Molecular detection and genetic characterization of Mycoplasma gallisepticum and Mycoplasma synoviae in selected chicken breeds in South Africa.

BACKGROUND: The impact of chickens on maintaining the economy and livelihood of rural communities cannot be overemphasized. In recent years, mycoplasmosis has become one of the diseases that affect the success of South African chicken production. Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the most prevalent strains of Mycoplasma in South Africa. MG and MS are significant respiratory pathogens affecting the productivity of chickens. The present study aimed to molecularly detect using qPCR and characterize the presence of MG and MS using phylogenetic analysis. The phylogenetic analysis was utilized to clarify general evolutionary relationships between related taxa of different MG and MS observed in tracheal swabs from South African chicken breeds. METHODS: Forty-five tracheal swabs of the Lohmann Brown (n = 9), Rhode Island Red (n = 9), Ovambo (n = 9), Venda (n = 9), and Potchefstroom Koekoek (n = 9) breeds were collected from symptomatic chickens present in the commercial farm. To detect MG and MS, DNA was extracted from tracheal swabs and faecal samples, and qPCR was performed with a 16 s rRNA (310 bp) and vlhA (400 bp) gene fragment. Following the sequencing of all the amplicons, MG, and MS dendrograms showing the evolutionary relationships among the five South African chicken breeds and the GeneBank reference population were constructed. RESULTS: The qPCR revealed the presence of MG and MS in 22% (2/9) of the tracheal swab samples tested for MS only in Rhode Island Red breeds; 66.6% (6/9) and 33% (3/9) of the tested samples in Ovambo breeds; and 11.1% (1/9) and 44.4% (4/9) of the tested samples in Venda breeds. No MG or MS were detected in the Lohmann Brown or Potchefstroom Koekoek breed. Furthermore, qPCR revealed the presence of MG in pooled faecal samples from Lohmann Brown and Ovambo breeds. Eight different bacterial isolates were recognized from both samples. Four isolates were of the 16 s ribosomal ribonucleic acid (rRNA) gene (named PT/MG51/ck/00, PT/MG48/ck/00, PT/MG41/ck/00 and PT/MG71/ck/00) gene of Mycoplasma gallisepticum, and the other was Mycoplasma Synoviae variable lipoprotein hemagglutinin A (vlhA) gene (named PT/MSA22/ck/01, PT/MS41/ck/01, PT/MS74/ck/01 and PT/MS46/ck/01) which were available in GenBank. These isolates were successfully sequenced with 95-100% similarity to the isolates from the gene bank. CONCLUSION: The study revealed the presence of both MG and MS in the chicken breeds sampled. Furthermore, the different breeds of chicken were found to be susceptible to infection under the intensive or commercial management system. Therefore, continuous surveillance is encouraged to prevent the spread and outbreak of MG and MS in the poultry industry in South Africa.

Genotyping Mycoplasma gallisepticum by multilocus sequence typing.

Mycoplasma gallisepticum causes chronic respiratory disease and reproductive disorders in many bird species, resulting in considerable economic losses to the poultry industry. Maintenance of M. gallisepticum-free flocks is the most adequate method to control infection. To this end, monitoring systems and vaccination programs with live vaccine strains are applied worldwide. There is strong demand for efficient epidemiological investigation tools to distinguish M. gallisepticum strains in order to control disease. Up to now, multilocus sequence typing (MLST) has been regarded as gold standard for genotyping bacteria due to its good reproducibility and high discriminatory power. The aim of this study was to develop an MLST assay which can determine phylogenetic distances between M. gallisepticum strains. After analysing more than 30 housekeeping genes, six loci (atpG, dnaA, fusA, rpoB, ruvB, uvrA) were selected for the MLST assay due to their genomic location and high diversity. Examination of 130 M. gallisepticum strains with this MLST method yielded 57 unique sequence types (STs) with a 0.96 Simpson's index of diversity. Considering the large number of STs and high diversity index, this MLST method was found to be appropriate to discriminate M. gallisepticum strains. In addition, the developed method was shown to be suitable for epidemiological investigations, as it confirmed linkage between related strains from outbreaks in different farms. Besides, MLST also suggested high impact of extensive international trade on the spread of different M. gallisepticum strains. Furthermore this method can be used for differentiation among vaccine and field strains.

