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Mycobacterium avium subspecies paratuberculosis diagnosis and geno-typing: Genomic insights
ARTICLE IN PRESS Microbiological Research 164 (2009) 330—337
www.elsevier.de/micres
Mycobacterium avium subspecies paratuberculosis diagnosis and geno-typing: Genomic insights J.S. Sohala, S.V. Singha,, Swati Subodhc, Neelam Sheoranb, K. Narayanasamyb, P.K. Singha, A.V. Singha, A. Maitrad a
Central Institute for Research on Goats, Makhdom-281 122, PO-FARAH, Mathura, UP, India Institute of Molecular Medicine, Okhla Industrial Area, Phase–III, New Delhi, India c Thrombosis Research Institute, Bangalore, India d McGill University Health Centre, Research Institute Rs1-105, Montreal, Que., Canada H3G 1A4 b
Received 6 January 2007; accepted 19 March 2007
KEYWORDS Mycobacterium avium subspecies paratuberculosis; Genotyping; PCR-RFLP; IS elements
Summary Effective control of paratuberculosis and investigations of potential link to Crohn’s disease have been hampered by the lack of effective assays for easy and accurate diagnosis of Mycobacterium avium subspecies paratuberculosis (Map). Map is extremely fastidious and depends on iron chelator (Mycobactin). Map strains from humans and sheep are very difficult to isolate and may require years to emerge. Therefore, small numbers of Map isolates have been maintained in available collections. This situation has limited the study of biodiversity of Map. Though, much is known about environmental and host factors that contribute to paratuberculosis disease, but little is known about bacterial genetic mechanism of infection. Diagnostic and strain typing markers still demand improvements. Complete genome sequence of Map K10 strain is available in public domain for comparative genomics with other mycobacteria and clinical isolates of Map. It is anticipated that the genome sequence will help in carrying molecular diagnosis and strain typing with respect to Map forward at rapid pace. This paper reviews the current diagnostic and strain typing markers, which may be useful in typing of clinical isolates in near future. & 2007 Elsevier GmbH. All rights reserved.
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+91 565 2736262 (R), 09719072856 (Mobile); fax: +91 565 2763246. E-mail addresses: [email protected], [email protected] (S.V. Singh).
Introduction Viewed in terms of its historical and contemporary disease burden Mycobacteria are undeniably most successful human and animal pathogens.
0944-5013/$ - see front matter & 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2007.03.005
ARTICLE IN PRESS Mycobacterium avium subspecies paratuberculosis diagnosis Irrespective of intense research, paratuberculosis caused by Mycobacterium avium subspecies paratuberculosis (Map), still remains single most important cause of morbidity in ruminants leading to huge economic losses, world over. JD is endemic in cattle in developed countries and variable prevalence of Map has been reported from clinically (5–55.0%; Taylor et al., 1981; Giese and Ahrens, 2000) and sub-clinically (2–12.0%; Sweeney et al., 1992; Streeter et al., 1995; Jakobsen et al., 2000) ill cows. Herd prevalence of bovine paratuberculosis estimated for European countries ranged from 3.5% to 71.0% (South West of England – 3.5%, Belgium – 8.0%, Czech Republic – 12.0%, Italy 13.3%, Denmark, 70.0%, The Netherlands 31.0–71.0% by Cetinkaya et al. (1996), Boelaert et al. (2002), Kennedy and Benedictus (2001), Jakobsen et al (2000) and Muskens et al. (2000). Using protoplasmic antigen from ‘Bison Type’ genotype of Map, Singh et al. (2005) reported sero-prevalence of paratuberculosis in bovines and goats as, 29.05% (buffaloes, 28.67% and cattle, 29.86%) and 23.63%, respectively, in Northern India. Cumulative sero-prevalence in Utter Pradesh and Punjab states of Northern India was 32.9% and 23.0%, respectively. Using sensitive Gold standard method (culture), high prevalence of Map has been recorded in India from cattle milk and buffalo tissues, young and adult goats and sheep. A number of Map-specific genomic sequences such as the insertion sequence IS900 (Green et al., 1989), F57 element (Poupart et al., 1993), and hsp X gene (Ellingson et al., 1998), have been identified. Of these multi-copy IS900 is a target of choice and is the most widely used in characterization of Map. There are concerns