Genetic characterization of three unique operational taxonomic units of Eimeria from chickens in Australia based on nuclear spacer ribosomal DNA

Available online at www.sciencedirect.com

Veterinary Parasitology 152 (2008) 226–234 www.elsevier.com/locate/vetpar

Genetic characterization of three unique operational taxonomic units of Eimeria from chickens in Australia based on nuclear spacer ribosomal DNA§ Cinzia Cantacessi a, Shane Riddell b, Genevieve M. Morris a, Timothy Doran b, Wayne G. Woods a, Domenico Otranto c, Robin B. Gasser a,* b

a Department of Veterinary Science, The University of Melbourne, 250 Princes Highway, Werribee, Victoria 3030, Australia Commonwealth Scientific and Industrial Research Organisation, Australian Animal Health Laboratory, Geelong, Victoria 3220, Australia c Department of Veterinary Public Health, Faculty of Veterinary Medicine, Valenzano, 70010 Bari, Italy

Received 31 October 2007; received in revised form 12 December 2007; accepted 18 December 2007

Abstract Coccidiosis of chickens is one of the commonest and economically most important parasitic diseases of poultry worldwide. Given the limitations of traditional approaches, molecular tools have been developed for the specific diagnosis of coccidiosis. Recently, a polymerase chain reaction (PCR)-based capillary electrophoresis (CE) method, employing genetic markers in the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA, was established for both analytical and diagnostic purposes. The application of this method to investigate the epidemiology of coccidiosis and genetic structures of Eimeria populations on commercial chicken establishments has discovered genetic variants of Eimeria (i.e., new operational taxonomic units OTU-X, OTU-Y and OTU-Z) which were (based on CE analysis) distinct from those of species of Eimeria identified previously in chickens in Australia. The present characterization of these OTUs, based on their ITS-2 sequences and phylogenetic analyses of selected sequence data, provides first evidence to support that OTU-X represents a population variant of Eimeria maxima, and that OTU-Y and OTU-Z represent cryptic species of Eimeria. Further biological and genetic studies are needed to rigorously test these proposals and establish the specific status of these OTUs and their importance as pathogens in chickens. An understanding of the epidemiology of these population variants or cryptic species in Australia is central to designing and implementing effective vaccination and control strategies. # 2008 Elsevier B.V. All rights reserved. Keywords: Eimeria; Chicken; Coccidiosis; Polymerase chain reaction (PCR); Capillary electrophoresis; Nuclear ribosomal DNA; Operational taxonomic unit (OTU)

1. Introduction

§

Note: Nucleotide sequence data reported in this paper have been deposited in the GenBankTM database under accession numbers AM922227–AM922258. * Corresponding author. Tel.: +61 3 97312000; fax: +61 3 97312366. E-mail address: [email protected] (R.B. Gasser). 0304-4017/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2007.12.028

Coccidiosis of chickens is an enteric disease caused by one or more species of Eimeria (Protozoa: Apicomplexa: Eimeriidae) and is one of the commonest and economically most important diseases of poultry worldwide. This disease causes annual production losses estimated at billions of dollars, as well as high morbidity (due to acute, bloody enteritis) and mortality

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rates (e.g., Shirley et al., 2005). While the control of coccidiosis has relied mainly on the preventative use of anticoccidial drugs (coccidostats), together with the induction of species-specific natural immunity in chicken flocks (Shirley et al., 2004, 2005), this broadly used approach is costly and has precipitated severe problems with drug resistance in Eimeria populations (Williams, 1998). Consequently, live, virulent vaccines have been utilized to protect chicken flocks against coccidiosis, particularly in intensive establishments (Williams, 2002). Also, attenuated or precocious, live vaccines are now finding widespread application in many countries (Shirley et al., 2004, 2005). In addition to the use of preventative chemotherapy and/or vaccination, specific diagnosis plays a critical role in the control and surveillance of coccidiosis (Morris and Gasser, 2006). Traditionally, diagnosis has been achieved by detecting and/or enumerating Eimeria oocysts excreted in the faeces from infected chickens and by measuring oocyst/sporocyst dimensions, and (at necropsy) assessing the site and extent of the pathological lesions caused by Eimeria in the intestine of chickens (Long and Joyner, 1984). However, these approaches are unreliable, particularly given that multiple species of Eimeria can simultaneously infect the chicken host and because there can be an ‘‘overlap’’ in the sizes of oocysts and in the affected regions of the intestines for some species (Long and Joyner, 1984). Therefore, molecular tools have been developed for the specific diagnosis of coccidiosis and the analysis of genetic structures of Eimeria populations (Morris and Gasser, 2006). Recently, a practical, rapid and low-cost PCR-based capillary electrophoretic (CE) method, employing genetic markers in the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA, was established for the specific diagnosis of coccidiosis in chickens (Gasser et al., 2005). The method employs a single pair of primers in a standard PCR for the simultaneous identification of all recognized species of Eimeria of chickens by electrophoresis in an automated electrophoresis apparatus (Gasser et al., 2005). Given its diagnostic and analytical characteristics, the CE-based approach has been employed effectively to conduct epidemiological surveys and to explore, for the first time, the abundance/intensity and dynamics of Eimeria infections in poultry establishments in Australia (Morris et al., 2007a,b), thus logically complementing prevention, vaccination and control programmes. In addition, given its analytical capacity, the application of the CE method has also provided detailed insights into the genetic make-up of Eimeria populations in selected poultry establishments in Australia. Importantly, in