Application of high-resolution melting-curve analysis on pvpA gene for detection and classification of Mycoplasma gallisepticum strains.

Mycoplasma gallisepticum (MG) is an avian species pathogen which causes heavy economic losses in the poultry industry. The purpose of this study was to determine genomic diversity of 14 MG field strains from chicken, Chuker partridge and peacock collected during 2009-2012 in Iran by polymerase chain reaction and partial sequencing of the pvpA gene. A High-Resolution Melting (HRM) technique was also developed and applied to differentiate between field and vaccine strains. Sequencing of the pvpA gene revealed a 51 nucleotide deletion, within DR-1 and DR-2, among MG strains from chicken and partridge whilst 63 nucleotides were deleted in MG strain from peacock. One nucleotide substitution was also observed among chicken MG strains. Phylogenetic analysis of the sequences clustered all of the Iranian MG strains into two clades or phylogeny groups; the strains from chicken and partridge in one group (group 1) and the strain from peacock into another group (group 4). HRM analysis has also produced comparable outcome to those of sequencing; four distinct melting curves which correspond to the three MG strains from chicken, Chukar partridge and peacock and ts-11 vaccine strain. Overall, findings of this study point towards a single source of infection for the chicken and partridge MG strains and likelihood of the strains being native and endemic in Iran. Peacock considered as an exotic species in Iran, hence the genetic distance for the pvpA gene. MG can be transmitted easily among different avian species and this distinct peacock strain may pose a threat to poultry industry. Our findings also show that molecular variation among pvpA gene of MG strains could be revealed using the relatively rapid and affordable HRM technique.

Core Genome Multilocus Sequence Typing: a Standardized Approach for Molecular Typing of Mycoplasma gallisepticum.

Mycoplasma gallisepticum is the most virulent and economically important Mycoplasma species for poultry worldwide. Currently, M. gallisepticum strain differentiation based on sequence analysis of 5 loci remains insufficient for accurate outbreak investigation. Recently, whole-genome sequences (WGS) of many human and animal pathogens have been successfully used for microbial outbreak investigations. However, the massive sequence data and the diverse properties of different genes within bacterial genomes results in a lack of standard reproducible methods for comparisons among M. gallisepticum whole genomes. Here, we proposed the development of a core genome multilocus sequence typing (cgMLST) scheme for M. gallisepticum strains and field isolates. For development of this scheme, a diverse collection of 37 M. gallisepticum genomes was used to identify cgMLST targets. A total of 425 M. gallisepticum conserved genes (49.85% of M. gallisepticum genome) were selected as core genome targets. A total of 81 M. gallisepticum genomes from 5 countries on 4 continents were typed using M. gallisepticum cgMLST. Analyses of phylogenetic trees generated by cgMLST displayed a high degree of agreement with geographical and temporal information. Moreover, the high discriminatory power of cgMLST allowed differentiation between M. gallisepticum strains of the same outbreak. M. gallisepticum cgMLST represents a standardized, accurate, highly discriminatory, and reproducible method for differentiation among M. gallisepticum isolates. cgMLST provides stable and expandable nomenclature, allowing for comparison and sharing of typing results among laboratories worldwide. cgMLST offers an opportunity to harness the tremendous power of next-generation sequencing technology in applied avian mycoplasma epidemiology at both local and global levels.

Sequencing analysis of Mycoplasma gallisepticum wild strains in vaccinated chicken breeder flocks.

Mycoplasma gallisepticum (MG) infection is still of continuing economic concern in commercial broiler breeder chicken flocks in Egypt. MG infection continues to emerge despite the application of vaccination programs in breeder flocks. This prompted flock surveillance including MG isolation and molecular characterization of the circulating MG strains. The present study was concerned with 15 broiler breeder flocks of different ages (5-51 weeks). Three flocks were apparently healthy and 12 flocks were diseased. The aim of the study was to characterize the MG strains recovered from tracheal swabs. Four positive MG DNA extracts identified by rt-PCR and confirmed by isolation were subjected to sequencing of the mgc2 gene and intergenic spacer region (IGSR). The current molecular study demonstrated the presence of 3 different wild-type MG strains (RabE1-08, RabE2-09 and RabE3-09) in vaccinated diseased flocks, while the fourth strain (RabE4-08), which was isolated from a nonvaccinated apparently healthy breeder flock, scored 100% of homology and similarity to the F-strain vaccine by the sequence analysis of mgc2 and IGSR. It can be assumed that the vaccine F strain, which is supposed to replace field strains not only failed to do that, but also infected nonvaccinated flocks. Accordingly, there is a need to revise the control program including vaccine strategy in parallel with biosecurity measures.