about its specificity and validity as a direct proxy for Map. Furthermore, as some members of the Mycobacterium tuberculosis complex, do not contain IS 6110 (Agasino et al., 1998). It is possible that some strains of Map are either low copy number or IS 900 negative, providing additional arguments for the need to develop molecular tests that rely on genomic characteristics other than mobile elements. Even 17 years after the IS 900 was discovered, the promise of a fast, affordable, specific and sensitive PCR for application to clinical samples remains elusive goal. In case of F57 and hsp X, their specificity for Map has not been rigorously evaluated in large numbers of clinical isolates. Commercially available serological assays for bovine paratuberculosis though convenient but suffer from poor specificity and sensitivity, especially during sub-clinical phases of the disease. Other limiting factor for Map studies is the lack of convenient strain typing methods. Most widely used
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method for typing Map isolates is RFLP based on IS 900 and IS1311 PCR-REA (Whittington et al., 2000; Stevenson et al., 2002). These methods are either applicable to cultivable strains or slow, technically demanding and have low-resolution power. Pulsefield gel electrophoresis of Map isolates provides more refined data for epidemiological interpretation (Stevenson et al., 2002). Unfortunately, the technique also requires large quantities of DNA and applicable to cultivable strains. rRNA gene and spacer region analysis has also been tested for the ability to distinguish Map strains. The RFLP analysis of the 5S rRNA gene region (Chiodini, 1990) identified indistinguishable restriction fragments of the 5S rRNA gene region in all Map isolates studied. An analysis of the spacer region of the rDNA indicated lack of discrimination as it revealed an identical sequence for all Map isolates tested (Scheibl and Gerlach, 1997). Recently developed technique to type Map isolates based on Multiplex PCR for IS 900 loci (MPIL), have almost similar discriminatory power (Motiwala et al., 2004) to IS 900 RFLP. Amplified fragment length polymorphism (AFLP) appears to have greater resolving power than MPIL and RFLP but suffers from the limitation that the allelic variation is indexed at anonymous bi-allelic sites or locations. Additionally, repeatability and interlaboratory variation limits its application in multiple site analyses. Due to disadvantages of the current methods, rapid and robust molecular methods are required to be assessed as alternatives for studying Map strain diversity. Since there is high degree of similarity at genomic levels between Map and other members of Mycobacterium avium complex (MAC), ability to differentiate between these closely related organisms is essential for rational clinical and epidemiological assessment. History clearly documents that we cannot detect all the infected animals simply. Though diagnostic tests have improved over the years but still there is room for improvement. This paper reviews the post-genomic identified targets of Map diagnosis and strain typing and also describes the future strategies to diagnose and type Map isolates.
Genome overview Map K10 genome is a single circular chromosome of 4,829,781 base pairs with G+C content of 69.3% and encodes 4350 predicted ORFs, 45 tRNAs and one rRNA operon (Table 1). In silico analysis identified 43000 genes homologous to human pathogen (Mtb), there is truncation in the Ent E
ARTICLE IN PRESS 332 Table 1.
J.S. Sohal et al. Summary of Mycobacterial genomes
Property
Map
Mav
Mtb
M. bovis
M. leprae
M. smegmatis
Genome size (bp) G+C content (%) Protein coding (%) ORFs Average gene length (bp) TRNAs rRNA operon
4,829,781 69.30 91.30 4350 1015 45 1
5,475,738 68.99 NA NA NA 45 1
4,411,532 65.61 90.80 3959 1012 45 1
4,345,492 65.63 90.59 3953 995 45 1
3,268,203 57.79 49.50 1604 1011 45 1
6,988,209 67.40 92.42 6897 936 47 2
domain of a salicyl-AMP ligase (MbtA), the first gene in the mycobactin biosynthesis gene cluster, providing a possible explanation for mycobactin dependence of Map. Approximately 1.5% (or 72.2 kb) of the Map genome is comprised of repetitive DNA-like insertion sequences, multigene families, and duplicated housekeeping genes. There are a total of 19 different IS elements with 58 copies in Map K10 genome.
