WO1998053074A1 - Large subunit ribosomal dna of neospora species - Google Patents

Abstract

An isolated nucleic acid molecule encoding the large subunit (LSU) ribosomal DNA (rDNA) of Neospora spp, the nucleic acid molecule having a sequence substantially as set out in Figure 2, or functionally equivalent sequence thereof, or a sequence that hybridises thereto; and methods of detecting Neospora spp using polymerase chain reaction (PCR) amplification.

Description

Large subunit ribosomal DNA of Neospora species

Technical Field

The present invention is directed to the large subunit (LSU) ribosomal DNA of Neospora species and uses thereof. Background Art

Translation, which occurs at the ribosome, is one of the most fundamental, yet essential processes which occurs in any cell. Research on ribosome structure, along with antibiotic inhibition and binding studies, have provided the focus for much of the knowledge available on the process of translation. Comparative sequence analysis and the identification of compensatory base changes was first used to elucidate the secondary structure of transfer ribonucleic acid (tRNA) and this approach has subsequently led to the proposal of a universally accepted secondary structure model for both the small subunit (SSU) and large subunit (LSU) ribosomal RNAs. Although approximately 100 sequences are available for the SSU rRNA of Apicomplexa, there has been little advance in our understanding of the LSU rRNA structure. The latest release of the LSU ribosomal database contains 429 sequences, yet only 10 (6 nuclear and 4 mitochondrial) are derived from parasites of the phylum Apicomplexa. Three of these nuclear gene sequences (all Toxoplasma gondii) were previously contained in the 1994 release of the database.

The primary and secondary structure of the LSU rRNAs of T. gondii and Plasmodium falciparum have been discussed in some detail and are available from the rRNA WWW Server at URL http://rrna.uia.ac.be/lsu/. These rRNAs conform to the universally accepted core secondary structure proposed for the LSU rRNA which consists of a central multi-branched loop from which helices emanate. The structures branching from the loop are labelled A to I, and within these branches the helices are numbered from 5' to 3¹. Within the core structure are scattered variable domains known as expansion segments. The origin of these segments is unknown but they always occur at the same place in the LSU structure although they vary in size between taxa. In T. gondii there have been recognised 81 helices and 12 expansion segments (domains called Dl to D12) in the 28S rRNA. Over the last 10 years or so, phylogenetic analyses of the SSU ribosomal RNA have provided important information on the evolutionary molecular biology, molecular phylogeny, speciation and classification of a huge range of taxa including many parasites. Many of these studies were aided specifically by the knowledge of the predicted secondary structure of the ribosomal RNAs.

Neospora caninum is a cyst-forming coccidian parasite which is recognised as being closely related to T. gondii. It causes neuromuscular disease in dogs and is now also recognised as a significant cause of abortion and neonatal mortality in livestock such as cows and goats. The genome organisation of Neospora has not been extensively investigated, although current evidence from rDNA comparisons show Neospora and Toxoplasma to be genetically very similar.

Competitive PCR (cPCR) involves the co-amplification, by PCR, of an internal reference standard (also frequently called the competitor sequence or PCR MIMIC) along with the target sequence of interest in the same reaction. The PCR MIMIC (PM) is typically a synthetic molecule that may be made in a wide variety of ways. Competitive PCR has found a practical use in the quantification of a number of infectious micro-organisms in biological specimens.

Competitive PCR may be used in one of two ways in order to determine the amount of target sequence in a biological specimen. In the first instance, the PM may be titrated in the presence of a constant amount of target sequence and the equivalence point determined where the PM and the target generate PCR products of similar yield. Alternatively, serial dilutions of target sequence are mixed with a fixed amount of PM and the reactions are subject to PCR. The concentration of the target sequence is determined by comparing the yield of PCR products obtained with a calibration curve constructed at the same time using material containing known amounts of the target sequence.

The present inventors have now determined the primary structure of the LSU rDNA of N. caninum. Comparison with a consensus sequence derived for the LSU rDNA of T. gondii demonstrated that the D2 domain

(Cl/Cl¹ region) can serve as a target for the development of a species-specific PCR for the detection of rDNA from Neospora. The present inventors have now identified a new genetic marker that can be vised to distinguish between Neospora spp and Toxoplasma spp. Furthermore, useful PCR MIMICs have been developed for use in cPCR assays for Neospora. Disclosure of Invention

In a first aspect, the present invention consists in an isolated nucleic acid molecule encoding the large subunit (LSU) ribosomal DNA (rDNA) of Neospora spp, the nucleic acid molecule having a sequence as set out in Figure 2, or a functionally equivalent sequence thereof, or a sequence that hybridises thereto.

Preferably, the Neospora spp is Neospora caninum. In a preferred embodiment, sequences which hybridise to the sequence shown in Figure 2 hybridise under stringent conditions. When used herein, stringent conditions are those that (a) employ low ionic strength and high temperature for washing, for example, 0.015 M NAC1/0.0015 M sodium citrate/0/1% NaDodS0₄ at 65°C; (b) employ during hybridisation a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C; or (c) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardfs solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS and 10% dextran sulfate at 42°C in 0.2 x SSC and 0.1% SDS. In a second aspect, the present invention consists in nucleic acid primers which are complementary to and specific for the LSU ribosomal DNA of Neospora spp. When the primers are used in a polymerase chain reaction (PCR) assay, the primers bind to LSU ribosomal DNA of Neospora spp present in a sample so as to allow the amplification of a nucleic acid molecule according to the first aspect of the present invention.

Preferably, the primers include the sequences selected from: NF6 5'-GTCCCTCGTGGACCC; and

GA1 5'-AACCTCTCTCAGAGATCG.

More preferably, the primers are NF6 and GAl. In a third aspect, the present invention consists in nucleic acid primers useful for the detection of Neospora spp by PCR in assays of clinical specimens, the primers including the sequences selected from: TimllF 5'-GGTACGTCTGTTTCAGTG; Timlδ 5'-CTCTCTCACCAGGTTTAG; Timl2 5'-GACCTAAAGGATCGATAG;

GA5 5'-CTATCGATCCTTTAGGTC; and GA6 5'-GCACGTGCACTCCGCATTTG.

More preferably, the primers are TimllF, Timl5, Timl2, GA5, or GA6.

The present inventors have found that the primer pairs of TimllF and GAl; Timl2 and GAl; Timlδ and Timl2; or GA5 and GA6 are particularly suitable to amplify the LSU rDNA of Neospora spp using PCR.

In a further preferred embodiment, the present invention consists in a unique Neospora species-specific PCR product of 270 bp obtained using the primer GAl with primer NF6 under standard PCR conditions.