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addition to the seven recognized species of Eimeria of chickens, two genetic variants (operational taxonomic units, OTU-X and OTU-Y) of Eimeria have been recorded (Morris et al., 2007a) and, more recently, a third (OTU-Z) was discovered (W. Woods, unpublished data) using the CE method. Extending recent findings, the aims of the present study were to (i) sequence the ITS-2 region and investigate sequence heterogeneity within this region for each of these three OTUs, (ii) compare the sequences representing each OTU with the ITS-2 sequences representing the seven recognized species of Eimeria (i.e., E. acervulina, E. brunetti, E. maxima, E. mitis, E. necatrix, E. praecox and E. tenella) of chickens in Australia, and (iii) initially test the proposal that each of the OTUs represents a new species using the ITS-2 sequence data sets. 2. Materials and methods 2.1. Propagation of Eimeria Each isolate of each of the seven species and three OTUs (OTU-X, OTU-Y and OTU-Z) of Eimeria was isolated and passaged separately in specific pathogen free (SPF) chickens (6 weeks of age), maintained in custom-built isolators under stringent conditions to prevent cross-contamination, as described previously (Woods et al., 2000). Originally, individual species of Eimeria were identified based on the morphometry of sporulated oocysts, prepatent period, location of gross lesions in the intestine(s) and/or sequencing of the 18S rRNA (Woods et al., 2000). For the present study, the identity of each species and OTU of Eimeria was defined (upon isolation) and continually verified (during passage) based on its electrophoretic profile from the CE analysis of oocyst samples (see below). An oocyst sample was considered monospecific or pure only if its CE profile matched precisely its reference profile defined previously (cf. Gasser et al., 2005; see Fig. 1). The following monospecific isolates of Eimeria from Queensland or Victoria, Australia, were employed in this study: A14 and A34 (E. acervulina), B16 (E. brunetti), M6 and M19 (E. maxima), m10 (E. mitis), N42 (E. necatrix), P11 (E. praecox) and T8 (E. tenella); single samples of OTU-X and OTU-Y both originated from Nagambie (Victoria), and OTU-Z was from Kelinda (New South Wales) (Table 1). 2.2. Isolation of genomic DNA Eimeria oocysts were isolated from individual faecal samples from chickens by flotation on saturated NaCl

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Fig. 1. Profiles from automated capillary electrophoretic (CE) analysis of ITS-2 amplicons derived from (monospecific) isolates of Eimeria acervulina, E. brunetti, E. maxima, E. mitis, E. necatrix, E. praecox and E. tenella from chickens in Australia. Dark-shaded areas represent the diagnostic peaks for each of the species. Profiles (pX, pY and pZ) representing unique operational taxonomic units of Eimeria (i.e., OTU-X, OTU-Y and OTU-Z, respectively) are displayed. Size in bp indicated at the top of the image.

