Use of molecular diversity of Mycoplasma gallisepticum by gene-targeted sequencing (GTS) and random amplified polymorphic DNA (RAPD) analysis for epidemiological studies

Naola M. Ferguson1, Diego Hepp2,3, Shulei Sun1, Nilo Ikuta2,3, Sharon Levisohn4, Stanley H. Kleven1 and Maricarmen García1

1 Department of Avian Medicine, Poultry Diagnostic and Research Center, The University of Georgia, Athens, GA 30602-4875, USA
2 Universidade Luterana do Brasil, Canoas, Rio Grande do Sul, Brazil
3 Simbios Biotecnologia, Canoas, Rio Grande do Sul, Brazil
4 Division of Avian and Aquatic Diseases, Kimron Veterinary Institute, Beit Dagan 50250, Israel

Correspondence
Maricarmen García
mcgarcia{at}uga.edu


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A total of 67 Mycoplasma gallisepticum field isolates from the USA, Israel and Australia, and 10 reference strains, were characterized by gene-targeted sequencing (GTS) analysis of portions of the putative cytadhesin pvpA gene, the cytadhesin gapA gene, the cytadhesin mgc2 gene, and an uncharacterized hypothetical surface lipoprotein-encoding gene designated genome coding DNA sequence (CDS) MGA_0319. The regions of the surface-protein-encoding genes targeted in this analysis were found to be stable within a strain, after sequencing different in vitro passages of M. gallisepticum reference strains. Gene sequences were first analysed on the basis of gene size polymorphism. The pvpA and mgc2 genes are characterized by the presence of different nucleotide insertions/deletions. However, differentiation of isolates based solely on pvpA/mgc2 PCR size polymorphism was not found to be a reliable method to differentiate among M. gallisepticum isolates. On the other hand, GTS analysis based on the nucleotide sequence identities of individual and multiple genes correlated with epidemiologically linked isolates and with random amplified polymorphic DNA (RAPD) analysis. GTS analysis of individual genes, gapA, MGA_0319, mgc2 and pvpA, identified 17, 16, 20 and 22 sequence types, respectively. GTS analysis using multiple gene sequences mgc2/pvpa and gapA/MGA_0319/mgc2/pvpA identified 38 and 40 sequence types, respectively. GTS of multiple surface-protein-encoding genes showed better discriminatory power than RAPD analysis, which identified 36 pattern types from the same panel of M. gallisepticum strains. These results are believed to provide the first evidence that typing of M. gallisepticum isolates by GTS analysis of surface-protein genes is a sensitive and reproducible typing method and will allow rapid global comparisons between laboratories.


Abbreviations: CDS, coding DNA sequence; DR, direct repeat; GTS, gene-targeted sequencing; p, passage; RAPD, random amplified polymorphic DNA

The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this work are AY556071–AY556382.

Six dendrograms constructed for GTS analysis are available as supplementary data with the online version of this paper.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Mycoplasma gallisepticum is a major problem in the poultry industry worldwide, causing chronic respiratory disease of chickens and turkeys. Control of M. gallisepticum has generally been based on eradication of the organism from breeder flocks and maintenance of mycoplasma-free status in the breeders and their progeny by implementation of biosecurity. The rapid and widespread expansion of poultry in restricted geographical areas and the consequent re-emergence of mycoplasma infection have necessitated a re-evaluation of the strategies utilized to control mycoplasma infections of poultry. In areas where complete eradication is difficult to attain, vaccination with live vaccines is utilized as an alternative control strategy (Whithear, 1996; Kleven, 1997). Consequently, with the increased use of vaccination, more powerful tools to trace the source of contamination and to differentiate vaccine strains from circulating field isolates are required to aid better understanding of the epidemiology of the disease and to improve control strategies.