Comparison of multiple genes and 16S-23S rRNA intergenic space region for their capacity in high resolution melt curve analysis to differentiate Mycoplasma gallisepticum vaccine strain ts-11 from field strains.

Mycoplasma gallisepticum (MG) is an important avian pathogen causing significant economic losses in the global poultry industry. In an attempt to compare and evaluate existing genotyping methods for differentiation of MG strains/isolates, high resolution melt (HRM) curve analysis was applied to 5 different PCR methods targeting vlhA, pvpA, gapA, mgc2 genes and 16S-23S rRNA intergenic space region (IGSR). To assess the discriminatory power of PCR-HRM of examined genes and IGSR, MG strains ts-11, F, 6/85 and S6, and, initially, 8 field isolates were tested. All MG strains/isolates were differentiated using PCR-HRM curve analysis and genotype confidence percentage (GCP) values of vlhA and pvpA genes, while only 0, 3 and 4 out of 12 MG strains/isolates were differentiated using gapA, mgc2 genes and IGSR, respectively. The HRM curve analysis of vlhA and pvpA genes was found to be highly correlated with the genetic diversity of the targeted genes confirmed by sequence analysis of amplicons generated from MG strains. The potential of the vlhA and pvpA genes was also demonstrated for genotyping of 12 additional MG strains from Europe and the USA. Results from this study provide a direct comparison between genes previously used in sequencing-based genotyping methods for MG strain identification and highlight the usefulness of vlhA and pvpA HRM curve analyses as rapid and reliable tools specially for diagnosis and differentiation of MG strains used here.

Multiple host transfers, but only one successful lineage in a continent-spanning emergent pathogen.

Emergence of a new disease in a novel host is thought to be a rare outcome following frequent pathogen transfers between host species. However, few opportunities exist to examine whether disease emergence stems from a single successful pathogen transfer, and whether this successful lineage represents only one of several pathogen transfers between hosts. We examined the successful host transfer and subsequent evolution of the bacterial pathogen Mycoplasma gallisepticum, an emergent pathogen of house finches (Haemorhous (formerly Carpodacus) mexicanus). Our principal goals were to assess whether host transfer has been a repeated event between the original poultry hosts and house finches, whether only a single host transfer was ultimately responsible for the emergence of M. gallisepticum in these finches, and whether the spread of the pathogen from east to west across North America has resulted in spatial structuring in the pathogen. Using a phylogeny of M. gallisepticum based on 107 isolates from domestic poultry, house finches and other songbirds, we infer that the bacterium has repeatedly jumped between these two groups of hosts but with only a single lineage of M. gallisepticum persisting and evolving in house finches; bacterial evolution has produced monophyletic eastern and western North American subclades.

The development and application of a Mycoplasma gallisepticum sequence database.

Molecular analysis was conducted on 36 Mycoplasma gallisepticum DNA extracts from tracheal swab samples of commercial poultry in seven South African provinces between 2009 and 2012. Twelve unique M. gallisepticum genotypes were identified by polymerase chain reaction and sequence analysis of the 16S-23S rRNA intergenic spacer region (IGSR), M. gallisepticum cytadhesin 2 (mgc2), MGA_0319 and gapA genetic regions. The DNA sequences of these genotypes were distinct from those of M. gallisepticum isolates in a database composed of sequences from other countries, vaccine and reference strains. The most prevalent genotype (SA-WT#7) was detected in samples from commercial broilers, broiler breeders and layers in five provinces. South African M. gallisepticum sequences were more similar to those of the live vaccines commercially available in South Africa, but were distinct from that of F strain vaccine, which is not registered for use in South Africa. The IGSR, mgc2 or MGA_0319 sequences of three South African genotypes were identical to those of the ts-11 vaccine strain, necessitating a combination of mgc2 and IGSR targeted sequencing to differentiate South African wild-type genotypes from ts-11 vaccine. To identify and differentiate all 12 wild-types, mgc2, IGSR and MGA_0319 sequencing was required. Sequencing of gapA was least effective at strain differentiation. This research serves as a model for the development of an M. gallisepticum sequence database, and illustrates its application to characterize M. gallisepticum genotypes, select diagnostic tests and better understand the epidemiology of M. gallisepticum.