(specific gravity 1.2), washed twice by centrifugation (1500  g) in 10 ml volumes of water and made up to a final aqueous suspension (2 ml volume). The oocysts were then purified over a sucrose-gradient (see Gasser et al., 1987) to remove faecal components inhibitory to the PCR, washed and then resuspended in 0.5 ml of water. The same volume of glass beads (0.5 mm in diameter, Sigma) was added and the tube vortexed vigorously for 5 min to disrupt the oocysts. Proteinase K (150 mg/ml) and sodium dodecyl-sulphate (5%, w/v) were added, the tube was incubated at 37 8C for 12–16 h and then vortexed again. After centrifugation at 12,000  g for 5 min, genomic DNA was columnpurified from the supernatant (Wizard DNA Clean-Up, Promega, USA) and quantified using a spectrophotometer (NanoDrop Technologies, USA). 2.3. PCR-coupled capillary electrophoretic (CE) analysis The CE method was carried out essentially as described recently (Gasser et al., 2005). In brief, the ITS-2 region (plus flanking sequence; 330–580 bp, depending on species) was PCR-amplified from individual genomic DNA samples using oligonucleotide primers WW2 (forward: 50 -ACGTCTGTTTCAGTGTCT-30 ; 50 -endlabelled with the fluorescent dye 5-carboxyfluorescein, FAM) and WW4r (reverse: 50 -AAATTCAGCGGGTAACCTCG-30 ; unlabelled) designed specifically to the 5.8S and 28S rRNA gene

sequences of members of the genus Eimeria (Woods et al., 2000). Primer WW4r is specific for the family Eimeriidae, and WW2 is specific for the genus Eimeria. PCR was performed in 25 ml volumes containing 50 pmol primer, 200 mM of each dNTP, 3.5 mM MgCl2 and 1 U GoTaq DNA polymerase (Promega, USA). Approximately 10–20 ng of genomic DNA were added to the PCR, and samples without DNA were also included as negative controls. The following thermocycling conditions were used: 94 8C, 30 s (denaturation); 55 8C, 30 s (annealing); 72 8C, 60 s (extension) for 35 cycles in a thermal cycler (480, PerkinElmer, USA). The quality and amount of individual amplicons were verified on ethidium bromide-stained agarose gels (Gasser et al., 2005). The amplicons were then diluted 1/30 with water, and 1 ml of each mixed with 10 ml of loading solution containing Hi Di Formamide and the LIZ 500 size standard (1:0.006 ratio) (Applied Biosystems, USA). Samples were denatured at 95 8C for 5 min, and 1 ml volumes electrokinetically injected into POP-7TM polymer matrix capillaries at 2 kV for 10 s and run in a 3730 DNA Analyzer (Applied Biosystems, USA) at 230 V for 1 h. Electrophoretic profiles for individual samples were captured, and the program Genemapper (v.3.7, Applied Biosystems, USA) employed to automatically process and analyse the chromatograms. Flagged data were then examined manually, and the final selections exported into a file in the program Excel1 (Microsoft Office Professional Edition 2003). Each of the seven recognized species

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Table 1 Summary of ITS-2 sequence data derived from reference isolates representing all recognized species of Eimeria (cf. Gasser et al., 2005) and three operational taxonomic units (OTUs; OTU-X, OTU-Y and OTU-Z), defined based on capillary electrophoretic profiles (see Fig. 1) Eimeria

Isolates (number of Length clones sequenced) (bp) and geographical origin

Eimeria A14 (14) acervulina Queensland A34 (12) Queensland

Number of sequence types (average p-distance)

350–356 2 (0.01) 350–356 2 (0.01)

Sequence type G + C Variable Singleton Informative Transition/ Accession (number of clones content sites sites sites transversion number with this type; ratio length in bp) Ea1 Ea2 Ea1 Ea2

(10; 350) (4; 356) (9; 350) (3; 356)

38.8 38.8 38.8 38.8

28

–

–

1.61

28

–

–

1.61

AM922227 AM922228 AM922227 AM922228

Eimeria brunetti

B16 (16) Queensland

373–413 3 (0.22)

Eb1 (6; 413) Eb2 (6; 373) Eb3 (4; 404)

32.5 44.0 43.3

159

154

5

1.71

AM922229 AM922230 AM922231

Eimeria maxima

M6 (14) Victoria

298–317 5 (0.18)

307) 298) 317) 309) 302) 307) 298) 317) 309) 302)

41.5 43.5 41.8 41.6 43.3 41.5 43.5 41.8 41.6 43.3

90

26

1.40

298–317 5 (0.18)

(2; (2; (3; (3; (4; (2; (2; (2; (2; (6;