In a fourth aspect, the present invention consists in a method of obtaining an isolated nucleic acid molecule encoding the LSU ribosomal

DNA of Neospora spp, the method including amplifying the nucleic acid molecule by PCR using primers according to the second or third aspects aspect of the present invention.

It will be appreciated that the primers according to the second or third aspects of the present invention can be used to identify or diagnose the presence of Neospora spp in a sample using PCR. Depending on the selection of primers, an assay based on PCR which is sensitive and specific for Neospora spp can be developed. The assay has the potential to provide the differential diagnosis between Neospora spp and Toxoplasma spp or other related or non-related microorganisms.

One of the flaws of utilising PCR as a diagnostic procedure is the interpretation of a negative result obtained through PCR. Two possible interpretations are possible for a negative result: a) the sample truly is negative for the PCR assay in that it (the sample) contains no target DNA; b) another alternative is that there has been a failure in the PCR reaction.

Consequently, in order to rule out the second of these alternatives during a diagnostic procedure, a positive control is necessary for inclusion as an internal standard. The present methods preferably require a nucleic acid molecule containing LSU sequence flanked by parasite-specific primers to be synthesised and cloned into a plasmid vector such as pGEM-T. The recombinant molecule may then be produced in large quantities using standard genetic engineering techniques for plasmid production, purified and seeded into a PCR reaction at known concentration in order to act as an internal standard for the PCR. Knowing the full sequence information of Neospora spp LSU has allowed the generation of suitable positive control internal sequences for use in PCR tests.

In a fifth aspect, the present invention consists in an internal reference standard, competitor sequence or PCR MIMIC for use in a Competitive PCR (cPCR) for Neospora spp, the internal reference standard, competitor sequence or PCR MIMIC having a nucleotide sequence including the following priming sites:

IP1 S'-CATGTGGATATTTTGCAGTCCCTCGTGGACCC and

IP2 5'-AAACTCCTGGAAGTTAAAGAACCTCTCTCAGAGATCG. In a preferred embodiment, the internal reference standard, competitor sequence or PCR MIMIC has the following sequence:

CATGTGGATATTTTGCAGTCCCTCGTGGACCCTTATATCTTTGTTCTTTCC

TTTTCCTTGTGGCTGAGGAGTGTTCTTGTTTCCGAGCTCCACTTTCGAGTA

CTCGGTTTCTGTGATGCTGGCTTAATCGGTTCCAACCGACCCGTCTTGAA ACACGGACCAAGGAGTCTAACATATGTGCGAGTATGCGGGTTTTACTCCT

GTATGCGCAATGAAAGTGAGAGTAGGGAGΛTTTTGGCTTTGCCATTCTTC

GCACCTACGACCGACCACGATCTCTGAGAGAGGTTCTTTAACTTCCAGGA

GTTT.

In order to ensure that a PCR assay for Neospora spp is working, the internal reference standards, competitor sequences or PCR MIMICs according to the fifth aspect of the present invention can be included in the method according to the fourth aspect of the present invention.

The present inventors have also devised internal reference standards. competitor sequences or PCR MIMICs for T. gondii. These internal reference standards, competitor sequences or PCR MIMICs can be included in PCR assays for Neospora spp and will allow ability to differentiate between

Neospora spp and T. gondii. The sequences are as follows: a) priming sites IP4 and IP4

IP3 (5'-TCCATTGGAGAGATTTGCGTTCCTTGTGGACCG); and IP4 (5'-AAACTCCTGGAAATCAGTAAACCTCTCTCAGAGATCG). b) DNA sequence of IP3/IP4 internal standard

TCCATTGGAGAGATTTGCGTTCCTTGTGGACCGTTATATCTTTGTTCTTT CCTTTTCCTTGTGGCTGAGGAGTGTTCCTGTTTCCGAGCTCCACTTTCGAG TACTCGGTTTCTGTGATGCTGGCTTAATCGGTTCCAACCGACCCGTCTTG AAACACGGACCAAGGAGTCTMACATATGTGCGAGTATGCGGGTTTTACT CCTGTATGCGCAATGAAAGTGAGAGTAGGGAGATTTTGGCTTTGCCATTC TTCGCACCTACGACCGACCACGATCTCTGAGAGAGGTTTACTGATTTCCA GGAGTTT.

It will be appreciated, however, that other internal reference standards, competitor sequences or PCR MIMICs could be developed from the sequences provided. For example, sequences including the primer pairs

IP1/IP2 having positioned therebetween an amplifiable string of nucleotides would also be suitable for this purpose. Additional internal reference standards, competitor sequences or PCR MIMICs may also include nucleotides positioned at the 5' and/or 3' ends of the primer pairs IP1/IP2. It will also be appreciated that internal standards for other microorganisms including Hammondia liammondi can be devised and used in PCR assays for Neospora spp.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

In order that the present invention may be more clearly understood, preferred forms will be described with reference to the following example and accompanying drawings.

Brief Description of Drawings

Figure 1 shows the location of primer sequences used for the PCR isolation of the LSU rDNA of N. caninum or T. gondii. Primer sequences are given in the Materials and Methods. Arrows indicate the direction of the primer extension reaction.

Figure 2 shows the sequence of an isolated nucleic acid molecule encoding the LSU ribosomal DNA of Neospora spp.

Figure 3 shows the comparison of the LSU rDNA of N. caninum and T. gondii. The LSU rDNA of N. caninum (N.c.) was aligned with that of T. gondii (GenBank accession number L25365). Only bases which are different between the sequences are indicated. In regions where the four T. gondii sequences disagree (GenBank accession numbers L25365, X75429, X75430 and X75453), a consensus sequence was generated (T.g.). ![Timl5/GAl] represents the fragment that was independently amplified by PCR and sequenced from Neospora (NCI, NC-Liverpool and SweBl strains) and

Toxoplasma (RH, ME49 strains) and compared to X75429, X75430 and X75453. ²{deletion} represents the region of rDNA that is deleted in X75429. ³ {replacement} represents the region of rDNA that is replaced in sequence X75430. Numbers given at the beginning and end of the sequence alignment indicate the base position in the alignment. Areas of inferred rRNA secondary structure which are discussed in the text are indicated between

< > . Dashes (-) represent gaps introduced into the sequence to maximise the alignments. Dots (.) in L25365 and T.g. represent a position where the nucleotide present at that position is identical to N.c. Modes for Carrying Out the Invention MATERIALS AND METHODS

Parasites

Tachyzoites of N. caninum (NC-Liverpool strain) or T. gondii (RH strain) were propagated by in-vitro culture on a Vero monolayer as described [4). Tachyzoites were purified by filtration through a 3 micron nucleopore filter [7]. DNA was prepared from tachyzoites by standard procedures involving lysis in a Tris buffer containing SDS, EDTA and proteinase K, followed by phenol/chloroform extraction and ethanol precipitation. Extraction of DNA