Several techniques have been developed for differentiation of M. gallisepticum strains, including protein profile analysis (Khan et al., 1987), restriction fragment length polymorphism (RFLP) (Kleven et al., 1988), ribotyping (Yogev et al., 1988), strain-specific DNA probes (Khan et al., 1989) and PCR with strain-specific primers (Nascimento et al., 1993). However, none of these methods has been as widely used as random amplified polymorphic DNA (RAPD) (Charlton et al., 1999b; Fan et al., 1995; Geary et al., 1994). The RAPD method has been successfully utilized to identify vaccine strains in both experimental and field conditions (Ley et al., 1997b; Kleven & Fan, 1998; Turner & Kleven 1998), as well as for tracking epidemiologically related isolates in the field (Kempf, 1998; Ley et al., 1997a; Charlton et al., 1999a; Levisohn & Kleven, 2000). However, the use of the RAPD method has not allowed for inter-laboratory comparisons or long-term epidemiological studies due to difficulty in standardizing and unifying protocols among laboratories. The RAPD technique has intrinsic problems of reproducibility because numerous experimental parameters, such as MgCl2 concentration, Taq polymerase concentration and source, template DNA concentration, and thermocycler program and model, all affect the reproducibility of the technique in different laboratories and over time in the same laboratory (Tyler et al., 1997). Recently sequencing methods have been introduced as a new approach for studying the molecular epidemiology of bacterial pathogens (Enright & Spratt, 1999). Multilocus sequence typing of housekeeping genes has been demonstrated to be a highly transferable typing method, readily applicable to a wide variety of bacteria, which has contributed to the understanding of global epidemiology and population structure of infectious diseases (Maiden et al., 1998).

In this study we present what is believed to be the first attempt to use a gene-targeted sequencing (GTS) approach to identify and differentiate among M. gallisepticum strains. Four genes were chosen for GTS analysis: three genes encoding previously characterized M. gallisepticum surface proteins and one gene encoding a predicted M. gallisepticum surface protein. The four gene sequences were identified in the genome of M. gallisepticum Rlow strain (Papazisi et al., 2003). The gapA gene encodes a protein shown to be involved in the cytadhesion process (Goh et al., 1998) identified as genome coding DNA sequence (CDS) MGA_0934. The mgc2 gene encodes a second cytadhesin protein also known to play a role in the attachment process (Hnatow et al., 1998) identified as genome CDS MGA_0932. The pvpA gene encodes a putative accessory cytadhesin that exhibits size variation among M. gallisepticum strains (Boguslavsky et al., 2000; Liu et al., 2001) and was identified as an interrupted CDS in the M. gallisepticum Rlow genome sequence by Papazisi et al. (2003). A 37 nt internal duplication within the pvpA gene resulted in a reading frame shift predicting two coding sequence fragments identified as genome CDSs MGAL_0256 and MGAL_0258, encoding the C-terminal and N-terminal ends of the pvpA gene products, respectively (Papazisi et al., 2003). The gene encoding a predicted conserved surface lipoprotein, originally recognized by Nascimento et al. (1991), was identified as genome CDS MGA_0319 (Papazisi et al., 2003).

Gene-targeted sequencing analysis of M. gallisepticum was conducted to: (1) evaluate the reproducibility and discriminatory capability of surface-protein genes as a tool for typing M. gallisepticum isolates; (2) compare the discriminatory power of GTS versus RAPD typing method; and (3) establish relationships among M. gallisepticum isolates from different outbreaks and geographical areas.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
M. gallisepticum isolates.
Sixty-seven M. gallisepticum isolates were analysed: fifty-four from the USA, five from Australia and eight from Israel. The isolates from the USA were obtained from the depository at the Poultry Diagnostic and Research Center (PDRC), University of Georgia. These isolates were from 16 different states, isolated from 1973 to 2001. They were from broiler breeders, commercial layers, turkey breeders, meat-type turkeys, house finches and an American goldfinch. The Israeli isolates were obtained from the depository of the Division of Avian and Aquatic Diseases, Kimron Veterinary Institute (Beit Dagan, Israel). These strains were isolated from M. gallisepticum outbreaks in broiler breeders, turkey breeders and meat-type turkeys. The outbreaks had no known epidemiological link, as was supported by initial RAPD typing performed in Israel. The Australian isolates were acquired courtesy of K. G. Whithear, from the University of Melbourne (Victoria, Australia) and included strain AU083CK80, the pathogenic field isolate from which the ts-11 vaccine was derived by mutagenesis (Soeripto et al., 1989).

