Patent Grant
8206908
This application is a national stage application filed under 35 U.S.C. 371 of International Application No. PCT/IB2006/003870, filed September 25, 2006.
Rickettsiae are intracellular pathogenic bacteria responsible for various diseases on Humans and animals. Rickettsiae are transmitted by arthropods, most frequently ticks, lice and mites, and cause major illnesses such as epidemic typhus or Rocky Mountain spotted fever. The genus Ehrlichia comprises other species pathogenic for humans and mammals such as E. chaffeensis, responsible for Human monocytic ehrlichiosis, E. canis, the causing agent of canine monocytic ehrlichiosis.
Another species, Ehrlichia ruminantium, formerly known as Cowdria ruminantium, is the causing agent of heartwater or cowdriosis, an economically important disease of domestic ruminants. Heartwater can cause up to 80% mortality in susceptible animals. E. ruminantium is transmitted by Amblyomma ticks and is present in Sub-Saharan Africa and surrounding islands, including Madagascar. Heartwater is also present in several Caribbean islands and is threatening the American mainland.
Vaccination against heartwater has long been based on “infection and treatment”. Naïve animals are inoculated with blood containing virulent organisms, a procedure which carries a high risk of uncontrolled clinical reactions and the inadvertent spread of undesirable parasites and viruses. A first generation cowdriosis inactivated vaccine based on cell-cultured derived elementary bodies was developed. Although representing a considerable improvement and the first heartwater vaccine acceptable for widespread use, the level of protection conferred is still not fully satisfactory. Indeed, all animals develop a clinical reaction at challenge despite vaccination. Furthermore, livestock also faces challenge by genetically and antigenically diverse strains.
Diversity of E. ruminantium is a key problem which has been recognized for a long time, but insufficient information is available for optimum vaccine formulation and specific diagnostic. Serological diagnostic tests of heartwater using crude antigens from whole bacteria detect false positive reactions due to common antigenic determinants
The diversity of E. ruminantium was demonstrated at the antigenic level by cross-immunisation studies. Variable antigens were identified by ELISA and immunoblot using cross-absorbed immune sera.
Genetic diversity was later demonstrated when sequencing the Map 1 gene which showed a high degree of sequence heterogeneity concentrated in three hypervariable regions. Genomic polymorphism was also detected using RAPD and RFLP markers. This DNA polymorphism was shown to correlate with antigenic polymorphism.
ELISA-based and serological diagnostics have been developed using the Map 1 and the GroEL (WO 9914233) antigens. Other peptides for serological diagnostic have been described (US 2002004051, US 20020132789, WO 02/066652). Although they have dramatically improved specificity, they still display cross reaction with E. canis and E. chaffeensis. The map1 gene initially considered as a good marker for geographic diversity, was recently shown not to be geographically constrained. Furthermore, the life span of anti-Map 1 antibodies is rather short.
PCR-based diagnostic methods represent methods of choice for the sensitive and specific detection of Ehrlichia in clinically reactive or asymptomatic carrier ruminants, as well as in vectors. However, in the field, hosts and vectors can be co-infested by several parasites and the diversity of pathogen species is further complicated by the existence of extensive intra-species diversity. Thus, it is important to provide means and diagnostic tools allowing not only to identify E. ruminantium but also to differentiate between different strains.
Sequences allowing differential diagnostic of E. ruminantium strain Gardel and E. ruminantium strain Welgevonden have been previously described by the inventors. They have shown, through complete genome sequencing and comparative genomic analysis that several genes were only found in either strain Gardel or strain Welgevonden, without counterpart in the other strain, and that several other genes, while being present in both strains differed between them by one or several mutations, such as large insertions and/or deletions that result in a frameshift and/or in a truncated version of the original gene. These genes were therefore primary targets to develop specific, multitarget diagnostic methods to differentiate between these two strains (WO 2006/045338; Frutos et al., Journal of Bacteriology. 188: 2533-2542, 2006).