Large sequence polymorphisms (LSPs) Recent work in mycobacterial genomics has revealed LSPs as major contributor of genetic diversity. LSPs have been employed for inferences of phylo-genetics (Brosch et al., 2002; Mostowy et al., 2002) and biological properties such as virulence (Kato-Maeda et al., 2001; Pym et al., 2002) of Mycobacterium tuberculosis (Mtb) complex, thus it is hypothesized that LSPs are important sources of genetic variability among MAC organisms. In silico comparisons of the genome sequences of Map K10 (NC002944) and M. avium subsp. avium (Mav) strain 104 (http://www. tigr.org) genomes have identified DNA sequences that are present in the former but missing in the latter (Bannantine et al., 2002, 2004; Paustian et al., 2004). Semret et al. (2005) by in silico analysis initially identified 17 LSPs varying in length from 2.9 to 66 kb that were unique to Map K10. These Map unique LSPs were designated as LSPPs. Microarray comparisons by Semret et al. (2004) using M. avium subsp. avium strain 104 as the reference identified regions designated them as LSPAs, specific for Mav and absent from Map isolates. LSPs and Map diagnosis Semret et al. (2005) tested the distribution of 17 LSPPs (Map specific) and 3 LSPAs (absent in Map) by PCR-based strategy across a panel of 383 M. avium complex isolates. Screening panel of 383 MAC isolates (107 Map+276 non-Map), revealed that LSPP2, LSPP4, and LSPP15 could not be amplified
Table 2.
Map-specific LSPPs
LSP
Sensitivity (%)
Specificity (%)
LSPP 2 LSPP 4 LSPP 15
41.1 68.2 90.7
100 100 100
from any of the 276 non-Map isolates, demonstrating 100% specificity of these LSPPs for this subspecies (Map). Of the Map specific LSPPs specific for LSPP15 consistently gave positive results (high sensitivity) across Map isolates (Table 2), while amplification of other LSPPs was variable (Semret et al., 2005). Among the LSPAs absence of LSPA8 was 100% specific for Map (Semret et al., 2005, 2006). This sequence was consistently missing from Map isolates and is present in every other M. avium complex strain tested (100% sensitive). This is akin to the RD1 region of the M. tuberculosis complex, which permits accurate differentiation between virulent Mycobacterium bovis and BCG strains (Talbot et al., 1997). Others LSPAs, LSPA13 and LSPA14 were also found to be absent in Map, but their absence may not be considered 100% specific for Map, since these sequences were invariably missing from some of non-Map isolates (Semret et al., 2005). Microarray-based genomic comparison by Paustian et al. (2005) also identified genomic regions divergent between Map and other members of the M. avium complex as potentially unique to Map. These regions represent LSPPs described by Semret et al. (2005). LSPs and Map strain typing The ability to both detect and differentiate between types of strains is of obvious importance to guide control programs. Semret et al. (2006), proposed a simple PCR-based strategy to rapidly type the Map isolates based on the presence or absence of LSPs. They studied the distribution of LSPs across a panel of strains and were able to assign unique genomic signatures to host-associated variants. The results show that, LSPA 4-II
ARTICLE IN PRESS Mycobacterium avium subspecies paratuberculosis diagnosis and LSPA 18 were present in S (Sheep) strains but missing from the two C (Cattle) strains. Another sequence, LSPA 20, was present in the C strains but missing from the S strains. Similar type of strategy of distribution of polymorphisms has been applied to differentiate members of M. tuberculosis complex (Talbot et al., 1997; Parsons et al., 2002). Findings of Semret et al. (2006) for the regions missing from C strains of Map are in agreement with representational difference analysis (RDA)-based study of Dohmann et al. (2003), that identified 3 loci missing from type II (C type) strains. The 233bp locus identified as pig-RDA10 (AY266300) by Dohmann et al. (2003), forms part of the 16-kb region called as LSPA 18 by Semret et al. (2006). The 197-bp locus identified as pig-RDA20 (AY266301) by Dohmann et al. (2003), is located within the 26-kb segment called as LSPA 4-II by Semret et al. (2006).