116

M19 (14) Victoria

Ema1 Ema2 Ema3 Ema4 Ema5 Ema1 Ema2 Ema3 Ema4 Ema5

116

90

26

1.40

AM922232 AM922233 AM922234 AM922235 AM922236 AM922232 AM922233 AM922234 AM922235 AM922236

(2; (2; (2; (5;

398) 408) 426) 415)

38.7 39.4 37.3 42.9

97

84

9

1.21

AM922237 AM922238 AM922239 AM922240

Eimeria mitis m10 (11) Queensland

398–426 4 (0.12)

Emi1 Emi2 Emi3 Emi4

Eimeria necatrix

N42 (13) Victoria

528–538 3 (0.02)

En1 (2; 538) En2 (8; 528) En3 (3; 531)

46.5 46.6 45.9

27

27

–

2.18

AM922241 AM922242 AM922243

Eimeria praecox

P11 (10) Victoria

425–462 2 (0.02)

Ep1 (3; 462) Ep2 (7; 425)

35.3 35.7

25

–

–

1.55

AM922244 AM922245

Eimeria tenella

T8 (18) Queensland

511–514 3 (0.01)

Et1 (6; 514) Et2 (5; 512) Et3 (7; 511)

47.7 47.3 47.3

11

11

–

1.38

AM922246 AM922247 AM922248

OTU-X

OTU-X (17) Victoria

312–327 4 (0.18)

OTUx1 OTUx2 OTUx3 OTUx4

312) 319) 323) 327)

42.3 38.2 40.8 41.8

110

100

7

1.56

AM922249 AM922250 AM922251 AM922252

OTU-Y

OTU-Y (18) Victoria

357–426 3 (0.21)

OTUy1 (6; 357) OTUy2 (5; 360) OTUy3 (7; 426)

46.7 46.4 37.7

114

114

–

1.34

AM922253 AM922254 AM922255

OTU-Z

OTU-Z (21) New South Wales

439–451 3 (0.08)

OTUz1 (11; 439) 35.1 OTUz2 (6; 437) 35.2 OTUz3 (4; 451) 34.8

51

51

–

1.58

AM922256 AM922257 AM922258

(7; (2; (2; (6;

Numbers of clones sequenced, and lengths and characteristics of the sequences as well accession numbers are listed.

was identified based on the diagnostic peaks defined previously (Gasser et al., 2005), with minor adjustment due to the use of a different analyser (Morris et al., 2007b). Each peak represented the position of the sense-strand for each denatured amplicon. The intensity scale (vertical axis) of the

chromatograms was adjusted, such that background fluorescence was displayed; hence, no peaks remained undetected. All data were stored in Excel1 spreadsheets. The smallest amount of genomic template from which the ITS-2 can be amplified and which was detectable on an agarose gel has been estimated at

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5–10 pg (equivalent to 5–50 oocysts) (Woods et al., 2000). 2.4. Sequencing of the ITS-2 nuclear ribosomal DNA region and analyses of sequence data For each species and OTU of Eimeria, the ITS-2 region was amplified by the PCR from 25 to 50 ng of genomic DNA from individual oocyst isolates, cloned and sequenced. Amplicons produced using unlabelled WW2 and WW4r primers using the Expand High Fidelity PCR system (Roche Pty Ltd., Australia) to minimize PCR-induced errors. In brief, the PCR was conducted in 50 ml consisting of 50 pmol of each primer, 200 mM of each dNTP, 2.5 mM MgCl2 and 1 U Expand High Fidelity Polymerase mix (Roche Pty Ltd., Australia). Purified amplicons were then cloned into the plasmid vector pGEM-T1 Easy (Promega, USA). Escherichia coli (strain JM109) (108 colony forming units/mg) was transformed with recombinant plasmids by heat shock and then grown overnight at 37 8C on Luria Bertani (LB) plates containing 10 mg/ml ampicillin, 0.5 mM isopropyl-b-D-thiogalactopyranoside (IPTG) and 80 mg/ml X-gal (5-bromo-4-chloro-3indolyl-b-galactosidase) for subsequent blue/white colony screening. Plasmid DNA was isolated from recombinant clones and column-purified (Wizard1, Promega, USA) from overnight liquid cultures, and inserts sequenced in both directions using vector oligonucleotide primers (M13 and SP6), employing Big Dye Terminator v.3.1 chemistry in an automated sequencer (AB3730xl, Applied Biosystems, USA). For each species and OTU, 10–21 clones were sequenced per amplicon; an ITS-2 sequence type was defined only if it occurred multiple times for each species and isolate. Sequences were aligned using ClustalW (http://www.ebi.ac.uk/clustalw/) (Thompson et al., 1994) and adjusted manually. The ITS-2 sequences representing each of the seven species and three OTUs of Eimeria known to infect chickens in Australia have been deposited in the GenBank under accession numbers AM922227–AM922258. For individual species and OTUs, all sequences were aligned and compared using the program MEGA v3.1 (Kumar et al., 2004). Pairwise comparisons of nucleotide variation ( p-distance) were made using the formula p-distance = 1  (M/L) (Chilton et al., 1995), where M is the number of alignment positions at which the two sequences have a base in common, and L is the total number of alignment positions over which the two sequences are compared. Also, numbers of variable, singleton and parsimony-informative sites as well as the