Reference M. gallisepticum strains used in this study included: 6/85 laboratory strain (29 p) (Evans & Hafez, 1992) and 6/85 vaccine (Intervet Inc., Millsboro, DE); ts-11 laboratory strain (99 p) (Whithear et al., 1990) and ts-11 vaccine (Merial, Gainesville, GA); R (13, 73 and 154 p) (Rodriguez & Kleven, 1980; Levisohn et al., 1986); S6 (20, 106 and 259 p) (Zander, 1961; Levisohn et al., 1986); HF-51 (91 p) (Luttrell et al., 1996); K503 (66 p), K703 (38 p), K730 (477 p) (Yoder, 1986); A5969 (192 p) (Van Roekel & Olesiuk 1952); F laboratory strain (16, 124 and 255 p) (Adler et al., 1960; Luginbuhl et al., 1967; Levisohn et al., 1986) and F vaccine (Schering Plough Animal Health, Millsboro, DE). The passage levels of A5969 and F laboratory strains are recorded from the time of deposit at the PDRC depository.

PCR amplification and sequencing of targeted genes.
Nucleic acid was extracted from 150 to 250 µl of a culture grown in modified Frey's medium or frozen Frey's medium stock cultures stored with 5 % (v/v) glycerol. Genomic DNA was extracted using the QIAamp DNA Mini Kit (QIAGEN), following the manufacturer's recommendations. Primer sequences, location and expected amplification product size for the targeted genes, gapA, MGA_0319 mgc2 and pvpA, are shown in Table 1. Primer positions given are based on the M. gallisepticum Rlow genome sequence (AE015450). All amplifications were performed in a PTC-200 DNA Engine MJ thermocycler (MJ Research) at 94 °C for 3 min, and 40 cycles of 94 °C for 20 s, 55 to 60 °C for 40 s, 72 °C for 60 s, and 72 °C for 5 min. The optimal annealing temperature utilized to amplify the MGA_0319 and pvpA genes was 55 °C, to amplify the mgc2 gene an annealing temperature of 58 °C was utilized, and 60 °C was utilized to amplify the gapA gene. PCR products were detected with UV light in a 2 % agarose gel containing 1 µg ethidium bromide ml–1. The amplified gene fragment was sequenced using an Applied Biosystems Prism 377 automated sequencer (PE Applied Biosystems). Each amplification product was sequenced in both directions with the forward and reverse amplification primers (Table 1). Complete overlapping of complementary sequences, editing and consensus construction was performed using the SEQMAN program (in LASERGENE; DNASTAR). Sequencing of all amplification products was performed at the Integrated Biotechnology Laboratories located at the University of Georgia. Sequencing of reference strains and Israeli isolates was also carried out at the Sequencing Unit, Weizmann Institute (Rehovot, Israel).


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Table 1. Location, product sizes and sequence positions for primers used in GTS analysis

 
Sequence stability of targeted genes.
In order to evaluate the stability of the four targeted gene regions, amplification and sequencing was performed for several in vitro passages of reference strains R and S6, vaccine strains F, ts-11 and 6/85, and vaccine strains as obtained from the manufacturers.

RAPD analysis.
The primers used in RAPD analysis were described by Fan et al. (1995). The RAPD reaction conditions were performed as described by Ley et al. (1997a). RAPD pattern analysis was performed by visual comparison using digitally recorded photographs of ethidium bromide stained agarose gel. All RAPD analyses were performed at PDRC University of Georgia. Each RAPD analysis gel was standardized by comparison of the M. gallisepticum unknown isolate to reference strains. Isolates were considered identical when major band differences could not be visualized. RAPD patterns for reference strains and isolates were designated with letters of the alphabet A to Z followed by double letter designations as new patterns appeared during the analysis. RAPD types were not designated with letters F, R and S to avoid confusion with M. gallisepticum reference strains F, R and S6.

GTS analysis.
To conduct GTS analysis all consensus sequences were edited to start at an equivalent coding sequence position using the EDITSEQ program (in LASERGENE; DNASTAR). Nucleotide positions of analysed gene fragments are given in Table 1 based on the genome sequence of M. gallisepticum Rlow strain. Alignments of individual gene and multiple gene sequences were constructed by the CLUSTAL V method with a gap penalty of 10 using the MEGALIGN program (in LASERGENE; DNASTAR). Dendrograms were constructed from the CLUSTAL V alignments by the neighbour joining method and 1000 bootstrap replicate analysis using the MOLECULAR EVOLUTIONARY GENETIC ANALYSIS (MEGA) software available at www.megasoftware.net for individual genes and for multiple genes sequence alignments. Distinct groups of closely related sequences were identified as clusters in the dendrogram and designated with roman numbers (I, II, III). Sequences within a cluster with <100 % nucleotide identity were identified as different sequence types by lower-case letters (a, b, c). For GTS analysis of individual and multiple genes each isolate received a sequence type designation indicating the cluster and sequence type they belong to. Because sequences from the same isolate did not segregate into the same cluster in each individual and multiple gene analysis, the same isolate did not necessarily receive the same cluster designation. The total number of sequence types identified by GTS analysis of individual and multiple genes was estimated and isolates were grouped based on RAPD and GTS types.