Map unique IS elements Genome sequencing of Map K10 had identified three more Map unique IS elements; ISMav2, ISMAP02 and ISMAP04 present in 3, 6 and 4 copies, respectively, in Map K10 genome in addition to previously known IS 900 (Li et al., 2005). Small-scale studies using these newly identified Map unique IS elements have provided encouraging results. Strommenger et al. (2001) had shown that ISMav2 has potential to act as Map diagnostic Marker, but has not yet been adequately evaluated at large scale. Stabel and Bannantine (2005) evaluated the sensitivity and specificity of the ISMAP02 element in both conventional and realtime PCR tests for the detection of Map in bovine fecal samples in comparison to the sensitivity and specificity of the IS900 element target. They found that ISMAP02 is 100% specific for Map. The sensitivity of detection for the ISMAP02 element in either conventional or real-time PCR format was less than 100 fg DNA or 102 CFU/ml. The results were comparable to those obtained for the IS900 element. PCR analyses of ISMAP02 by Paustian et al. (2004), also demonstrated that the sequence was present in all 39 isolates of Map examined but was absent from all other mycobacterial species and subspecies tested. This analysis further substantiates the specificity of this sequence. To date, there is no data available on the use of ISMAP04 as specific diagnostic marker for Map.
Variable number tandem repeat (VNTR) analysis Tandem repeat sequences often represent polymorphic structures in the genomes of microbes.
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VNTR typing offers a promising PCR-based study for typing of mycobacteria based on variation in repeat numbers. Overduin et al. (2004) explored the strain typing ability of VNTRs by multilocus variable number tandem repeat analysis (MLVA) and compared with IS 900 RFLP. By testing 20 VNTR loci across 49 Map isolates, they identified 5 polymorphic VNTRs. These 5 polymorphic VNTR loci yielded 6 VNTR types for 49 Map isolates and were able to subdivide the most predominant IS 900 RFLP type, R01, into six subtypes. Interesting finding was all human isolates revealed the same MLVA type as found for the majority of the cattle strains. Biet et al. (2005) identified 8 polymorphic MIRU-VNTR loci by screening 72 Map isolates; they found 12 different MIRU-VNTR types for these 8 polymorphic repeat elements. In total, MIRU-VNTR grouped 69 isolates into 9 clusters whereas 3 MIRU-VNTR patterns were unique. MIRU-VNTR typing sub-divided the major RFLP type into 11 different MIRU-VNTR types, thus providing an additional method to study the diversity of Map.
Short sequence repeats (SSRs) and Map typing SSRs in bacterial DNA have been used as markers for the differentiation and sub-typing strains of several bacterial species, including M. tuberculosis (Gascoyne-Binzi et al., 2001; Kremer et al., 1999). It has been recognized that regions of mono-, di-, and tri-nucleotide tandem repeats are often the most diverse in a bacterial genome, while complex longer repeats generally have lower levels of diversity (Keim et al., 2000). Results of multi locus short sequence repeat (MLSSR) sequencing by Amonsin et al. (2004), enabled facile and reproducible high-resolution sub-typing of Map isolates for molecular epidemiologic and population genetic analyses. They identified 11 poly-morphic short sequence repeats (SSRs), with an average of 3.2 alleles per locus. This analysis differentiated the 33 Map isolates into 20 distinct MLSSR types. MLSSR analysis easily and reproducibly differentiated strains representing the predominant MPIL genotype and AFLP genotypes. Further analysis of Map isolates using SSR analysis by Motiwala et al. (2004), enabled the differentiation of the Map isolates in clade A18 (by MPIL analysis) into seven distinct alleles. The analysis revealed eight subtypes among the 33 species of animals, suggesting the interspecies transmission of specific strains. Also SSR analysis of Map isolates by Ghadiali et al. (2004), from CD patients identified two alleles,
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J.S. Sohal et al.
both of which clustered with strains derived from animals with JD (Fig. 1).
Single nucleotide polymorphisms (SNPs) and Map typing SNPs are the most abundant form of DNA polymorphism. Compared with other molecular markers, SNPs are slow molecular clocks, making them valuable for phylogenetic and evolutionary analysis. The large-scale studies on the SNP markers for Map isolates are lacking that has been used for studying evolution, pathogenesis and epidemiology of in clinical isolates of M. tuberculosis (Alland et al., 2003). Marsh et al. (2005) studied SNPs using PCR and sequencing-based comparison of S and C Map strains at 30 loci across 29 Map genes. They found 8 (Table 3) contained a total of 11 SNPs in S strain compared to C strain. Sequences from both the strains were used to query the Map K10 genome. The C strain was identical and 11 SNPs were confirmed in S strain. The efforts of
Marsh et al. (2005) can be extended on Map isolates from different host species and different geographical regions to identify more SNP markers to type the Map strains.