Genomic DNA was purified from 10⁸ tachyzoites by lysis in 1% SDS, 100 mM EDTA, 10 mM Tris pH 9 containing 100 μg/ml proteinase K at 56 °C for 2 hours. DNA was further purified by phenol/chloroform extraction; centrifiigation through a cesium chloride gradient (density 1.68 gm per ml); followed by dialysis against 10 mM Tris pH 7.5, 0.1 mM EDTA. Isolation and characterisation of LSU rDNA A standard polymerase chain reaction (PCR) protocol was used throughout (unless otherwise specified) which consisted of an initial denaturation at 94°C; then thirty cycles of 95 °C (denaturation) for one minute; 52°C (primer annealing) for one minute; 72°C (primer extension) for two minutes. The PCR was terminated with a final primer extension reaction at 72°C for five minutes. A Hybaid omnigene thermocycler was used.

LSU rDNA of N. caninum was isolated as a series of overlapping fragments by PCR (Figure 1). PCR fragments were obtained by primer combinations TimllF (5'-GGTACGTCTGTTTCAGTG) and GAl (5'-AACCTCTCTCAGAGATCG); Timlδ (5'-CTCTCTCACCAGGTTTAG) and GAl;

Tim δ and Timl2 (5'-GACCTAAAGGATCGATAG); and GA5 (5¹- CTΛTCGATCCTTTAGGTC) and GA6 δ'-GCACGTGCACTCCGCATTTG.

The PCR products were purified by a QIAquick purification column (Qiagen, USA); ligated into the plasmid vector pGEM-T (Promega, USA) and transformed into Escherichia coli (DHδalpha). Transformants containing insert DNA were identified by growth on L-agar plates containing ampicillin, IPTG and Xgal; grown in L-broth containing ampicillin and plasmid DNA prepared from them using the Qiagen miniprep kit. Inserts were sequenced in both directions by cycle sequencing (Sequitherm, Epicentre, USA) using IRD-41 fluorescein labelled M13 forward or reverse primers (LiCOR, USA). The products of the cycle sequencing were run and visualised on a LiCOR 4000L automated DNA sequencing machine. At least 6 clones were sequenced in both directions for each PCR product cloned (i.e. 12 sequencing reactions in total) and a consensus sequence was obtained for each fragment using AssemblyAlign™ . A consensus sequence for the LSU rDNA was then obtained by compiling the sequence from the overlapping fragments. Using this approach 3,499 nucleotides of the LSU rDNA of N. caninum were obtained. During early studies on comparing LSU rDNA from N. caninum and T gondii, comparisons were made using the L2563δ entry for the RH strain [2] since this represents the only sequence of T. gondii that has been fully described in the scientific literature. It became apparent, however, that discrepancies existed between this sequence and others for the LSU rDNA of T. gondii in GenBank™ (accession numbers X75429, RH strain; X75430, Sailie strain; P strain, X754δ3). Furthermore, a number of anomalies were observed between all the T. gondii sequences. In order to help resolve some of these discrepancies, LSU rDNA of T. gondii was PCR amplified from T. gondii RH strain DNA (described in [δ)) using primers Tim3 and Tim6. Tim3 (δ¹- CCGCTGCAGAGGTGAACCTGCGGAAGGATC-3') is the reverse complement of primer called P3 [5], Tim6 sequence (δ'-

CCGCTGCAGAGGATAATCGCTCTACAA) was taken from the region called "oligo". In brief, Tim3 anneals to the 3¹ end of the SSU while Tim6 anneals to the 5S rDNA. Both Tim3 and Tim6 have Pstl sites at their 5' ends and these Pstl sites were used to clone the PCR product into the plasmid vector pUCl9.

The ligation products were electroporated into E. coli (strain DHδalpha) and transformants were selected on L- agar plates containing ampicillin, IPTG and Xgal. Individual white colonies were cultured in liquid media, plasmid DNA isolated and the insert DNAs were examined by restriction analysis using Pstl. One positive clone obtained from this process was selected and called pRHT3T6. This single clone was sequenced by cycle sequencing and an ABI automated sequencer using a combination of the primers described above.

The primers Timlδ and GAl were used to amplify a DNA fragment covering the D2 and D3 expansion segments of the LSU rDNA from RH and ME49 strains of T gondii; NCl and NC-SweBl strains of N. caninum and

Hammondia hammondi. All strains of Neospora and Toxoplasma were grown in-vitro and DNA prepared as described above. The PCR products were cloned into pGEM-T, sequenced and aligned to available data for N. caninum (NC-Liverpool) and T gondii (X7δ429, X7δ430 and X7δ453) by Clustal W. A comparison of the sequences derived from the D2 expansion segment (Cl/Cl' region) of Neospora and Toxoplasma revealed nucleotide sequences which were incorporated into a species-specific primer designed for N caninum (NF6; 5'-GTCCCTCGTGGACCC). A similar primer was designed against the T gondii sequence (TF6; δ'-GTTCCTTGTGGACCG) but subsequently proved not to be species-specific. PCR amplification of

Neospora, Toxoplasma, or Hammondia DNA was performed using either NF6 or TF6 with GAl.

Toxoplasma DNA from 23 independent strains (21 of them are described in [6]; RH and RH88 DNAs are described in [δ]) were subject to PCR using primers GA7 (δ'-ATTCGCTTTACCTGA) and GA8 (5'-

CGATCTCTGAGAGAGGTT) . Preparation of competitor molecule

Primers IPl (δ'-CATGTGGATATTTTGCAGTCCCTCGTGGACCC) and IP2 (δ'-AAACTCCTGGAAGTTAAAGAACCTCTCTCAGAGATCG) were used to amplify, by PCR, a 306 bp fragment from genomic DNA of N. caninum .

IPl represents a composite of primers NS2 (δ'-CATGTGGATATTTTGCA) and NF6 (δ'-GTCCCTCGTGGΛCCC) whereas primer IP2 represents a composite of primers NRl (5'-AAACTCCTGGAAGTTAAAG) and GAl (δ'-AACCTCTCTCAGAGATCG). Under these conditions, priming sites for NS2 and NRl are placed on either side of a 270 bp fragment derived from the

D2 domain of the large subunit (LSU) ribosomal (r) DNA (which is amplified by primers NF6 and GAl). The 306 bp PCR product generated was purified using a Qiagen column and the sequence determined by cycle sequencing using primer NS2 or NRl. The PCR product was cloned into the plasmid vector pGEM-T and transformed into Escherichia coli by standard calcium chloride transfection techniques. Plasmid DNA was extracted from recombinant bacterial clones by the procedure of alkaline lysis, purified by a Qiagen column, and the DNA concentration determined by optical density.