Sequences of 67 isolates and 10 reference strains were submitted to GenBank under the following accession numbers: MGA_0319, AY556071–AY556148; gapA, AY556149–AY556226; mgc2, AY556227–AY556304; pvpA, AY556305–AY556382.

Discrimination index of gene size polymorphism, GTS and RAPD analysis.
The overall discriminatory power of a typing method is defined as its ability to distinguish between different strains. This can be expressed as an index that measures the probability that two unrelated strains will be placed into different groups, and is calculated using Simpson's diversity or discrimination (D) index, which takes into account the number of types defined by the method and the relative frequencies of these types (Hunter & Gaston, 1988). A D index of >0·90 is considered adequate, and a D index >0·95 is considered as a good typing discrimination power. For each gene fragment, isolates with identical sequences (100 % identity) were considered as the same sequence type. Discrimination indices were estimated for RAPD and GTS analysis of individual and multiple genes, and for gene size polymorphism of pvpA and mgc2 genes.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Sequence stability of targeted genes
Targeted gene stability was evaluated by sequencing several in vitro passage levels of reference strains R and S6, and vaccine strains F, ts-11 and 6/85. Sequences of reference strain R passaged 13, 73 and 154 times, were 100 % identical for all targeted genes. Likewise, sequences of each of the targeted genes in strain S6, at passage levels 20, 106, 259, were 100 % identical as were sequences in vaccine strain ts-11 passaged 99 times and commercial vaccine 6/85 passaged 29 times. The MGA_0319, mgc2 and pvpA gene sequences of F strain, passaged 16, 124 and 255 times, and the commercial vaccine, were 100 % identical. However, two nucleotide differences (99·4 % identity) were found in the gapA gene of F strain passaged 124 times as compared to F strain passaged 16 and 255 times, and the commercial F strain vaccine. Nucleotide changes of F strain 124 p were located at nucleotide positions 2920 (G->C) and 2928 (A->C) of the gapA genome CDS MGA_0934.

RAPD analysis
A total of 77 isolates of M. gallisepticum, including field isolates and reference strains, were typed by RAPD analysis. As previously reported by Fan et al. (1995) and Ley et al. (1997a, b), M. gallisepticum reference strains, K503CK74 (RAPD type G), K703CK75 (RAPD type H), K730CK75 (RAPD type I) (Fig. 1a, lanes 1 to 3), 6/85 (RAPD type M), HF-51 (RAPD type N), ts-11 (RAPD type L), F (RAPD type B) and R (RAPD type C) (Fig. 1a, lanes 8 to 12), were readily differentiated using Fan primers. RAPD analysis of M. gallisepticum isolates from the USA, Australia and Israel were also typed using the Fan primer set. Eighteen different RAPD types were observed for the fifty-four USA isolates analysed. These were designated RAPD types B, E, J–Q, T–Z and AA. Among the fifty-four USA isolates, three were identified as RAPD type B, characteristic of vaccine strain F; seven were RAPD type M, characteristic of vaccine strain 6/85; and seven were RAPD type L, characteristic of vaccine strain ts-11. The remaining 37 isolates all presented RAPD patterns different from the M. gallisepticum reference strains. Fig. 1(a) shows an example of M. gallisepticum isolates characterized as RAPD types K, W, E and X (lanes 4 to 7), and Fig. 1(b) shows an example of M. gallisepticum isolates characterized as RAPD type O (lanes 16 to 20) and RAPD type Z (lanes 21 to 23). All eight Israeli isolates showed different RAPD types, designated AF to AM. Of the five Australian isolates, four showed unique RAPD types designated AB to AE while strain AU083CK80, the parent strain of ts-11, typed similar to the vaccine strain (RAPD type L). Results of the RAPD typing for the 77 M. gallisepticum isolates are presented in Table 2.