transition/transversion (s/v) ratios were determined by pairwise comparison among the sequences representing each of the species or OTUs. Phylogenetic analyses of sequence data were conducted using the neighbor-joining (NJ) and maximum parsimony (MP) methods, employing PAUP* v4.0b10 (Swofford, 2002), as described recently (Hu et al., 2005). Characters were weighted equally and treated as unordered. An heuristic search with tree bissection-reconnection (TBR) branch swapping was used to infer the shortest trees. The length, consistency index, excluding uninformative characters, and retention index of the most parsimonious trees were recorded. A bootstrap analysis (using 1000 replicates) was conducted using heuristic searches and TBRbranch swapping with the MulTrees option to determine the relative support for clades in the consensus tree. An independent phylogenetic analysis of the same data was carried out using Bayesian inference, employing the program MrBayes v3.1.2 (Ronquist and Huelsenbeck, 2003). Each analysis was run for 2 million generations, with default prior probabilities and topologies being sampled every 100th generation. The consensus tree was constructed using the ‘sumt’ command with the variables burnin = 2000. Nodal support was assessed based on consensus posterior probabilities. 3. Results The CE profiles representing individual species and OTUs are shown in Fig. 1. The profile pX (representing OTU-X) was similar to that of E. maxima and consisted of two dominant peaks; band migration was retarded with respect to the E. maxima profile. Profiles pY (OTUY) and pZ (OTU-Z) contained bands that were within the size range of bands displayed for E. brunetti, E. praecox, E. acervulina and E. mitis (see Fig. 1). ITS-2 inserts from a total of 178 clones representing the seven species and three OTUs of Eimeria were sequenced and the sequence data subjected to analyses (Table 1). For each species and OTU, there was significant sequence heterogeneity in the ITS-2 within an isolate. For each E. acervulina and E. maxima, there was concordance in sequence types between the two amplicons sequenced (see Table 1). The number of ITS2 sequence types per isolate, sequence lengths, mean G + C content, number of variable, singleton and informative sites, pairwise distance ( p-distance) and transition/transversion ratio (s/v) calculated for each species or OTU are given in Table 1. The total number of ITS-2 sequence types determined per species ranged

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from two (for E. acervulina and E. praecox) to five (for E. maxima) (Table 1). Sequence lengths varied from 298 bp (E. maxima) to 538 bp (E. necatrix), and the mean G + C content ranged from 35.1% (within OTU-Z) to 47.4% (within E. tenella). The average p-distance ranged from 0.01 for E. tenella to 0.22 for E. brunetti, and the overall s/v from 1.21 in E. mitis to 2.18 in E. necatrix (Table 1). For the OTUs, four main sequence types (312–327 bp; mean G + C content of 40.8%) were recorded for OTU-X, whereas three were determined for both OTU-Y (357–

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426 bp; mean G + C content of 43.3%) and OTU-Z (439–451 bp; mean G + C content of 35%) (see Table 1). The average p-distance within each OTU was 0.18 (OTU-X), 0.21 (OTU-Y) and 0.08 (OTU-Z) (cf. Table 1). Pairwise comparisons of sequence variation in the ITS-2 region (for sequences which could be aligned with confidence) revealed that the sequence types of OTU-X were most similar (average p-distance: 0.19) to those of isolate M6 representing E. maxima (Table 1 and Fig. 2). The NJ and MP analyses of the these sequence

Fig. 2. Alignment of the five ITS-2 sequence types (Ema1–Ema5; accession numbers AM922232–AM922236) determined for E. maxima and of the four types (OTUx1–OTUx4; accession numbers AM922249–AM922252) for OTU-X. A dot indicates a position identical to that in the top sequence, and a dash indicates a deletion/insertion event.