Evaluation of Mycoplasma gallisepticum K-strain as a live vaccine in chickens.

We evaluated the pathogenicity of three live Mycoplasma gallisepticum (MG) vaccine candidates by infection via aerosol of 3-wk-old chickens with log phase broth cultures (trial 1). Two of the candidates (K3020 and K4649A) colonized only 10% and 20% of the chickens, respectively, unlike K2101 (K-strain), which was reisolated from all of the vaccinated chickens tested. K-strain inoculation did not result in significant air sac or tracheal lesions in chickens at 10 and 39 days postinfection (P < or = 0.05). The efficacy of K-strain as a live vaccine was evaluated in trial 2, by challenge of vaccinated chickens with virulent R-strain via aerosol at 6 wk postvaccination. K-strain vaccination resulted in significant protection from air sac and tracheal lesions (P < or = 0.05). The K-strain was further investigated to evaluate transmissibility (trial 3), colonization and persistence of infection following aerosol administration (trial 4), genetic and phenotypic stability following back passage through chickens (trial 5), and vertical transmission (trial 6). The K-strain had a low rate of horizontal transmission; it remained primarily in the respiratory system of inoculated birds and persisted in the upper respiratory tract for the duration of the trial 4 (5 mo). There was no increase in virulence of K-strain when it was back passaged five times through chickens, and no vertical transmission of K-strain was detected. K-strain showed great potential as a safe and effective live MG vaccine.

Extensive variation in surface lipoprotein gene content and genomic changes associated with virulence during evolution of a novel North American house finch epizootic strain of Mycoplasma gallisepticum.

Mycoplasma gallisepticum, a significant respiratory and reproductive pathogen of domestic poultry, has since 1994 been recognized as an emergent pathogen of the American house finch (Carpodacus mexicanus). Epizootic spread and pathognomonic characteristics of house finch-associated Mycoplasma gallisepticum (HFMG) have been studied as a model of an emergent to endemic pathogen in a novel host. Here we present comparative analysis of eight HFMG genomes, including one from an index isolate and seven isolates separated spatially and temporally (1994-2008) across the epizootic, and notably having differences in virulence. HFMG represented a monophyletic clade relative to sequenced poultry isolates, with genomic changes indicating a novel M. gallisepticum lineage and including unique deletions of coding sequence. Though most of the HFMG genome was highly conserved among isolates, genetic distances correlated with temporal-spatial distance from the index. The most dramatic genomic differences among HFMG involved phase-variable and immunodominant VlhA lipoprotein genes, including those variable in presence and genomic location. Other genomic differences included tandem copy number variation of a 5 kbp repeat, changes in and adjacent to the clustered regularly interspaced short palindromic repeats, and small-scale changes affecting coding potential and association of genes with virulence. Divergence of monophyletic isolates from similar time/space in the epizootic indicated local diversification of distinct HFMG sublineages. Overall, these data identify candidate virulence genes and reveal the importance of phase-variable lipoproteins during the evolution of M. gallisepticum during its emergence and dissemination in a novel host in nature, likely mediating an important role at the interface between pathogen virulence and host immunity.

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.

Effects of vaccination with F-strain Mycoplasma gallisepticum on egg production and quality parameters of commercial layer hens previously vaccinated with 6/85-strain Mycoplasma gallisepticum.