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Fig. 1. RAPD analysis of M. gallisepticum reference strains and field isolates with the primers described by Fan et al. (1995). (a) Lanes 1 to 3, reference strains K503, K703, K730; lanes 4 to 7, isolates K3020TK90, K4705CK99, K435TK73 and K4931TK00, characterized as RAPD types K, W, E and X, respectively; lanes 8 to 12, reference strains 6/85, HF51, ts-11, F and R, with RAPD types M, N, L, B and C; lane 13, negative control; lane 14, molecular mass marker. (b) Lane 15, reference strain HF51; lanes 16 to 20, isolates K4158CTK96, K4110ATK96, K4110BTK96, K4110FTK96 and K4181BTK96, characterized as RAPD type O; lanes 21 to 23, isolates K5029BCK00, K5037ACK00 and K5039HCK00, characterized as RAPD type Z; lanes 24, 25, 26 and 27, reference strains 6/85, t-s11, R and F, respectively; lane 28, negative control; lane 29, molecular mass marker.

 

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Table 2. RAPD and GTS analysis of M. gallisepticum reference strains and field isolates

 
GTS analysis
Dendrograms were constructed from individual and multiple gene sequence alignments. A total of six dendrograms constructed for GTS analysis are available with the online version of this paper. Dendrograms 1-gapA, 2-MGA_0319, 3-mgc2 and 4-pvpA were constructed from individual gene alignments, and dendrograms 5-mgc2/pvpA and 6-gapA/MGA_0319/mgc2/pvpA were constructed from alignments of combined mgc2 and pvpA sequences or all four targeted genes, respectively. Table 2 shows the M. gallisepticum strains and isolates listed by RAPD type with the corresponding sequence types for individual and multiple GTS analysis.

GTS of multiple genes and RAPD analysis enabled correlation of epidemiologically linked isolates. Twenty-three of the fifty-four (43 %) USA isolates were epidemiologically related to several M. gallisepticum poultry outbreaks and were differentiated by both RAPD and GTS analysis. Poultry outbreak related isolates were separated into six RAPD types J, O, P, U, Y and Z, and into ten different sequence types, VIIa, VIIb, XIa, XIb, Xa, XVa, XVb, XIIIa, XIIIb and XIVa, as shown by gapA/MGA_0319/mgc2/pvpA GTS analysis (Table 2).

Sixteen of the fifty-four (30 %) USA field isolates were characterized as similar to vaccine strains F, ts-11 or 6/85 by RAPD (types B, L and M) and GTS analysis of multiple genes (Table 2). Epidemiological evidence indicated that some of the isolates within the RAPD group M (K3944TK95, K5111ATK01) and RAPD group L (K5109BCK01, K5109DCK01, K5155CCK01) originated from flocks vaccinated with 6/85 and ts-11, respectively. However, from the sixteen vaccine-like isolates, three F-strain-like (K4781ATK99, K5058ETK01, K5104TK01), three ts-11-like (K4688CKC, K4688CKF, K4688CKG) and five 6/85-like (K4029TK95, K4043TK95, K4421ATK97, K4423BTK97, K4465TK97) isolates originated from non-vaccinated flocks. GTS analysis revealed that F-strain-like isolates K4781ATK99 and K5058ETK01 exhibited the same nucleotide polymorphism in the gapA gene sequence as F strain p124 (Table 2, sequence types IIa and IIb).

All six house finch isolates (RAPD type N), with the exception of isolate K4409HF97, were identical by RAPD and GTS analysis of individual and multiple genes (Table 2). The only difference between isolate K4409HF97 and the other five finch isolates was a 231 nt deletion in the pvpA gene (sequence type IIIb). Nonetheless this isolate was identified as sequence type Va, similar to the other finch isolates, by GTS analysis of multiple genes (Table 2).

Even though the Israeli isolates did not share 100 % sequence identity with each other, cluster analysis consistently resulted in the segregation of these isolates into a separate cluster away from the USA and Australian isolates. Israeli isolates were differentiated as two sequence types by gapA GTS (VIa and Ib), two sequence types by MGA_0319 GTS (IXa and IXb), two sequence types by pvpA GTS (IXa and IXb), three sequence types by mgc2 GTS (VIIIa, VIIIb, VIIIc), five sequence types by mgc2/pvpA GTS, six sequence types by gapA/MGA_0319/mgc2/pvpA GTS analysis, and eight RAPD types (AF–AM) (Table 2).