The inventors have now found that the use of a particular combination of some of the target genes described in WO 2006/045338 allowed not only to discriminate between strains Gardel and Welgevonden, but also in a more general way, to detect specifically E. ruminantium and to discriminate between a broad range of strains of E. ruminantium other than Gardel and Welgevonden including strains for which no genomic sequence data are available.
An object of the invention is thus the use of the following set of genes:
Erum1, defined by the sequence SEQ ID NO: 6
Erum2, defined by the sequence SEQ ID NO: 3
Erum3, defined by the sequence SEQ ID NO: 1
Erum4, defined by the sequence SEQ ID NO: 4
Erum5, defined by the sequence SEQ ID NO: 2
Erum6, defined by the sequence SEQ ID NO: 5
Erum7, defined by the sequence SEQ ID NO: 13
Erum8, defined by the sequence SEQ ID NO: 15
Erum9, defined by the sequence SEQ ID NO: 14
Erum10, defined by the sequence SEQ ID NO: 8,
as targets for the strain-specific detection of Ehrlichia ruminantium.
The reference sequences used herein to define the target genes Erum 1-5 and Erum 7-9 are those identified in the Gardel strain; the reference sequences used herein to define the target genes Erum6 and Erum10 are those identified in the Welgevonden strain.
However, it is to be understood that each of these genes actually exists under different allelic forms, depending on the strain of Ehrlichia ruminantium. The allelic forms that will be considered herein, having in view strain-specific detection, are in particular those resulting from large insertions and/or deletions that lead to a frameshift or to a truncated version of the original gene.
The invention thus provides a method for the strain-specific detection of Ehrlichia ruminantium wherein said method comprises determining, for each of the genes Erum 1 to Erum10 defined above, whether said gene is present in the bacteria to be tested, and under which allelic form.
Advantageously, the method of the invention is carried out by performing PCR amplification of all the target genes Erum 1 to Erum10, and checking, for each of these genes, the presence of one or more amplification product(s), and the size of said amplification product(s).
Within the target genes Erum 1 to Erum10, preferred target regions are as follows:
For Erum 1, the target region can consist of the whole sequence SEQ ID NO: 6, or of a portion thereof; in particular the target region can be defined within the portion spanning from nucleotide 1 to nucleotide 173 of SEQ ID NO: 6.
For Erum 2, the target region can consist of the whole sequence SEQ ID NO: 3, or of a portion thereof; in particular the target region can be defined within the portion spanning from nucleotide 1 to nucleotide 218 of SEQ ID NO: 3.
For Erum 3, the target region can consist of the whole sequence SEQ ID NO: 1, or of a portion thereof; in particular the target region can be defined within the portion spanning from nucleotide 1 to nucleotide 509 of SEQ ID NO: 1.
For Erum 4, the target region can consist of the whole sequence SEQ ID NO: 4, or of a portion thereof; in particular the target region can be defined within the portion spanning from nucleotide 56 to nucleotide 698 of SEQ ID NO: 4.
For Erum 5, the target region can consist of the whole sequence SEQ ID NO: 2, or of a portion thereof; in particular the target region can be defined within the portion spanning from nucleotide 1 to nucleotide 239 of SEQ ID NO: 2.
For Erum 6, the target region can consist of the whole sequence SEQ ID NO: 5, or of a portion thereof; in particular the target region can be defined within the portion spanning from nucleotide 3 to nucleotide 130 of SEQ ID NO: 5.
For Erum 7, a preferred target region is located within the portion spanning from nucleotide 1 to nucleotide 1981 of SEQ ID NO: 13; another preferred target region is located within the portion spanning from nucleotide 2378 to nucleotide 3252 of SEQ ID NO: 13.
For Erum 8, a preferred target region is located within the portion spanning from nucleotide 1 to nucleotide 926 of SEQ ID NO: 15; another preferred target region is located within the portion spanning from nucleotide 1816 to nucleotide 3570 of SEQ ID NO: 15.
For Erum 9, a preferred target region is located within the portion spanning from nucleotide 1 to nucleotide 1307 of SEQ ID NO: 14; another preferred target region is located within the portion spanning from nucleotide 151 to nucleotide 1836 of SEQ ID NO: 14.