As N. caninum is frequently misdiagnosed as T. gondii we developed in parallel a PCR that will detect specifically T. gondii DNA. A competitor molecule (hereafter called IP3/IP4) was also made using the process described here from primers IP3

(δ'-TCCATTGGAGAGATTTGCGTTCCTTGTGGACCG) and IP4 (δ'-AAACTCCTGGAAATCAGTAAACCTCTCTCAGAGATCG). IP3 represents a composite of primers TS4 (5'-TCCATTGGAGAGATTTGC) and TF6 (5'- GTTCCTTGTGGACCG) whereas primer IP4 represents a composite of primers TRl (δ'-AAACTCCTGGAAATCAGTA) and GAl (δ'-AACCTCTCTCAGAGATCG). Under these conditions, priming sites for TS4 and TRl are placed on either side of a 270 bp fragment derived from the D2 domain of the LSU rDNA of T gondii (which is amplified by primers TF6 and GAl). IP3/IP4 is, therefore, compatible with the T. gondii specific primers TS4 and TRl and PCR amplification of IP3/IP4 with TS4 and TRl generates a PCR product of 307 bp. Polymerase chain reaction

The reaction conditions for the cPCR comprised δ μl of test sample; lxPCR Buffer; 1.7δ mM MgCl₂; δ0 μM of NS2 and NRl (or TS4 and TRl) and

0.8 units of Taq DNA polymerase in a total reaction volume of 50 μl. PCR was performed on a Hybaid omnigene with the following thermal cycle program: 9δ°C for 5 minutes; 5 cycles of 94°C for 30 seconds, 60°C for lδO seconds, 72°C for 30 seconds; lδ cycles of 88°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds; 10 cycles of 88°C for 30 seconds, δ4°C for 30 seconds, 72°C for 30 seconds; 2δ cycles of 86°C for 30 seconds, 54°C for 30 seconds, 72°C for 30 seconds; and finally 72°C for 10 minutes. Under these conditions of PCR, NS2 and NRl generates a PCR product of 146 bp from N. caninum genomic DNA, whereas TS4 and TRl generates a PCR product of 18δ bp from T gondii DNA. A hot start PCR was used to prevent PCR amplification of non-specific products.

The products of the PCR were run on a 2% agarose gel which included either a 100 bp molecular weight ladder or a Hpaϊl digest of pUCl3 as markers.

Infection of Mice

Purified tachyzoites were concentrated by centrifugation and counted using a hemocytometer. Female, in-bred Balb/C mice (sourced from ARC, Perth, Western Australia or Gore Hill Research Laboratories, NSW) approximately 20 g in body weight, were housed in groups of 6-12 in plastic box cages and provided feed and water ad libitum. Defined doses of tachyzoites were inoculated subcutaneously into mice. The brains of N. caiunum infected mice were removed at either 9, lδ or 18 days post infection (dpi); DNA was prepared from them using a QIAamp tissue Kit (Qiagen) as recommended by Jenkins et al. [8] and subject to cPCR using primers NS2 and NRl in the presence of the IP1/IP2 PM. Brains from uninfected mice were used as negative controls. Preparation of DNA from Mice Brains

Whole mouse brain was weighed, homogenised and digested according to the manufacturers instructions (QIAamp tissue kit, Qiagen) with 180 μl ATL buffer for every 2δ mg brain tissue. The lysate was incubated at δδ°C overnight after which 400 μl of lysate solution was removed (equivalent to δ0 mg of tissue) and further purified by column chromatography according to the manufacturers instructions. The remaining lysed mouse brain was stored at 4°C. DNA was eluted off the column in a volume of 400 μl. Four or 8 μl of

DNA was then used in a cPCR.

Throughout this study the nomenclature described in De Rijk et al. (1997) [1] has been used to describe the secondary structure of the rRNA. The model on which this secondary structure is based has received universal acceptance, although it is acknowledged that variations on the theme will occur between taxa. The only exception is the use of the term "D2 domain" [2]. This corresponds to the Cl/Cl' region described in [1] but the structure is poorly defined in the Apicomplexa because of lack of sequence data. RESULTS

Isolation and characterisation of LSU rDNA

A large number of nucleotides (3,499) of the LSU rDNA of N. caninum were obtained with just δ ambiguous bases (GenBank accession number AF001946). These bases were located to positions at the terminal loop of D14

(probably a T or C); two were located in the single stranded region between Ell-1 and E12; one was located in the terminal loop of G7 (probably a C) and the final unresolved base lie in the single stranded region between Hl-1 and Hl-3. An alignment (using GJAP of the GCG package) of the N. caninum sequence with that of L2δ63δ of T. gondii revealed the sequences to be approximately 98% identical. The sequences differed at 21 "regions" of the sequence alignment and Figure 3 summarises these differences.

In an attempt to determine whether the nucleotide sequence differences observed between N. caninum and T. gondii were real and reproducible differences, the sequences were compared in the 21 regions to those additional LSU sequences available for T gondii (GenBank accession numbers X75429, RH strain; X7δ430, Sailie strain; P strain, X7δ4δ3). In fifteen of these regions (terminal loops of B13 and Dδ; stems of Dlδ, D17¹, E13\ E14', E12', E18, E21, E23¹ and E18'; bulges or single stranded regions in E14, E12', E20/E21, G16' and 13'; and various regions between E20 and E20-2) a nucleotide sequence difference between N. caninum and T gondii was not supported, in that the sequences of X7δ429, X7δ430 and X75453 were identical to that determined here for N. caninum. Using this information, a consensus sequence was generated for the LSU rDNA of T. gondii (Figure 2). Six regions supported a consistent nucleotide difference between the

LSU sequence of N. caninum and the consensus sequence derived for T. gondii. A single insertion of one base and non-similarity of two others occurs in the terminal loop of B13-1; a single transversion occurs in the stem of D20 (A>C); a single transversion occurs in the single stranded region between Gδ- 1' and Gδ-2 (T> A); a single transition occurs in the stem of Hl-1 (T>C); a single transition occurs between Hl-1 and Hl-3 (C>T) plus a single insertion of a C. However the most divergent region was the region defined by Cl/Cl' which corresponds to the D2 expansion segment or domain.

Experiments showed that the primers Timl5 and GAl amplified most of this region of the rDNA from Neospora, Toxoplasma and Hammondia DNA.