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data (accession numbers AM922249–AM922252 and AM922232–AM922236), with the sequence types representing E. acervulina as the outgroup, both showed (with strong bootstrap support) that sequence type OTUx2 clustered directly with sequence type Ema5, whereas sequence types OTUx1, OTUx3 and OTUx4 grouped to the exclusion of other sequence types of isolate M6 (Fig. 3). The topologies of the NJ and MP trees (which were the same) were concordant with that inferred using the Bayesian method, with node supports of 0.86–1.00 (see Fig. 3). In contrast to the findings for OTU-X, none of the sequence types of OTU-Y or OTUZ could be aligned with confidence with the sequence types representing any of the seven recognized species of Eimeria, thus preventing phylogenetic analyses. While selected tracts in the ITS-2 sequences representing OTU-Y had some similarity to those of E. brunetti (data not shown), the average p-distance (0.33) among sequence types of this species or OTU-Y was considered as too great for the data to be subjected to phylogenetic analyses.

Fig. 3. The neighbor-joining (NJ) tree based on the analysis of ITS-2 sequence data for OTU-X (i.e., OTUx1–OTUx4) and representative data for E. maxima (i.e. Ema1–Ema5), employing E. acervulina (i.e., Ea1–Ea2) as the outgroup. Bootstrap values obtained using the NJ and the maximum parsimony (MP) methods as well as nodal support obtained using the Bayesian method are indicated at the first, second and third positions (in round brackets). Accession numbers of the sequences used in the analyses are indicated to the right (in brackets).

4. Discussion In molecular-epidemiological surveys of various poultry establishments for coccidiosis in 2006, three new OTUs of Eimeria were detected which were each distinct in CE profile from any of the seven recognized species recorded in Australia (see Morris et al., 2007a; W. Woods, unpublished data). The CE profiles representing these OTUs (OTU-X, OTU-Y and OTUZ) were designated pX, pY and pZ, respectively (see Fig. 1). One or more of these profiles have since been detected on different establishments at different time points of the year in the states of Victoria and New South Wales in Australia (W. Woods, unpublished data). Oocysts from chickens known to be infected with OTU-X, OTU-Y or OTU-Z were isolated from faecal samples. Each OTU was then propagated by experimental infection and serial passage in SPF chickens. The location of each OTUs was in the upper small intestine (W. Woods, unpublished data). The shedding of Eimeria oocysts in the faeces commenced 130, 132 and 140 h after oral inoculation of the chickens with OTU-X, OTU-Y and OTU-Z, respectively. Thereafter, oocysts were isolated from faecal samples from these experimentally infected chickens and subjected to CE analysis. The CE profile of each oocyst isolate taken after each propagation or passage was identical to that of the original oocyst isolate used for the first inoculation. The profile pX (representing OTU-X) was similar to that of E. maxima and consisted of two dominant peaks; band migration was retarded with respect to the profile for E. maxima. Profiles pY (OTUY) and pZ (OTU-Z) contained bands that were within the range of E. brunetti, E. praecox, E. acervulina and E. mitis. However, these three profiles did not match any of profiles determined previously in Australia for the seven recognized species (Gasser et al., 2005). The ‘genetic uniqueness’ of OTU-X, OTU-Y and OTU-Z was independently verified using an optimized DNA fingerprinting approach (unpublished). Having demonstrated the uniqueness of OTU-X, OTU-Y and OTU-Z based on CE profiles (Fig. 1), the next step was to characterize these OTUs by their ITS-2 sequences and to compare them genetically with the seven recognized species of Eimeria from chickens. The ITS-2 of nuclear ribosomal DNA was used, because it provides the specific identification of Eimeria from chickens based on length in CE analysis and sequence types (Woods et al., 2000; Gasser et al., 2001, 2005). Consistent with the CE profiles (see Fig. 1), sequencing revealed that each of the three OTUs had multiple, dominant ITS-2 sequence types (Table 1). Four main