Conclusions Though the present phase is beginning of the post-genomic phase in Map research, but some initiatives taken in this field can guide in designing our future strategies to combat this devastating disease of animals and human beings. This paper dwelt extensively on the diagnostic and strain typing strategies to overcome limitations of the present methods. Studies indicate that many LSP regions are heterogeneously distributed across geographically diverse isolates, so lacked the specificity required for diagnostics. However, subset of LSPs, appear highly specific (LSPP2, LSPP4, LSPP15) and sensitive (LSPP15) for Map and may prove useful in the Clinical isolate
Cattle 8g-4ggt 9g-4ggt 13g-5ggt 14g-5ggt
Sheep
P3ggt 15g-3ggt P7g-3ggt
10g-5ggt 15g-5ggt
3 Primer based Test for LSP 8LSP LSP 8 Present
11g-5ggt 12g-5ggt Goat
7g-4ggt 7g-5ggt
LSP 8 Absent
Non-Map Isolate
Map Isolate 3 Primer Test for LSPA 4-II, LSPA18 and LSPA 20
LSPA4-IIandLSPA18Present
LSPA 20 Present
Human
Figure 1. SSR, allele distribution across various host species suggests strain sharing and interspecies transmission among Map strains. Table 3.
Map S type
Map C type
Figure 2. Diagnostic algorithm for detecting and typing Map isolate based on the presence/absence of LSPs.
Eleven SNPs differentiating S and C Map strains
Gene
S strain to C strain
S strain amino acid
C strain amino acid
Change
Hsp65
T to C A to G C to G T to C T to G C to T C to T T to G T to C G to A G to A
Thr Glu Leu Leu Ala Arg Ser Ile Val Gln Pro
Thr Glu Val Pro Ala Trp Ser Met Ala Gln Pro
Conserved Conserved Neutral to neutral Neutral to neutral Conserved Basic to neutral Conserved Neutral to polar Neutral to neutral Conserved Conserved
SodA DnaA DnaN RecF GyrB
InhA pks8
ARTICLE IN PRESS Mycobacterium avium subspecies paratuberculosis diagnosis
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Forward Primer 1 LSP
Flanking Region
Reverse Primer 1
Forward Primer 2
Reverse Primer 2
Flanking Region
Figure 3. Four primers-based strategy for testing the presence/absence of LSP. The Forward Primer (green) and Reverse Primer 2 (yellow) are in genomic regions flanking the LSP and Forward Primer 2 Reverse Primer 1 (red) is an internal primer within LSP. A positive PCR with both pairs of Forward Primer and Reverse Primer will indicate the presence of LSP and positive PCR with Forward Primer 1 and Reverse Primer 2 will indicate the absence of the LSP region.
development of effective diagnostics for JD. LSPA8 have immediate applicability in the diagnostics of Map. The simple PCR strategy to diagnose and characterize Map isolates based on LSPs proposed by Semret et al. (2006) (Fig. 2) is valuable. The newly discovered IS elements (ISMav2, ISMAP02 and ISMAP04) are of particular interest for their use as specific potential diagnostic targets due to their absence in other mycobacteria and in recent future these elements can offer an alternative or additional PCR-based method for the rapid detection of Map in biological samples. Since IS elements are mobile and many of the LSPPs have characteristics of mobile genetic elements and include genes annotated as encoding transposes and phage-like integrases (Semret et al., 2005). Consistent with their mobile nature, they may be variably present in Map. Thus a diagnostic test targeting different markers (LSPs and IS elements) can be designed either in multiplex form or singly to assure that the isolate is not Map. Preliminary studies using tandem repeat DNA elements (VNTRs, SSRs) provide a promising molecular sub-typing approach. The analysis using these elements enables the genetic characterization of Map isolates from different host species and provide evidence for the host specificity of some Map strains as well as sharing of strains between wild, domesticated animal species and human beings. But the studies conducted above, do not involve large number of Map isolates. Further analysis on extended panel of Map isolates may yield an epidemiological tool with an higher resolution, enabling a clear interpretation of comparative DNA fingerprinting of Map isolates from animal and human sources. Studies have shown that 2 LSPs can be employed to type Map isolates (LSPA4 and LSPP20). More number of such sequences can be explored to type host associated variants of Map from diverse geographical regions. Based on the presence/absence of particular LSP
and VNTR/SSR type, unique molecular signatures can be assigned to different host-associated variants of Map for epidemiological studies. In our efforts to characterize Indian Map isolates, we have designed a four primer-based strategy (Fig. 3) to look at the presence/absence of particular LSP (unpublished data). This study will also identify more of such sequences specific for particular hostassociated variant of Map.