Comparison of the sequence data generated for the D2 and D3 expansion segments of N. caninum (NC-Liverpool, NCI and NC-SweBl) and T. gondii (RH and ME49) (amplified and sequenced using the primers Timlδ and GAl; Figure 1) showed that five nucleotides reliably differed between the two taxa providing total support for the accuracy of the consensus sequence derived for T gondii (Figure 2). All five base positions were located in the D2 expansion segment. The rest of the sequences were identical apart from two nucleotides (one nucleotide position in X7δ430 and one in X7δ429), Four of the nucleotides that differed between the N. caninum and T. gondii sequences in this region were the result of transitions (either T>C or C>T), compared to one transversion (C>G). Of importance is the observation that the sequence of this region for the bovine strain of N. caninum (NC-SweBl) is identical to the other two strains of N. caninum isolated from dogs (NCI and NC-Liverpool).

PCR primers NF6 and TF6 were designed based on the observed nucleotide differences between Neospora and Toxoplasma in the Timl5/GAl sequence in an attempt to generate species-specific PCR primers. The primer combination GAl and NF6 under PCR conditions that utilise a primer annealing temperature of 5δ°C produce a unique species-specific PCR product of 2δ0 bp from N. caninum DNA. No product is obtained using these two primers with Toxoplasma, or Hammondia DNA unless the primer annealing temperature is lowered to δ2°C. The primer combination TF6 and GAl generated no PCR products from Neospora DNA using a primer annealing temperature of 55°C. Products of approximately 2δ0 bp, however, were obtained from T. gondii and H. hammondi DNA respectively. Since this result indicates a very close genetic relationship between Toxoplasma and H. hammondi, the Timlδ/GAl of H. hammondi was amplified and sequenced. The priming site for TF6 was identical in both T. gondii and H. hammondi.

Intriguingly, during these comparisons of the LSU sequences of T gondii, two major discrepancies were noticed between the sequences (along with a number of minor base changes). In the first instance, the sequence alignments showed the presence of a deletion

(δ' TTTTATCAGGTAAAGCGAATGATTAGAGGCATCGGGGGCGCG) in sequence X7δ429 which differs from the sequences of Gagnon et al. (1996) [2] and X7δ430 and X7δ453. This deletion may potentially lead to a truncation of region D14 [1] also known as helix 26 [2]. Consequently, this region was sequenced in the pRHT3T6 clone derived from the RH strain of T. gondii [5]. The sequence data generated was identical to the sequence of X7δ429 in that it contained an identical deletion. PCR using DNA from a large number of virulent and avirulent Toxoplasma strains using primers GA7 (whose priming site occurs in the "deleted" sequence and GA8 (the complement of GAl), however, gave a product of approx. 200 bp from all

DNAs studied which is the fragment size predicted from the DNA sequence data. The generation of a PCR product using primers GA7 and GA8 clearly shows that the "deleted" sequence is present in rDNA from a wide variety of different Toxoplasma strains. The second major change was a substitution of bases 3044 to 3195 of

Gagnon et al. (1996) [2] with the sequence

5'CCTTGTCAACCCAGCCTATGAACCATTTACGCCGATCGACTTTTCGA in X75430 (Sailie). At this location the other sequences available for T gondii are in agreement. This region was also isolated by PCR and sequenced from pRHT3T6 and found to be identical to the sequences of

X75429, X7δ430 and X7δ4δ3. Thus the substitution observed in X7δ430 does not resemble any other sequence; a FastA search of the GenBank™ database revealed no direct homologous sequence, although some resemblance to 12S rRNA sequences were disclosed. This sequence had no homology to the T. gondii 12S mitochondrial sequence.

Development of internal reference standard, competitor sequence or PCR MIMIC

In order to ensure that the PCR reaction has not failed during a diagnostic procedure, a positive control is necessary for inclusion as an internal standard. Knowing the full sequence information of Neospora spp

LSU allowed the present inventors to the generate suitable positive control internal sequences for use in PCR tests.

The following primers were designed and developed for use as internal standards for diagnostic PCR assays: IPl (δ'-CATGTGGATATTTTGCAGTCCCTCGTGGACCC);

IP2 (δ'-AAACTCCTGGAAGTTAAAGAACCTCTCTCAGAGATCG); IP3 (δ'-TCCATTGGAGAGATTTGCGTTCCTTGTGGACCG); and IP4 (δ'-AAACTCCTGGAAATCAGTAAACCTCTCTCAGAGATCG).

Primers IPl and IP2 produce a PCR product of 306 bp from N. caninum DNA whereas IP3 and IP4 produce a PCR product of 307 bp from T gondii

DNA. The sequences of IP1/IP2 and IP3/IP4 are shown below. DNA Sequences of the PCR MIMICs developed are described below: a) DNA sequence of IP1/IP2 PCR product δ'CATGTGGATATTTTGCAGTCCCTCGTGGACCCTTATATCTTTGTTCTTTC CTTTTCCTTGTGGCTGAGGAGTGTTCTTGTTTCCGAGCTCCACTTTCGAGT ACTCGGTTTCTGTGATGCTGGCTTAATCGGTTCCAACCGACCCGTCTTGA AACACGGACCAAGGAGTCTAACATATGTGCGAGTATGCGGGTTTTACTCC TGTATGCGCAATGAAAGTGAGAGTAGGGAGATTTTGGCTTTGCCATTCTT CGCACCTACGACCGACCACGATCTCTGAGAGAGGTTCTTTAACTTCCAGG AGTTT b) DNA sequence of IP3/IP4 PCR product

5'CCATTGGAGAGATTTGCGTTCCTTGTGGACCGTTATATCTTTGTTCTTTC CTTTTCCTTGTGGCTGAGGAGTGTTCCTGTTTCCGAGCTCCACTTTCGAGT ACTCGGTTTCTGTGATGCTGGCTTAATCGGTTCCAACCGACCCGTCTTGA AACACGGACCAAGGAGTCTMACATATGTGCGAGTATGCGGGTTTTACTC CTGTATGCGCAATGAAAGTGAGAGTAGGGAGATTTTGGCTTTGCCATTCT

TCGCACCTACGACCGACCACGATCTCTGAGAGAGGTTTACTGATTTCCAG GAGTTT.

In order to assess the potential suitability of IP1/IP2 as a PM for a cPCR assay, 30 pg of IP1/IP2 were added to individual reactions containing serial dilutions of genomic DNA from N. caninum and subject to PCR. The products of the cPCR were subject to agarose gel electrophoresis. Two bands were detected on the gel, the largest derives from the PM whereas the smaller one derives from the genomic DNA template. The yield of the two bands was dependent on the amount of genomic DNA included in the PCR reaction. In another experiment, each lane of the gel was scanned using a densitometer and the natural log of the relative density of the two PCR products in each track were plotted against the mass of the target sequence present in the PCR reaction. In one example, a calibration curve demonstrated an equivalence point reached with approximately 50 pg of IP1/IP2 and 800 ng of genomic DNA, implying both populations of DNA contain similar numbers of priming sites (e.g. that 0.05/800 or approximately 0.2 % of N. caninum genomic DNA represents ribosomal DNA).