For Erum 10, a preferred target region is located within the portion spanning from nucleotide 1 to nucleotide 598 of SEQ ID NO: 8; another preferred target region is located within the portion spanning from nucleotide 792 to nucleotide 3522 of SEQ ID NO: 8; still another target region is located within the portion spanning from nucleotide 599 to nucleotide 791 of SEQ ID NO: 8.
Various techniques for detection of target nucleic acid sequences based on PCR amplification are available in the art.
These methods include in particular combined PCR analysis, i.e. simultaneous gel visualization of ten individual PCR reactions, each one targeting only one of the genes Erum1 to Erum10 defined above. The ten target genes can also be analysed by multiplex PCR, by a single PCR reaction involving simultaneous amplification of all the genes using a mixture of primers and visualization of the pattern on electrophoresis gel, or by a combination of multiplex PCR reactions, each one concerning a subset of the target genes listed above.
Non-limitative examples of PCR primers allowing to carry out the method of the invention are given in Table 2 below. Other suitable PCR primers can easily be designed by one of skill in the art, on the basis of the information provided by the present invention. By way of non-limitative example of oligonucleotide design software suitable for obtaining PCR primers of the invention, one can mention the software Vector NTI Advance 9.0 (Invitrogene).
The invention also comprises diagnostic kits for discriminating between strains of E. ruminantium wherein said kits comprise PCR primers for all the target genes Erum 1 to Erum10.
The method of the invention is useful in particular to discriminate between strains of E. ruminantium other than strain Gardel and strain Welgevonden. It is also useful to discriminate between strain Gardel and strains of E. ruminantium other than strain Welgevonden, or conversely, between strain Welgevonden and strains of E. ruminantium other than strain Gardel. Furthermore, it also allows for discriminating between a virulent strain of E. ruminantium and its attenuated counterpart.
The method of the invention can be performed either on whole bacteria previously lysed, or on nucleic acid (genomic DNA, cDNA or mRNA) isolated from said bacteria. It is suitable for use at various stages of the life cycle of E. ruminantium, more specifically but not limited to the domestic-ruminants infectious stage, vector-interaction stage or reservoir animals-interaction stage. Preferred utilisations of the method of the invention include the detection of Ehrlichia ruminantium in a given territory, the strain specific identification of Ehrlichia ruminantium in a given territory, the discrimination between strains of Ehrlichia ruminantium in a given territory or between different geographical regions, the analysis of strain movements within a region or between geographically distinct regions, the differential presence of strains of Ehrlichia ruminantium according to vector species and/or populations or the early detection and risk assessment in regions where potential vectors are present but where the disease has not been recorded yet.
Specifically exemplified herein is the identification of E. ruminantium strains based on the specific amplification patterns of the ten target genes defined above.
For each strain, purified DNA was broken by sonication to generate fragments of differing sizes. After filling up the ends with Klenow polymerase, DNA fragments ranging from 0.5 kb to 4 kb were separated in a 0.8% agarose gel and collected after gelase (Epicentre) digestion of a cut agarose band. Blunt-end DNA fragments were inserted into pBluescript II KS (Stratagene) digested with EcoRV and dephosphorylated. Ligation was performed with the Fast-Link DNA Ligation kit (Epicentre) and competent DH 10B E. coli were transformed prior to colony isolation on LB-agar+Ampicillin+Xgal+IPTG. About 15000 clones were isolated for each strain of E. ruminantium. Plasmidic DNA from recombinant E. coli strains was extracted according to the alkaline lysis method and inserts were sequenced on both strands using universal forward and reverse M13 primers and the ET DYEnamic terminator kit (Amersham). Sequences were obtained with ABI 373 et ABI 377 automated sequencers (Applied Biosystems). Data were analysed and contigs were assembled using Phred-Phrap and Consed software packages (http://www.genome.washington.edu). Gaps were filled in through primer-directed sequencing using custom made primers. A total of about 20000 raw sequence runs were generated and analysed for each E. ruminantium strain to generate a full length consensus sequence with a coverage of 6× to 7×.
