 x 10(10) cfu. Immediately after aerosolization, concentrations of about 10(6) to 10(7) cfu/m3, 10(7) to 10(8) cfu/m3 and 10(8) to 10(9) cfu/m3 air of M. synoviae, M. gallisepticum and E. faecalis were found, respectively. At 25 min M. synoviae concentrations dropped below the detection level (<4 x 10(4) cfu), while 10(5) to 10(6) and 10(8) to 10(9) cfu were found for M. gallisepticum and E. faecalis, respectively. The average M. synoviae concentration during the experiment was estimated at 10(2) to 10(3) cfu/l. The M. gallisepticum and E. faecalis aerosol generated an average of approximately 10(3) to 10(4) cfu/l air and 10(5) to 10(6) cfu/l air, respectively. Thus mycoplasma and E. faecalis aerosols were successfully generated despite considerable initial loss as measured by culture. The loss was greater in the mycoplasma aerosols, especially those of M. synoviae.