A cPCR containing target and PM should fulfil the principle condition that the two sequences are PCR amplified with equal efficiencies by identical primers so that a plot of log (T₀/C₀) against log C₀ should be a straight line with a gradient of -1 [9] . Since this issue appears to be generally ignored by most workers, data generated for IP1/IP2 was subject to analysis. A calibration curve of log (T₀/C₀) against log C₀ showed linearity over the range of 5 to 3000 ng of genomic DNA. The shape of the curve was therefore consistent and compatible with its use as a calibration curve for the prediction of DNA concentrations in biological specimens. The range of linearity could be modified however, by varying the amount of PM included in the reaction.

Preliminary experiments demonstrated that the outcomes of the cPCR was dependent on the relative mass of the PM and genomic DNA put into the reaction. For example, in a typical experiment, varying masses of PM were subject to PCR in the presence of a fixed amount of Neospora genomic DNA (2δ6 pg). Incorporation of greater than 219 ng of PM caused saturation of the PCR in that only a 306 bp product (derived from the PM) was generated. No detectable PCR product was generated from the Neospora genomic DNA. Using less than 13 ng of PM resulted in the loss of the 306 bp product and the generation of only the 146 bp product from the Neospora genomic DNA. Thus this experiment defined the relative amount (between 13 and 879 ng) of PM needed for cPCR under these conditions.

Since one aim of these experiments was to design a cPCR which could be used to detect Neospora DNA in the brains and fetal tissues, for example, of animals such as mice, the effect of mouse brain DNA on the PCR was investigated. An excess of normal mouse brain DNA (0, 100 ng, 1 or δ μg) was mixed with 2δ6 pg of N. caninum DNA and 30 pg of PM and subject to PCR. Using these masses of PM and Neospora genomic DNA , it was predicted from experiments that only a 146 bp product from the Neospora genomic DNA should be generated. The control experiment where no mouse brain DNA was incorporated shows this to the case. However, 5 μg of mouse brain DNA completely inhibited PCR production and 1 μg inhibited the reaction by approximately 50%. PCR in the presence of 100 ng of mouse brain DNA gave a PCR product that was similar in intensity to the control. Hence it was concluded that it was preferably necessary to limit the amount of mouse brain DNA in the PCR. This information resulted in the development of the protocol described in the Materials and Methods for the detection of Neospora DNA in the brains of infected mice. In order to demonstrate the utility of the cPCR to detect Neospora DNA in mouse brains, mice were infected with culture-derived tachyzoites. NC- Liverpool induced severe clinical signs of neosporosis in the infected mice including discoordinated movement, hindlimb paralysis and coat ruffling with severe weight loss at days 17-28 after inoculation. DNA from the brains of mice sacrificed at 9 dpi did not support the generation of a 146 bp PCR product by cPCR using 30 ng PM; a 146 bp PCR product was produced however in the presence of the PM at lδ (in mice infected with 10⁶ tachyzoites) and 18 (in mice infected with 10⁵ tachyzoites) dpi. DISCUSSION

The primary structure of the LSU rDNA of N. caninum has been determined by PCR and DNA sequencing and it is highly similar to the LSU rDNA of T gondii. In T. gondii Gagnon et al. (1996) [2] recognised 81 helices and 12 expansion segments (called Dl to D12) in the 28S rRNA. No structure was suggested for the expansion segments. Similarly the Antwerp LSU ribosomal database contains a secondary structure prediction for T gondii LSU rRNA that contains at least 102 helices and presents a proposal for the secondary structure for the total LSU rRNA [1]. Given the universal acceptance of the secondary structure model upon which the LSU rRNA alignments are based in the Antwerp LSU ribosomal database, the nomenclature and model in use was adopted in this study for convenience. Furthermore, the secondary structure alignments were readily available from the Antwerp database.

A comparison of the LSU sequences of T. gondii showed sequence data that was inconsistent which made a comparison of LSU sequences of N. caninum and T. gondii difficult. For example, L2536δ contained 26 bases in the region between the stems of Dδ and Dδ' whereas the N. caninum and T. gondii sequences X7δ429, X7δ430 and X7δ4δ3 contain 27 bases. The sequences of D14 were inconsistent and the base pairing suggested by them were not consistent with the secondary structure model described in [1]. Similarly, the sequence of L25365 was missing TT in Dlδ, whereas the sequence determined for N. caninum was identical to that of X7δ429, X7δ430 and X7δ4δ3. The present inventors realised that several of the regions where the LSU sequences of T gondii differed were in helices, which are normally considered to be some of the most evolutionary conserved sites within the rRNA molecule. Figure 3 summarises these and many other differences found. Consequently, in Figure 2 the present inventors present a primary sequence for the LSU rDNA of T gondii which is a consensus of the available sequence data and is consistent with the universally accepted secondary structure model summarised in [1],

The LSU rRNA is the largest of the rRNAs, yet it has not been used extensively for studies on the phylogeny of parasites because few sequences are available and because their sequences probably have rates of evolution similar to those of the SSU rRNA. Specific regions (primarily the expansion or variable domains) of the LSU sequences have proven useful for distinguishing among a number of proposed alternatives of vertebrate phylogeny. The observations that the expansion segment D2 contains nucleotides that differ between Neospora and Toxoplasma shows that this region may be useful for investigating the relationships among these cyst- forming coccidia. Of specific note is that the data provided here, through a comparison of the D2 sequences derived from various Neospora and Toxoplasma strains, provides further support for the proposal that bovine and canine strains of Neospora are indeed the same species. The three Neospora strains studied all possess identical sequences for the D2 and D3 domains of the LSU. The results presented here therefore identifies a new genetic marker that appears suitable for use in the discrimination of Neospora from Toxoplasma. The similarity detected in primary structure of the LSU rDNA between

N. caninum and T gondii provides further evidence for a close taxonomic relationship between these two taxa. There are a number of biological characteristics that distinguish Toxoplasma from Hammondia which support the continued use of the name Hammondia. For example, Toxoplasma possesses a facultative heteroxenous life cycle whereas Hammondia is obligatory heteroxenous, and the cyst stages are found predominantly in skeletal muscle of the intermediate host in Hammondia and not in neural tissue. One would therefore predict that the credibility of the genus Neospora will require the determination of the complete natural life cycle of this parasite. The results presented, through a comparison of the D2 and D3 domains, implies that Hammondia is genetically more closely related to Toxoplasma than Neospora. If one accepts the validity of the genus Hammondia then, by implication, one must also accept the validity of the genus Neospora. Table 1. Location and identity of nucleotide differences observed through a comparison of LSU rDNA sequences of N. caninum (AF001946) and T. gondii (L2536δ). rRNA Secondary Agreement Nucleotide Identity of location structure difference Neospora location observed specific nucleotides