This study was conducted to determine the effect of overlaying (revaccinating) F-strain Mycoplasma gallisepticum at 22 or 45 wk of age on commercial leghorn hens previously vaccinated with 6/85-strain M. gallisepticum at 10 wk of age. The treatment groups included unvaccinated hens (group 1), hens receiving 6/85-strain M. gallisepticum only (group 2), and hens receiving 6/85-strain M. gallisepticum followed by F-strain M. gallisepticum at either 22 (group 3) or 45 (group 4) wk of age. There was no significant effect on egg production or egg size distribution between any of the treatment groups, unlike previous studies looking at F-strain vaccination only. Egg quality parameters, including eggshell strength, Haugh unit score, and blood-meat spot were similar between the different treatment groups. There was a difference in the rate of pimpling at postpeak production for the treatment group receiving F-strain M. gallisepticum at 22 wk of age, consistent with previously published results. This work suggests that hens previously vaccinated with 6/85-strain M. gallisepticum can be safely revaccinated with F-strain M. gallisepticum to increase protection from field strains while ameliorating the adverse effects associated with F-strain M. gallisepticum vaccination in layers post onset of lay.

Differentiation of Mycoplasma gallisepticum strains using PCR and high-resolution melting curve analysis.

Mycoplasma gallisepticum (MG) is an economically important pathogen of poultry worldwide, causing chronic respiratory disease in chickens and turkeys. Differentiation of MG strains is critical, especially in countries where poultry flocks are vaccinated with live vaccines. In this study, oligonucleotide primers were designed based on a region preceding the trinucleotide repeat of a member of the vlhA gene family, and amplicons of 145-352 bp were generated from cultures of 10 different MG strains, including the ts-11, F and 6/85 vaccine strains. High-resolution melting (HRM) curve analysis of the resultant amplicons could differentiate all MG strains. Analysis of the nucleotide sequences of the amplicons from each strain revealed that each melting curve profile related to a unique DNA sequence. The HRM curve profiles (for ts-11) remained consistent after at least five passages under laboratory conditions. PCR-HRM curve analysis of 33 DNA extracts derived from respiratory swabs, or mycoplasma cultures grown from respiratory swabs, of ts-11-vaccinated commercial or specific pathogen-free chickens identified all these specimens, according to their sequences, as ts-11. The potential of the PCR-HRM curve analysis was also shown in the genotyping of 30 additional MG isolates from Europe, the USA and Israel. The results presented in this study indicate that PCR followed by HRM curve analysis provides a rapid and robust technique for genotyping of MG isolates/strains using both MG cultures and clinical swabs.

Effects of time-specific F-strain Mycoplasma gallisepticum inoculation overlays on prelay ts-11-strain M. gallisepticum vaccination on blood characteristics of commercial laying hens.

Two trials were conducted to determine the effects of a prelay ts-11-strain Mycoplasma gallisepticum (ts-11MG) vaccination alone or in combination with subsequent time-specific F-strain M. gallisepticum (FMG) inoculations on the blood characteristics of commercial laying hens. The following 4 treatments were utilized: 1) sham vaccination at 10 wk of age, 2) vaccination of ts-11MG at 10 wk, 3) ts-11MG at 10 wk overlaid by FMG inoculation at 22 wk, and 4) ts-11MG at 10 wk overlaid by FMG at 45 wk. Parameters measured in both trials were whole blood hematocrit, plasma protein, serum cholesterol, serum triglycerides, and serum calcium. No significant age x treatment interactions and no significant age or treatment main effects were observed for any of the blood parameters investigated, except for serum calcium. At wk 22, serum calcium concentrations were increased by vaccination with ts-11MG at 10 wk, and levels were further increased when the ts-11MG vaccination at 10 wk was overlaid by an FMG inoculation at 22 wk. These results suggest that ts-11MG vaccination at 10 wk of age alone or combined with F-strain inoculum overlays at either 22 or 45 wk may be used without any consequential effects on hematocrit or the lipid and protein levels in the blood of commercial layers. Because elevations in serum calcium were not associated with changes in hen performance, as reported in a previous companion article, it is further suggested that prelay ts-11MG vaccination before FMG inoculation overlays during lay may provide adequate protection against field strain M. gallisepticum infections while being innocuous to layer performance.

Effects of time-specific F-strain Mycoplasma gallisepticum inoculation overlays on prelay ts-11-strain M. gallisepticum vaccination on digestive and reproductive organ characteristics of commercial egg-laying hens.