E. ruminantium strain Gardel and E. ruminantium strain Welgevonden are virulent pathogenic strains causing heartwater in Guadeloupe Island (French West Indies) and South Africa, respectively. The genome of E. ruminantium strains Gardel and Welgevonden is arranged as a circular chromosome of 1499920 bp and 1512977 bp, respectively. The respective G+C contents for the strains Gardel and Welgevonden is 27.51% and 27.48%. The genome of E. ruminantium strain Gardel comprises 948 coding sequences of an average size of 1018 bp which represent a total coding surface of 63% of the whole genome. The genome of E. ruminantium strain Welgevonden bears 957 genes of the same average size of 1018 bp. The genome surface of this strain devoted to coding sequences is 62%. Both genomes comprise 36 transfer RNAs (tRNA) and 3 ribosomal RNAs (rRNA).
The differential analysis of the whole genomes of E. ruminantium strains Gardel and Welgevonden showed the presence of coding sequences which are present in only one of the strains and not in the other. Some of the CDS which are unique to E. ruminantium strain Gardel and found only in the genome of this strain are presented in Table 1 (Seq ID NO 1 to Seq ID NO 5). One of the CDS which is unique to E. ruminantium strain Welgevonden and found only in the genome of this strain is presented in Table 1 (Seq ID NO 6). Since these sequences are unique to one or the other strain, they clearly represent targets for the differential detection of E. ruminantium strain Gardel versus E. ruminantium strain Welgevonden.
The differential analysis of the whole genomes of E. ruminantium strains Gardel and Welgevonden also showed the presence of coding sequences which are affected by one or several mutations in one of the two strains and for which a non-mutated, functionally active and normal allele is present in the genome of the other strain. Mutations yielded a stop codon which may result in shorter but still predicted CDS depending upon the size of the remaining fragments. Truncated genes resulting in a single CDS are denominated partial CDS, whereas those resulting in two or more predicted CDS are described as fragmented CDS. These coding sequences are presented in Table 1. One Such CDS in the genome of E. ruminantium strain Gardel which is affected by mutations and differs from its native counterpart in E. ruminantium strain Welgevonden is presented in Table 1 (SEQ ID NO 7). This is a truncated version of the native gene in E. ruminantium strain Welgevonden (Table 1, SEQ ID NO 8). The genome of E. ruminantium strain Welgevonden also bears mutated genes, with respect to their allelic variant counterparts in the genome of E. ruminantium strain Gardel. Three of these CDS which are affected by mutations generating a truncated version of the genes are presented in Table 1 (SEQ ID NO 9 to SEQ ID NO 12). The native full length allele of these CDS present in the genome of E. ruminantium strain Gardel are shown in Table 1 (SEQ ID NO 13 to SEQ ID NO 15). One series of CDS in E. ruminantium strain Welgevonden (SEQ ID NO 11 and SEQ ID NO 12), whose native full length alleles are found in the genome of E. ruminantium strain Gardel (Table 1, SEQ ID NO 15) was affected by mutations generating a frameshift.
Differential PCR identification of strains Gardel and Welgevonden of E. ruminantium was achieved using primers described in Table 2.
DNA is extracted from elementary bodies of E. ruminantium, as described by Perez et al. (1997). E. ruminantium strains are grown in BUEC cells as described above. Elementary bodies are purified from the culture supernatant by differential centrifugation and resuspended in 350 μl of PBS to which is added 150 μl of buffer containing 25 mM Tris-HCl (pH 8.0), 10 mM MgCl2 and 125 μg of DNase in order to remove contaminating host cell DNA. After incubation for 90 min. at 37° C., the reaction is stopped by addition of 25 mM EDTA. Elementary bodies are washed three times in water and lysed by overnight incubation at 55° C. in a solution of 100 mM Tris-HCl (pH 8.0), 150 mM NaCl, 25 mM EDTA, 1.5% SDS and 250 μg/ml of proteinase K. Bacterial DNA is extracted with phenol-chloroform, precipitated with cold ethanol an resuspended in sterile distilled water. Contamination with cell DNA is evaluate by slot blot hybridization using labeled bovine DNA as a probe and dilutions of bovine DNA (12.5 ng and 25 ng) as positive controls.