B13-1 terminal loop N N/A insertion of A; two transitions (C>T and T>C)

Cl to Cl' various N N/A four transitions, one transversion (see text and Fig. 2)

D5 terminal loop Y deletion of G N/A

D15 stem Y deletion of TT N/A

D17' stem Y insertion of T N/A

D20 stem transversion A>C

E13' stem Y TTT

E14 bulge Y N/A

E14' stem Y insertion of G N/A

E12' bulge Y insertion of C

E18 various Y (except one different at 6 N/A base) bases; deletion of 2. 4 not identical

E20 to E20-2 various Y 6 base N/A deletion

E20 to E21 single Y insertion of N/A stranded TGC

E21 stem Y insertion of A N/A

E23' stem Y deletion of G N/A

E18' stem Y transversion N/A of T>A

G5-1 to G5-2 single N N/A transversion stranded T>A

G16' bulge Y deletion of G N/A

Hl-1 bulge transition T>C

Hl-1 to Hl-3 N N/A one transition C>T; one insertion of C

13' bulge Y N/A N/A Table Legend

1. Using the secondary structure summarised in [1]. Cl/Cl¹ refers to D2 domain [2].

2. Type of secondary structure in which the nucleotide differences are observed.

3. Agreement between the N. caninum sequence with T gondii sequences X75429, X7δ430 and X7δ4δ3 at these locations. Y, yes; N, no.

4. Nucleotide difference observed in T. gondii sequence (L2δ36δ) compared to N. caninum sequence. δ. Identity of observed N. caninum specific nucleotides after comparison to

T gondii consensus sequence for LSU rDNA.

In recent years, several procedures have been described that allow the (semi-) quantification of PCR. The main aim of any quantitative PCR technique is to estimate the quantity of target DNA sequences present in the specimen or sample under study. Competitive PCR has become the most popular and widely used quantitative PCR procedure since it effectively controls for tube to tube variation in amplification efficiency and it is not necessary to restrict the reactions to the exponential phase of the PCR. Consequently this approach was adopted and a semi-quantitative PCR technique that can be used to determine the amount of Neospora DNA present in a biological specimen was developed.

Competitor molecules were constructed by PCR using primers that were composites of two primers. Species-specific priming sites normally located only in the ITSl were introduced onto either side of a small fragment of the LSU rDNA, resulting in the generation of a synthetic competitor molecule that could be amplified using the primers directed against the ITSl. Two PMs were made: IP1/IP2 containing Neospora LSU rDNA flanked by primers that amplify specifically Neospora ITSl (NS2 and NRl); and IP3/IP4 containing Toxoplasma LSU rDNA flanked by primers specific for the ITSl of

T gondii (TS4 and TRl).

A calibration curve of log (T₀/C₀) against log C₀ showed linearity over the range of δ to 3000 ng of genomic DNA using the cPCR described thus confirming the utility of the PM and the cPCR. Outside of this, an excess of either target or competitor saturated the PCR. Therefore δ to 3000 ng of genomic DNA represents the range over which quantitation of target sequence can be reliably performed using the conditions described, if the PCR products are analysed by agarose gel electrophoresis and ethidium bromide staining. This represents approximately δ x 10⁴ to 3xl0⁷ tachyzoites. There are however a number of alternative ways in which PCR products can be detected and quantified (e.g. incorporation of radioactive label into the PCR and autoradiography) that may potentially alter the range over which the competitive PCR is functional.

Competitive PCR was used to detect N. caninum DNA in the brains of infected mice. No DNA was detected at 9 dpi, but was present at 15 and 18 dpi depending on the dose of tachyzoites given. The detection of a significant increase in parasite DNA between days 9 and 15 post infection is correlated with the onset of severe weight loss due to neosporosis which becomes noticeable at around 14 dpi.

Jenkins et al. [8] suggested that a semi-quantitative PCR procedure has several advantages over histologic and pathologic procedures for estimating the number of parasites in a biological specimen. For example, parasite numbers may vary enormously between adjacent δ micron sections taken from tissue specimens. The present analyses of sections taken from the brains of aborted bovine foetuses have confirmed this is the case:- in 8 cases where PCR was able to detect parasite DNA in δ micron sections not all of the sections examined from the same specimen gave a positive result by single tube nested PCR. Consequently in the PCR procedure described here DNA was extracted from the entire organ under study (in this case the brain) in order to increase the chances of detecting parasite DNA. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. REFERENCES

[1] De Rijk, P., Van de peer, Y., Chapelle, S. & De Wachter, R. (1997)

Database on the structure of large ribosomal subunit RNA. Nucleic Acids

Research 25, 117-122. [2] Gagnon, S., Bourbeau, D. & Levesque, R.C. (1996) Secondary structures and features of the 18S, 5.8S and 26S ribosomal RNAs from the

Apicomplexan parasite Toxoplasma gondii. Gene 173, 129-13δ.

[3] Payne, S. & Ellis, J. (1996) Detection of Neospora caninum DNA by the polymerase chain reaction. International Journal of Parasitology 26, 347-3δl. [4] Barber, J., Trees, A.J., Owen, M. & Tennant, B. (1993) Isolation of

Neospora caninum from a British dog. Veterinary Record 133, δ31-δ32.

[δ] Brindley, P.J., Gazinelli, R.T., Denkers, E.Y., Davis, S.W., Dubey, J.P.,

Belfort, Jr, R., Martins, M-C, Silveira, C, Jamra, L., Waters, A.P. & Slier, A.

(1993) Differentiation of Toxoplasma gondii from closely related coccidia by riboprint analysis and a surface antigen gene polymerase chain reaction.

American Journal of Tropical Medicine & Hygiene 48, 447-456.

114, 165-171.

[6] Homan, W.L., Limper, L., Verlaan, M., Borst, A., Vercammen, M. & van

Knapen, F. (1997) Comparison of the internal transcribed spacer, ITSl, from Toxoplasma gondii isolates and Neospora caninum. Parasitology Research 83,

285-289.

[7] Stenlund, S., Bjorkman, C, Holmdahl, O.J.M., Kindahl, H. and Uggla,

A. (1997) Characterisation of a Swedish bovine isolate of Neospora caninum.

Parasitol. Res. 83, 214-219. [8] Jenkins, M., Trout, J. and Fayer, R. (1997) A semi-quantitative method for measuring Cryptosporidium parvum infection using polymerase chain reaction. J. Micro. Meth. 28, 99-107.