The Australian isolates could be differentiated from each other and from the remaining isolates by both RAPD and GTS analysis. Isolate AU083CK80, the parent strain from which the ts-11 vaccine was derived by mutagenesis (Soeripto et al., 1989), was identical to vaccine ts-11 by GTS and RAPD analysis. The remaining four Australian isolates (AU043CK94, AUO22CK96, AU019CK97, AU169CK99) were more similar to isolates from the USA than to the isolates from Israel. The four Australian isolates were readily differentiated by pvpA GTS analysis (types VIIb, IIc, IId, IIe), by mgc2/pvpA (types XId, IVa, VIIIc, VIIId), by gapA/MGA_0319/mgc2/pvpA (types IVc, IIId, VIIIc, VIIId) and by RAPD analysis (RAPD types AB–AE) (Table 2).

Gene size polymorphism analysis
Gene size polymorphism was not detected among the gapA and CDS MGA_0319 amplification products of the 77 M. gallisepticum isolates: all isolates yielded 332 and 590 bp PCR products with the gapA and lp primers, respectively. However, as previously reported (Boguslavsky et al., 2000; Liu et al., 2001; Pillai et al., 2003), the pvpA gene exhibited size polymorphism, with PCR products of 437, 578, 606 and 665 bp detected among M. gallisepticum reference strains and isolates examined (Table 3). The region of the pvpA gene amplified encodes the protein carboxy-terminus, where truncations of the pvpA protein have been reported to be located within the proline-rich direct repeat (DR) (Boguslavsky et al., 2000). Previous sequence analysis of M. gallisepticum reference strains R (RAPD type C) and A5969 (RAPD type A) demonstrated that these two strains possess both complete DR sequences, and yield a 665 bp PCR product. Isolates from the USA, characterized as RAPD types E, J, L and V, and Australian isolates, characterized as RAPD types AC, AD and AE, all produced a 665 bp PCR product where the complete pvpA gene DR region is present, as confirmed by sequencing analysis. Forty-two of the fifty-four USA isolates, characterized as RAPD types D, K, M–Q, T, U, W–Z and AA, and Australian isolate AU043CK94 (RAPD type AB) yield a 606 bp PCR product with the pvpA primers (Table 2). Sequence analysis indicated that all these isolates have a deletion of 59 nt located between the two DR sequences. Gene size polymorphism of the pvpA gene has been previously reported for M. gallisepticum atypical strains K503CK74 (RAPD type G), K703CK75 (RAPD type H) and K730CK75 (RAPD type I) (Boguslavsky et al., 2000). In this study we found that all Israeli isolates (RAPD types AF–AM) share the pvpA size polymorphism with the USA atypical M. gallisepticum strains. However, the full pvpA gene sequence of one Israeli isolate as compared to the USA atypical M. gallisepticum strain K703CK75 indicated that the latter has an additional 3 nt insertion near the DR region (Boguslavsky et al., 2000) not included by this GTS analysis. RAPD type B isolates, including vaccine strain F and house finch isolate K4409HF97 (RAPD type N), are among the other M. gallisepticum strains for which pvpA size polymorphism has been previously reported (Boguslavsky et al., 2000; Liu et al., 2001).


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Table 3. Gene size polymorphism and RAPD analysis of M. gallisepticum isolates

 
Gene size polymorphism was also evident among the mgc2 PCR products (Table 3). The most obvious size polymorphism was observed for RAPD type M isolates, including vaccine strain 6/85, isolate K4236TK96 (RAPD type Q) and isolate AU022CK96 (RAPD type AC); all these isolates produced a 761 bp PCR product with a 63 nt deletion in the DR region of the MGC2 protein carboxy-terminus (Hnatow et al., 1998). Forty-two isolates yielded a PCR product of 854 bp, twenty-three isolates yielded a PCR product of 857 bp, reference strain R yielded a 824 bp PCR product, while isolate K3020TK90 (RAPD type K) yielded a PCR product of 839 bp (Table 3).