Two trials were conducted to determine the effects of a prelay ts-11-strain Mycoplasma gallisepticum (ts-11MG) vaccination alone or in conjunction with F-strain M. gallisepticum (FMG) inoculation overlays at 2 different age periods during lay on the digestive and reproductive organ characteristics of commercial egg-laying hens. In each trial, the following 4 treatments were utilized: sham vaccination at 10 wk of age, ts-11MG vaccination at 10 wk of age, ts-11MG at 10 wk of age overlaid by FMG inoculation at 22 wk of age, and ts-11MG at 10 wk of age overlaid by FMG at 45 wk of age. Necropsies were performed at the end of both trials (58 wk of age), using 2 birds from each of 4 replicate units per treatment, to observe treatment effects on the following parameters: liver weight, liver lipid and moisture concentrations, incidence of fatty liver hemorrhagic syndrome, ovary weight, number of mature ovarian follicles, and the total and segmental weights, lengths, and histologies of the oviduct and small intestine. Treatments affected only vaginal length as a percentage of total oviduct length. Vaginas were relatively longer in hens that had only been vaccinated with ts-11MG at 10 wk in comparison to all the other treatment groups, including controls. Except for relative vaginal length, the digestive and reproductive organs of layers were not influenced by the ts-11MG and FMG treatment regimens imposed in this study. These results confirm that when coupled with FMG inoculations during lay, prelay ts-11MG vaccinations may be a practical substitute for prelay FMG inoculations for providing continual protection against field-strain M. gallisepticum infections in layers.

Effects of 6/85-strain Mycoplasma gallisepticum vaccination alone at ten weeks of age or in conjunction with F-strain Mycoplasma gallisepticum inoculation overlays at twenty-two or forty-five weeks of age on the reproductive and digestive organs of commercial egg-laying hens.

Two trials were conducted to determine the effects of a prelay 6/85-strain Mycoplasma gallisepticum (6/85MG) vaccination alone or in conjunction with time-specific F-strain M. gallisepticum (FMG) inoculation overlays on the gross reproductive and digestive organ characteristics of commercial egg-laying hens. In each trial, the following 4 treatments were applied: 1) sham vaccination at 10 wk of age; 2) vaccination of 6/85MG at 10 wk; 3) 6/85MG at 10 wk overlaid by FMG inoculation at 22 wk; and 4) 6/85MG at 10 wk overlaid by FMG at 45 wk. Two birds per isolation pen (experimental replicate unit) were necropsied at the end of both trials to observe the effects of treatment on liver weight, liver lipid and moisture concentrations, incidence of fatty liver hemorrhagic syndrome, ovary weight, mature ovarian follicle numbers, and the total and segmental weights, lengths, and histologies of the oviduct and small intestine. The applied treatments affected only liver moisture. Liver moisture content was greater in birds vaccinated with 6/85MG at 10 wk alone or in conjunction with FMG at 45 wk in comparison with sham vaccinated controls and birds that received a 6/85MG vaccination at 10 wk overlaid by an FMG inoculation at 22 wk. Prelay 6/85MG vaccinations may be a suitable substitute for prelay FMG inoculations, and FMG overlays during lay on prelay 6/85MG vaccinations may also provide continual protection against field-strain MG infections without eliciting any subsequent suppressive effects on performance, as noted in an earlier study.

Effects of 6/85-strain Mycoplasma gallisepticum inoculation alone at ten weeks of age or in conjunction with F-strain Mycoplasma gallisepticum inoculation overlays at twenty-two or forty-five weeks of age on the performance of commercial egg-laying hens.