[9] Raeymaekers, L. (1993) Quantitative PCR: theoretical considerations with practical implications. Anal. Biochem. 214, 582-δ85.

Claims

CLAIMS:

1. An isolated nucleic acid molecule encoding the large subunit (LSU) ribosomal DNA (rDNA) of Neospora spp, the nucleic acid molecule having a sequence substantially as set out in Figure 2, or functionally equivalent sequence thereof, or a sequence that hybridises thereto.

2. The nucleic acid molecule according to claim 1 wherein the Neospora spp is Neospora caninum.

3. A nucleic acid primer which is complementary to and specific for the LSU ribosomal DNA of Neospora spp according to claim 1 or 2. 4. The primer according to claim 3 including a sequence selected from the group consisting of:

GAl δ'-AACCTCTCTCAGAGATCG; and

NF6 δ'-GTCCCTCGTGGACCC. δ. The primer according to claim 4 selected from GAl and NF6. 6. A nucleic acid primer useful for the detection of Neospora spp by polymerase chain reaction (PCR) amplification assays of clinical specimens, the primer including a sequence selected from the group consisting:

TimllF δ'-GGTACGTCTGTTTCAGTG;

Timl5 δ'-CTCTCTCACCAGGTTTAG; Timl2 δ'-GACCTAAAGGATCGATAG;

GAδ δ'-CTATCGATCCTTTAGGTC; and

GA6 δ'-GCACGTGCACTCCGCATTTG.

7. The primer according to claim 6 selected from the group consisting of primers TimllF, Timlδ, Timl2, GAδ, and GA6. 8. An internal reference standard, competitor sequence or PCR MIMIC for use in a Competitive PCR (cPCR) for Neospora spp, the internal reference standard, competitor sequence or PCR MIMIC having a nucleotide sequence including the following priming sites:

IPl δ'-CATGTGGATATTTTGCAGTCCCTCGTGGACCC and IP2 δ'-AAACTCCTGGAAGTTAAAGAACCTCTCTCAGAGATCG.

9. The internal reference standard, competitor sequence or PCR MIMIC according to claim 7 being:

CATGTGGATATTTTGCAGTCCCTCGTGGACCCTTATATCTTTGTTCT

TTCCTTTTCCTTGTGGCTGAGGAGTGTTCTTGTTTCCGAGCTCCACTTTCG AGTACTCGGTTTCTGTGATGCTGGCTTAATCGGTTCCAACCGACCCGTCT

TGAAACACGGACCAAGGAGTCTAACATATGTGCGAGTATGCGGGTTTTA CTCCTGTATGCGCAATGAAAGTGAGAGTAGGGAGATTTTGGCTTTGCCAT

TCTTCGCACCTACGACCGACCACGATCTCTGAGAGAGGTTCTTTAACTTC

CAGGAGTTT.

10. Use of primer pairs TimllF and GAl; Timl2 and GAl; Timlδ and Timl2: GAδ and GA6; or NF6 and GAl for PCR amplification of the LSU rDNA of Neospora spp.

11. The use according to claim 10 wherein the primers are GAl and NF6 and the PCR amplification giving a Neospora species-specific PCR product of 270 base pairs. 12. The use according to claim 10 or 11 wherein the Neospora spp is

Neospora caninum.

13. The use according to any one of claims 10 to 12 further including the internal reference standard, competitor sequence or PCR MIMIC according to claim 8 or 9. 14. The use according to claim 13 further including an internal reference standard, competitor sequence or PCR MIMIC specific for Toxoplasma gondii having the following priming sites:

IP3 (5'-TCCATTGGAGAGATTTGCGTTCCTTGTGGACCG) and

IP4 (5'-AAACTCCTGGAAATCAGTAAACCTCTCTCAGAGATCG) . lδ. The use according to claim 13 further including an internal reference standard, competitor sequence or PCR MIMIC specific for Toxoplasma gondii having the following sequence:

TCCATTGGAGAGATTTGCGTTCCTTGTGGACCGTTATATCTTTGTTCTTTC

CTTTTCCTTGTGGCTGAGGAGTGTTCCTGTTTCCGAGCTCCACTTTCGAGT ACTCGGTTTCTGTGATGCTGGCTTAATCGGTTCCAACCGACCCGTCTTGA

AACACGGACCAAGGAGTCTMACATATGTGCGAGTATGCGGGTTTTACTC

CTGTATGCGCAATGAAAGTGAGAGTAGGGAGATTTTGGCTTTGCCATTCT

TCGCACCTACGACCGACCACGATCTCTGAGAGAGGTTTACTGATTTCCAG

GAGTTT. 16. A method of obtaining an isolated nucleic acid molecule encoding the

LSU ribosomal DNA of Neospora spp or part thereof, the method comprising amplifying the nucleic acid molecule by PCR using primers according to any one of claims 3 to 7.

17. The method according to claim 16 wherein the amplifying of the LSU ribosomal DNA of Neospora spp is used to identify or diagnose the presence of Neospora spp in a clinical sample.

18. The method according to claim 17 wherein differential diagnosis between Neospora spp and Toxoplasma spp or other related or non-related microorganisms is obtained.

19. The method according to any one of claims 16 to 18 wherein the Neospora spp is Neospora caninum.

20. The method according to any one of claims 16 to 19 further including the internal reference standard, competitor sequence or PCR MIMIC according to claim 8 or 9.

21. The method according to claim 20 further including an internal reference standard, competitor sequence or PCR MIMIC specific for

Toxoplasma gondii having the following priming sites:

IP3 (δ'-TCCATTGGAGAGATTTGCGTTCCTTGTGGACCG) and

IP4 (δ'-AAACTCCTGGAAATCAGTAAACCTCTCTCAGAGATCG) .

22. The method according to claim 20 further including an internal reference standard, competitor sequence or PCR MIMIC specific for

Toxoplasma gondii having the following sequence:

TCCATTGGAGAGATTTGCGTTCCTTGTGGACCGTTATATCTTTGTTCTTTC CTTTTCCTTGTGGCTGAGGAGTGTTCCTGTTTCCGAGCTCCACTTTCGAGT ACTCGGTTTCTGTGATGCTGGCTTAATCGGTTCCAACCGACCCGTCTTGA AACACGGACCAAGGAGTCTMACATATGTGCGAGTATGCGGGTTTTACTC

CTGTATGCGCAATGAAAGTGAGAGTAGGGAGATTTTGGCTTTGCCATTCT TCGCACCTACGACCGACCACGATCTCTGAGAGAGGTTTACTGATTTCCAG GAGTTT.