In order to determine if size polymorphism of both the pvpA and the mgc2 genes was feasible for typing M. gallisepticum isolates the pvpA/mgc2 PCR product sizes were compared to the corresponding RAPD types (Table 3). A total of 11 pvpA/mgc2 PCR size combination types were identified. Seven isolates showed unique PCR size combinations that correlated with specific RAPD types. These were RAPD type J isolates (K2101CK84, K4385TK97, K4424ATK97, K4902TK00), R-strain, RAPD type K isolate (K3020TK90) and RAPD type AC isolate (AU022CK96). On the other hand, a total of 70 isolates belonging to 32 different RAPD types were distributed within the nine remaining pvpA/mgc2 PCR size types (Table 3).

Discrimination index of gene size polymorphism, GTS and RAPD
To evaluate the capability of gene size polymorphism, GTS and RAPD analysis to discriminate between M. gallisepticum isolates, the discrimination index was estimated for each genotyping method. GTS analysis of individual genes gapA, MGA_0319, mgc2 and pvpA, identified 17, 16, 20 and 22 sequence types, while gene size polymorphism of the pvpA/mgc2 genes identified 11 types. D index values of 0·713 (GTS gapA), 0·874 (GTS MGA_0319), 0·915 (GTS mgc2), 0·920 (GTS pvpA) and 0·854 (pvpA/mgc2 PCR size) were estimated for GTS analysis of individual genes and for gene size polymorphism (Table 4). The estimated discrimination indices indicated that individual GTS analysis of mgc2 and pvpA genes provided adequate discriminatory power. However, GTS analysis of multiple genes provided better discriminatory power. GTS analysis of mgc2/pvpA identified 38 sequence types and GTS analysis of gapA/MGA_0319/mgc2/pvpA identified 40 different sequence types from a total of 77 sequences. Discrimination indices of 0·962 (GTS mgc2/pvpA) and 0·965 (GTS gapA/MGA_0319/mgc2/pvpA) were estimated for GTS of multiple genes. Genotyping by RAPD analysis identified 36 different pattern types with a D index of 0·958 (Table 4).


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Table 4. Comparison of RAPD, GTS and gene size polymorphism genotyping methods of M. gallisepticum

 
Overall, the discriminatory power of the GTS method using multiple genes was slightly higher than the discriminatory power of the RAPD method. However, not all M. gallisepticum isolates required GTS analysis of multiple genes in order to be genotyped. Thirty-six, thirty-two, twenty-one and fourteen of the seventy-seven isolates were readily differentiated by GTS analysis of individual pvpA, mgc2, MGA_0319 and gapA genes.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Reliable methods for the differentiation of M. gallisepticum strains play a pivotal role in understanding the epidemiology and spread of the disease because they generate the information necessary to identify and track new outbreaks. Ideally, methods of strain differentiation must have high enough discriminatory power to clearly differentiate unrelated strains, as well as to demonstrate the relationship of isolates from individuals infected through the same source. Genotyping methods should also have a high degree of reproducibility, which is particularly important during construction of reliable databases. Furthermore, genotyping methods should be easy to interpret and rapid to perform. Progress in the molecular biology of M. gallisepticum (Razin et al., 1998) and the availability of the complete genome sequence (Papazisi et al., 2003) have driven the idea to evaluate GTS as a typing tool for differentiating M. gallisepticum strains.

This study focused on the evaluation of GTS analysis of three known surface-protein genes (gapA, mgc2 and pvpA), and one predicted surface lipoprotein (MGA_0319) as a method for genotyping M. gallisepticum strains. Sequencing of targeted genes from selected in vitro passages of M. gallisepticum strains (R, F, S6, ts-11, 6/85) indicated that the gene regions analysed in this study were stable within passages of the same strains. Overall the gapA and MGA_0319 gene sequences were more conserved than the mgc2 and pvpA gene sequences. The genetic variability of the pvpA gene has been previously documented (Boguslavsky et al., 2000; Liu et al., 2001; Pillai et al., 2003); however, the genetic variability of the mgc2 gene has not been previously addressed.