The effects of 6/85-strain Mycoplasma gallisepticum (6/85MG) inoculation alone or in conjunction with a F-strain M. gallisepticum (FMG) overlay and its timing on the performance of commercial egg-laying hens were investigated. Control birds received sham inoculations at 10 wk of age. A second treated group of birds was inoculated with 6/85MG at 10 wk of age, a third treatment group of birds was inoculated with 6/85MG at 10 wk and received an overlay of FMG at 22 wk, and a fourth treatment group was inoculated with 6/85MG at 10 wk followed by a 45-wk overlay inoculation of FMG. Parameters investigated between 20 and 55 wk of age included BW, mortality, weekly and total egg production, egg weight, relative eggshell conductance, eggshell weight per unit of surface area, percentage shell weight, yolk/albumen ratio, and egg shape index. Hen age effects were reported for BW, egg weight, yolk/albumen ratio, and egg production. No treatment effects or hen age x treatment interactions were noted for those parameters except for yolk/albumen ratio, and no significant effects of any kind were noted for the remaining parameters examined. At wk 47, a significant treatment effect for yolk/albumen ratio was noted. The yolk/albumen ratio in the group of birds that received 6/85MG at 10 wk followed by an overlay of FMG at 22 wk was significantly lower than the sham control birds and those that were inoculated with 6/85MG at 10 wk followed by a 45-wk overlay inoculation of FMG. Prelay 6/85MG inoculations may be a suitable substitute for prelay FMG inoculations, and FMG overlays during lay on prelay 6/85MG inoculations may provide continual protection without eliciting any subsequent suppressive effects on performance.

Effects of supplemental dietary phytase and 25-hydroxycholecalciferol on the performance characteristics of commercial layers inoculated before or at the onset of lay with the F-strain of Mycoplasma gallisepticum.

The effects of dietary supplementation with phytase and 25-hydroxycholecalciferol on the performance characteristics of commercial layers that were inoculated prelay (12 wk of age) or at the onset of lay (22 wk of age) with F-strain Mycoplasma gallisepticum were assessed. Experimental layer diets, which included a basal control diet or the same diet supplemented with 0.025% phytase and 25-hydroxycholecalciferol, were fed from 20 through 58 wk of age. Weekly and total egg production were determined from 22 through 58 wk, and egg weight and various internal egg and eggshell quality characteristics were examined at 34, 50, and 58 wk of age. F-strain M. gallisepticum inoculation decreased egg production at the beginning of lay (wk 22 and 23) but increased post-peak lay at wk 45. However, there were no treatment effects of any kind on total egg production, egg weight, or any of the internal egg and eggshell characteristics examined during lay. In conclusion, dietary supplementation with phytase and 25-hydroxycholecalciferol did not affect layer performance or interact with the effects of F-strain M. gallisepticum inoculation; however, F-strain M. gallisepticum inoculation resulted in a shift in egg production from wk 22 to 45 without having an overall effect on total egg production.

Strain differentiating real-time PCR for Mycoplasma gallisepticum live vaccine evaluation studies.

Mycoplasma gallisepticum causes respiratory disease and production losses in poultry. Vaccination of poultry with M. gallisepticum live vaccines is an approach to reduce susceptibility to infection and to prevent the economic losses. The development and evaluation of live vaccines usually requires the involvement of several vaccine and challenge strains in the same experimental setup. Our goal was to develop a tool to allow the differentiation between a set of known M. gallisepticum strains in a quantitative manner. We developed 5 real-time PCR assays that absolutely differentiated between one of the five commercial and laboratory vaccine strains: F, ts-11, 6/85, K5831, K5054, and the challenge strain R low when tested on in vitro cultures. The assay K5831 vs. R low was also tested on specimens from live birds that were vaccinated with K5831 and challenged with R low, and successfully differentiated between the vaccine and the challenge strains in a quantitative manner. This preliminary in vivo application of the method also shed light on possible protection mechanisms for the M. gallisepticum K5831 vaccine strain.

Molecular characterization and typing of enrofloxacin-resistant clinical isolates of Mycoplasma gallisepticum.

Emergence of resistance to fluoroquinolones is mainly due to chromosomal mutations in genes encoding the subunits of the drug's target enzymes, DNA gyrase and topoisomerase IV, which are essential for DNA replication. The quinolone resistance-determining regions (QRDRs) of these genes were characterized in 25 Mycoplasma gallisepticum strains isolated from commercial poultry flocks during 1997-2007, which exhibited different levels of susceptibility to fluoroquinolones. All enrofloxacin-resistant isolates harbored amino acid substitutions in the QRDRs of each of three proteins (GyrA, GyrB, and ParC). Molecular typing of those strains by random amplification of polymorphic DNA and gene-targeted sequencing supports ongoing, stepwise selection of resistant strains from the existing reservoir of susceptible M. gallisepticum strains.