Acknowledgment Authors are grateful to Head, Institute of Molecular Medicine, New Delhi for extending necessary facilities for genomic studies of Indian Map isolates.
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www.elsevier.de/micres
Mycobacterium avium subspecies paratuberculosis diagnosis and geno-typing: Genomic insights J.S. Sohala, S.V. Singha,, Swati Subodhc, Neelam Sheoranb, K. Narayanasamyb, P.K. Singha, A.V. Singha, A. Maitrad a
Central Institute for Research on Goats, Makhdom-281 122, PO-FARAH, Mathura, UP, India Institute of Molecular Medicine, Okhla Industrial Area, Phase–III, New Delhi, India c Thrombosis Research Institute, Bangalore, India d McGill University Health Centre, Research Institute Rs1-105, Montreal, Que., Canada H3G 1A4 b
Received 6 January 2007; accepted 19 March 2007
KEYWORDS Mycobacterium avium subspecies paratuberculosis; Genotyping; PCR-RFLP; IS elements
Summary Effective control of paratuberculosis and investigations of potential link to Crohn’s disease have been hampered by the lack of effective assays for easy and accurate diagnosis of Mycobacterium avium subspecies paratuberculosis (Map). Map is extremely fastidious and depends on iron chelator (Mycobactin). Map strains from humans and sheep are very difficult to isolate and may require years to emerge. Therefore, small numbers of Map isolates have been maintained in available collections. This situation has limited the study of biodiversity of Map. Though, much is known about environmental and host factors that contribute to paratuberculosis disease, but little is known about bacterial genetic mechanism of infection. Diagnostic and strain typing markers still demand improvements. Complete genome sequence of Map K10 strain is available in public domain for comparative genomics with other mycobacteria and clinical isolates of Map. It is anticipated that the genome sequence will help in carrying molecular diagnosis and strain typing with respect to Map forward at rapid pace. This paper reviews the current diagnostic and strain typing markers, which may be useful in typing of clinical isolates in near future. & 2007 Elsevier GmbH. All rights reserved.
Corresponding author. Tel.: +91 565 2763260x269 (O),
+91 565 2736262 (R), 09719072856 (Mobile); fax: +91 565 2763246. E-mail addresses: [email protected], [email protected] (S.V. Singh).
Introduction Viewed in terms of its historical and contemporary disease burden Mycobacteria are undeniably most successful human and animal pathogens.