In addition to nucleotide sequence variability, gene size polymorphism of the pvpA and mgc2 genes was observed among M. gallisepticum strains. Notably, variation in the sequences and size of the pvpA and mgc2 genes was mostly located in the proline-rich, surface-exposed carboxy-terminal-encoding region of both genes. Size polymorphism of both pvpA and mgc2 genes was evaluated as a possible strategy to differentiate among M. gallisepticum isolates but only seven isolates showed unique PCR size combinations. Furthermore, short nucleotide insertions/deletions were difficult to detect on ethidium bromide stained agarose gels and sequencing of the PCR products was ultimately required. Therefore differentiation of isolates based on PCR size polymorphism of the pvpA and mgc2 genes was not considered as a reliable method to differentiate among M. gallisepticum isolates.

Sequence analysis of the four target genes allowed the establishment of sequence clusters that included USA isolates from known poultry outbreaks, isolates closely related to vaccine strains, and isolates from finches. Australian isolates separated into different gene clusters together with USA isolates, while Israeli isolates separated into a complete distinct cluster separate from the USA and Australian isolates.

Some USA isolates were identified as vaccine strains F, ts-11 and 6/85 by GTS analysis of multiple genes. In particular 6/85-like isolates from turkeys have been extensively genotyped by GTS analysis of the same genes presented in this study, and by RAPD analysis using four different primer sets (Kleven et al., 2004). Whether these isolates are 6/85-derived vaccine subpopulations or isolates closely related to the vaccine that evolved independently in the field is still not clear. To precisely determine the relation of these vaccine-like isolates to the commercially available vaccines, complete vaccine genome sequences and further analysis of the genetic stability of live M. gallisepticum vaccines in the field are needed.

Six house finch isolates were characterized as identical by RAPD and GTS analysis of individual and multiple genes indicating genetic homogeneity among these isolates, as previously reported (Ley et al., 1997a). This result is in contrast to findings in a recently reported study where 55 house finch isolates, with similar RAPD type, were grouped into 16 different genotypes by sequence analysis of the pvpA gene (Pillai et al., 2003). This result indicated that genetic variability among house finch isolates does exist, and this was recognized by examining a larger population of isolates. The identification of turkey isolate K5054TK01 as identical to the house finch isolates verified our earlier reports of a naturally occurring house-finch-like isolate in a turkey breeder flock (Ferguson et al., 2003). This isolate has been characterized by its low pathogenicity in turkeys and chickens, and its potential as a live M. gallisepticum vaccine for turkeys and chickens has been evaluated (Ferguson et al., 2004).

Overall, GTS analysis of multiple surface-protein genes was demonstrated to have better discriminatory power than RAPD analysis. The discriminatory power hierarchy, from highest to lowest, for GTS analysis of individual genes was ranked as: pvpA, mgc2, MGA_0319 and gapA. In addition to its proven discriminatory power, the inter-laboratory reproducibility of GTS analysis was validated by comparing sequence results from the M. gallisepticum reference strains and the Israeli isolates obtained independently in the laboratories in Israel and the USA.

The identification of gene polymorphism in bacteria by nucleotide sequence analysis of genes encoding antigenic surface proteins jointly with genes encoding housekeeping proteins has been proven useful in the surveillance of pathogenic bacteria (Byun et al., 1999; Maiden et al., 1998; van Loo et al., 2002; Kotetishvili et al., 2003). Particularly multilocus sequence typing (MLST) schemes where data can be stored, analysed and queried through a central web server have permitted local and global epidemiology studies of several infectious agents (Chan et al., 2001).

In this study we have demonstrated that GTS analysis of M. gallisepticum surface-protein genes is a reproducible typing method with satisfactory discriminatory power to separate isolates from unrelated outbreaks and to identify closely related isolates. Further development of a M. gallisepticum GTS database, including sequences from surface proteins as well as housekeeping genes, will provide a global alternative for typing M. gallisepticum isolates, and a resource to understand the evolutionary relationships of this poultry pathogen.


   ACKNOWLEDGEMENTS
 
This research was supported by the USA-Israel Agricultural Research and Development Fund (BARD) Grant IS-3126-99. We thank Sylva M. Riblet, Savitha Subramanian, Jennifer Shuster, Marta Jaramillo, Bill Hall, Ruth Wooten and Victoria Leiting for their excellent technical assistance and significant contribution making this study possible, and Dr Alejandro Banda for his valuable help with the sequence cluster analysis.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 21 September 2004; revised 3 January 2005; accepted 17 February 2005.



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