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sequence types (312–327 bp; mean G + C content of 41%) were recorded for OTU-X, whereas three were determined for both OTU-Y (357–426 bp; mean G + C content of 43%) and OTU-Z (439–451 bp; mean G + C content of 35%) (see Table 1). The average p-distance within each OTU was 0.18 (OTU-X), 0.21 (OTU-Y) and 0.08 (OTU-Z) (see Table 1). Pairwise comparisons of sequence variation in the ITS-2 showed that the sequence types of OTU-X were most similar (average p-distance: 0.19) to those of E. maxima and could be aligned with confidence with the latter. The phylogenetic analyses of the sequence data for OTU-X with selected sequences representing E. maxima from Australia and the outgroup (E. acervulina; accession numbers AM922227–AM922228) showed (with strong bootstrap support) that some sequence types clustered with E. maxima sequences, whereas others grouped to their exclusion (Fig. 3). By contrast, the sequence types representing OTU-Y could not be aligned with any confidence (i.e., homologous characters could not be defined with confidence) with any of the sequence types representing any of the seven species of Eimeria; those of OTU-Z showed no sequence similarity whatsoever to any other sequence of any other species. Therefore, it was not possible to conduct such an analysis to assess the relationships of these two OTUs. The recent discovery of OTU-X, OTU-Y and OTU-Z was possible because the oligonucleotide primer pair WW2–WW4r is specific in the PCR for the family Eimeriidae or the genus Eimeria (Woods et al., 2000). These OTUs are not necessarily linked to a specific geographical location, as they have been detected recently and independently in a number of commercial chicken production establishments in Victoria, New South Wales and/or Western Australia (W. Woods, unpublished data). In a recent study of a persistent coccidiosis problem (Morris et al., 2007a), OTU-Y was a predominant entity. Its prevalence increased to prominence after the treatment with an anticoccidial drug (Baycox1, Bayer), which may suggest that this species may have propagated opportunistically after the drug-induced decrease or removal of other Eimeria species from chickens. The detection of such new OTUs raises some concerns regarding the effectiveness of commonly used, traditional diagnostic approaches, and leads to an increased awareness of their potential disease and economic importance. While there is presently no detailed information on the biology of these new OTUs, preliminary infection experiments of each of them indicate clear distinctiveness in biology from the seven currently recognized species of Eimeria (as defined by their ITS-2 CE profiles; Gasser et al.,

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2005) (W. Woods, unpublished data). The molecular findings of the present study support the hypotheses that OTU-X represents a population variant of E. maxima and that OTU-Y and OTU-Z represent cryptic species of Eimeria. Further testing of these hypotheses through detailed genetic (using independent, less variable genetic markers) and biological investigations (life cycle, epidemiology and pathogenesis) of OTU-X, OTU-Y and OTU-Z will establish their specific status and importance as pathogens in chickens. Also, future work should include a systematic evaluation as to whether current vaccines (e.g., Eimeriavax1, Bioproperties, Pty Ltd., Australia) protects chickens against challenge infection with each of the OTUs. The discovery of these new OTUs demonstrates the advantages and benefit of the analytical/diagnostic CE approach. Since this technique does not rely on clinical signs for diagnosis, even relatively non-pathogenic species can be detected; this is important under intensive farming conditions, because all species may have a negative effect on growth rate and feed conversion efficiency (Gore and Long, 1982; FitzCoy and Edgar, 1992; Williams, 1998), particularly in chickens with mixed-species infections. While the development and preventative administration of anticoccidial drugs to control coccidiosis has facilitated modern, intensive farming of poultry (Chapman, 1997; Williams et al., 1999), there are significant problems with drug resistance and growing concerns regarding the use of drugs in food-producing animals (e.g., banning of the use of preventative anticoccidials in the European Union; see Bedford, 2000). In the future, a clear understanding of the biology and epidemiology of each species, population variant and cryptic species of Eimeria will be central to the design and implementation of effective vaccination and control strategies, particularly in a situation in which anticoccidial drugs are not permitted to be used preventively. More extensive surveys of different flocks in different poultry establishments using the CE approach should provide insights into the dynamics of Eimeria populations and coccidiosis, assisting or underpinning future prevention and control programmes. Acknowledgements Funding to RBG for project 03-15 from the Australian Poultry Cooperative Research Centre (CRC) is gratefully acknowledged. CC thanks Drs. Nathan Bott and Min Hu for assistance with phylogenetic analyses. SR was supported by an Australian Poultry CRC scholarship.

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