0944-5013/$ - see front matter & 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2007.03.005
ARTICLE IN PRESS Mycobacterium avium subspecies paratuberculosis diagnosis Irrespective of intense research, paratuberculosis caused by Mycobacterium avium subspecies paratuberculosis (Map), still remains single most important cause of morbidity in ruminants leading to huge economic losses, world over. JD is endemic in cattle in developed countries and variable prevalence of Map has been reported from clinically (5–55.0%; Taylor et al., 1981; Giese and Ahrens, 2000) and sub-clinically (2–12.0%; Sweeney et al., 1992; Streeter et al., 1995; Jakobsen et al., 2000) ill cows. Herd prevalence of bovine paratuberculosis estimated for European countries ranged from 3.5% to 71.0% (South West of England – 3.5%, Belgium – 8.0%, Czech Republic – 12.0%, Italy 13.3%, Denmark, 70.0%, The Netherlands 31.0–71.0% by Cetinkaya et al. (1996), Boelaert et al. (2002), Kennedy and Benedictus (2001), Jakobsen et al (2000) and Muskens et al. (2000). Using protoplasmic antigen from ‘Bison Type’ genotype of Map, Singh et al. (2005) reported sero-prevalence of paratuberculosis in bovines and goats as, 29.05% (buffaloes, 28.67% and cattle, 29.86%) and 23.63%, respectively, in Northern India. Cumulative sero-prevalence in Utter Pradesh and Punjab states of Northern India was 32.9% and 23.0%, respectively. Using sensitive Gold standard method (culture), high prevalence of Map has been recorded in India from cattle milk and buffalo tissues, young and adult goats and sheep. A number of Map-specific genomic sequences such as the insertion sequence IS900 (Green et al., 1989), F57 element (Poupart et al., 1993), and hsp X gene (Ellingson et al., 1998), have been identified. Of these multi-copy IS900 is a target of choice and is the most widely used in characterization of Map. There are concerns about its specificity and validity as a direct proxy for Map. Furthermore, as some members of the Mycobacterium tuberculosis complex, do not contain IS 6110 (Agasino et al., 1998). It is possible that some strains of Map are either low copy number or IS 900 negative, providing additional arguments for the need to develop molecular tests that rely on genomic characteristics other than mobile elements. Even 17 years after the IS 900 was discovered, the promise of a fast, affordable, specific and sensitive PCR for application to clinical samples remains elusive goal. In case of F57 and hsp X, their specificity for Map has not been rigorously evaluated in large numbers of clinical isolates. Commercially available serological assays for bovine paratuberculosis though convenient but suffer from poor specificity and sensitivity, especially during sub-clinical phases of the disease. Other limiting factor for Map studies is the lack of convenient strain typing methods. Most widely used
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method for typing Map isolates is RFLP based on IS 900 and IS1311 PCR-REA (Whittington et al., 2000; Stevenson et al., 2002). These methods are either applicable to cultivable strains or slow, technically demanding and have low-resolution power. Pulsefield gel electrophoresis of Map isolates provides more refined data for epidemiological interpretation (Stevenson et al., 2002). Unfortunately, the technique also requires large quantities of DNA and applicable to cultivable strains. rRNA gene and spacer region analysis has also been tested for the ability to distinguish Map strains. The RFLP analysis of the 5S rRNA gene region (Chiodini, 1990) identified indistinguishable restriction fragments of the 5S rRNA gene region in all Map isolates studied. An analysis of the spacer region of the rDNA indicated lack of discrimination as it revealed an identical sequence for all Map isolates tested (Scheibl and Gerlach, 1997). Recently developed technique to type Map isolates based on Multiplex PCR for IS 900 loci (MPIL), have almost similar discriminatory power (Motiwala et al., 2004) to IS 900 RFLP. Amplified fragment length polymorphism (AFLP) appears to have greater resolving power than MPIL and RFLP but suffers from the limitation that the allelic variation is indexed at anonymous bi-allelic sites or locations. Additionally, repeatability and interlaboratory variation limits its application in multiple site analyses. Due to disadvantages of the current methods, rapid and robust molecular methods are required to be assessed as alternatives for studying Map strain diversity. Since there is high degree of similarity at genomic levels between Map and other members of Mycobacterium avium complex (MAC), ability to differentiate between these closely related organisms is essential for rational clinical and epidemiological assessment. History clearly documents that we cannot detect all the infected animals simply. Though diagnostic tests have improved over the years but still there is room for improvement. This paper reviews the post-genomic identified targets of Map diagnosis and strain typing and also describes the future strategies to diagnose and type Map isolates.
Genome overview Map K10 genome is a single circular chromosome of 4,829,781 base pairs with G+C content of 69.3% and encodes 4350 predicted ORFs, 45 tRNAs and one rRNA operon (Table 1). In silico analysis identified 43000 genes homologous to human pathogen (Mtb), there is truncation in the Ent E
ARTICLE IN PRESS 332 Table 1.