PCR amplification of amplicons is performed by mixing 250 ng of E. ruminantium DNA, 2.5 U of Taq DNA polymerase, 200 nM of each dNTP, 1 μM of each, sense and antisense, primer and 3 mM MgCl2 in a final volume of 50 μl. Amplification is done under the following conditions: 5 min denaturation at 94° C., followed by 30 cycles of amplification with a 1-min denaturation, 45 sec of annealing at 45° C. and 2 min extension at 72° C. An extra extension step of 10 min at 72° C. is added after completion of the 30 cycles. PCR products, i.e. amplicons, are analysed by 1% agarose gel electrophoresis in Tris-borate-EDTA buffer.
The results are summarized in Table 3,
Legend of
As shown in Table 3,
The primer pairs P-1350-A+P-1350-B, P-4510-A+P-4510-B, P-5750-A+P-5750-B and P-7420-A+P-7420-B yielded PCR products of the respective expected size of 2791, 552+1071, 1361 and 1095 on strain Gardel and 2395, 492, 1178 and 1691 on strain Welgevonden, respectively (Table 3,
The use primers listed in Table 2 were used for the specific identification and discrimination of E. ruminantium strains other than strain Gardel and strain Welgevonden. The strains others than Gardel and Welgevonden presented in this example are strains Umpala (Mozambique), Senegal (Senegal), Bankouma (Burkina Faso), Bekuy (Burkina Faso), Lamba (Burkina Faso), Banan 1 (Burkina Faso) and Banan 2 (Burkina Faso). These strains are presented here to illustrate samples from different parts of Sub-Saharan Africa and the Caribbean.
DNA is extracted from elementary bodies of E. ruminantium and PCR amplification performed as described in Example 3.
The results are shown in Table 4,
Legend of
As shown in Table 4,
It is however the overall analysis of all the PCR patterns yielded by all the pairs of primers described in Table 2 which provides a strain specific diagnostic. The strains Bekuy and Lamba which were isolated in Burkina Faso from the nearby villages of Bekuy and Lamba, respectively, are most likely to be two isolates of the same strain. Furthermore, these strains display the same map-1 genotype determined by PCR amplification and sequencing of the map-1 gene. All the other strains display differing map-1 genotypes. This further indicates that strains Bekuy and Lamba are two isolate of the same strain. The identical overall pattern obtained for these two strains with all the pairs of primers described in Table 2 also further demonstrate the strain-specificity of the subject of the invention and its ability to identify different strains and separate isolates of the same strain.
642a
apresence of additional multiple bands is observed
The primers listed in Table 2 also allow the specific identification of attenuated variants of known strains of E. ruminantium.
The following variants were tested:
DNA is extracted from elementary bodies of E. ruminantium and PCR amplification performed as described in Example 3.
The results are shown in Table 5,
Legend of
As shown in Table 5,
To verify the specificity assessment of the primers listed in Table 2, they were tested on Rickettsiales belonging to other species and genera than E. ruminantium i.e. Ehrlichia canis, Anaplasma platys and Anaplasma marginale.
DNA extraction and PCR amplification were performed as described in Example 3.
The results are shown in
Legend of
The tools provided by the invention allow thus both for specific detection of E. ruminantium, even in presence of contaminating related Ricketssiales, for specific discrimination between different strains of E. ruminantium and for specific discrimination between a virulent strains and its vaccinal attenuated derivates. This in turn allows for monitoring of vaccination.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2006/003870 | 9/25/2006 | WO | 00 | 6/22/2009 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2008/038062 | 4/3/2008 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 5523217 | Lupski et al. | Jun 1996 | A |
| 7919436 | Frutos et al. | Apr 2011 | B2 |
| Number | Date | Country |
|---|---|---|
| WO 2006045338 | May 2006 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20090301880 A1 | Dec 2009 | US |