J.S. Sohal et al. Summary of Mycobacterial genomes
Property
Map
Mav
Mtb
M. bovis
M. leprae
M. smegmatis
Genome size (bp) G+C content (%) Protein coding (%) ORFs Average gene length (bp) TRNAs rRNA operon
4,829,781 69.30 91.30 4350 1015 45 1
5,475,738 68.99 NA NA NA 45 1
4,411,532 65.61 90.80 3959 1012 45 1
4,345,492 65.63 90.59 3953 995 45 1
3,268,203 57.79 49.50 1604 1011 45 1
6,988,209 67.40 92.42 6897 936 47 2
domain of a salicyl-AMP ligase (MbtA), the first gene in the mycobactin biosynthesis gene cluster, providing a possible explanation for mycobactin dependence of Map. Approximately 1.5% (or 72.2 kb) of the Map genome is comprised of repetitive DNA-like insertion sequences, multigene families, and duplicated housekeeping genes. There are a total of 19 different IS elements with 58 copies in Map K10 genome.
Large sequence polymorphisms (LSPs) Recent work in mycobacterial genomics has revealed LSPs as major contributor of genetic diversity. LSPs have been employed for inferences of phylo-genetics (Brosch et al., 2002; Mostowy et al., 2002) and biological properties such as virulence (Kato-Maeda et al., 2001; Pym et al., 2002) of Mycobacterium tuberculosis (Mtb) complex, thus it is hypothesized that LSPs are important sources of genetic variability among MAC organisms. In silico comparisons of the genome sequences of Map K10 (NC002944) and M. avium subsp. avium (Mav) strain 104 (http://www. tigr.org) genomes have identified DNA sequences that are present in the former but missing in the latter (Bannantine et al., 2002, 2004; Paustian et al., 2004). Semret et al. (2005) by in silico analysis initially identified 17 LSPs varying in length from 2.9 to 66 kb that were unique to Map K10. These Map unique LSPs were designated as LSPPs. Microarray comparisons by Semret et al. (2004) using M. avium subsp. avium strain 104 as the reference identified regions designated them as LSPAs, specific for Mav and absent from Map isolates. LSPs and Map diagnosis Semret et al. (2005) tested the distribution of 17 LSPPs (Map specific) and 3 LSPAs (absent in Map) by PCR-based strategy across a panel of 383 M. avium complex isolates. Screening panel of 383 MAC isolates (107 Map+276 non-Map), revealed that LSPP2, LSPP4, and LSPP15 could not be amplified
Table 2.
Map-specific LSPPs
LSP
Sensitivity (%)
Specificity (%)
LSPP 2 LSPP 4 LSPP 15
41.1 68.2 90.7
100 100 100
from any of the 276 non-Map isolates, demonstrating 100% specificity of these LSPPs for this subspecies (Map). Of the Map specific LSPPs specific for LSPP15 consistently gave positive results (high sensitivity) across Map isolates (Table 2), while amplification of other LSPPs was variable (Semret et al., 2005). Among the LSPAs absence of LSPA8 was 100% specific for Map (Semret et al., 2005, 2006). This sequence was consistently missing from Map isolates and is present in every other M. avium complex strain tested (100% sensitive). This is akin to the RD1 region of the M. tuberculosis complex, which permits accurate differentiation between virulent Mycobacterium bovis and BCG strains (Talbot et al., 1997). Others LSPAs, LSPA13 and LSPA14 were also found to be absent in Map, but their absence may not be considered 100% specific for Map, since these sequences were invariably missing from some of non-Map isolates (Semret et al., 2005). Microarray-based genomic comparison by Paustian et al. (2005) also identified genomic regions divergent between Map and other members of the M. avium complex as potentially unique to Map. These regions represent LSPPs described by Semret et al. (2005). LSPs and Map strain typing The ability to both detect and differentiate between types of strains is of obvious importance to guide control programs. Semret et al. (2006), proposed a simple PCR-based strategy to rapidly type the Map isolates based on the presence or absence of LSPs. They studied the distribution of LSPs across a panel of strains and were able to assign unique genomic signatures to host-associated variants. The results show that, LSPA 4-II
ARTICLE IN PRESS Mycobacterium avium subspecies paratuberculosis diagnosis and LSPA 18 were present in S (Sheep) strains but missing from the two C (Cattle) strains. Another sequence, LSPA 20, was present in the C strains but missing from the S strains. Similar type of strategy of distribution of polymorphisms has been applied to differentiate members of M. tuberculosis complex (Talbot et al., 1997; Parsons et al., 2002). Findings of Semret et al. (2006) for the regions missing from C strains of Map are in agreement with representational difference analysis (RDA)-based study of Dohmann et al. (2003), that identified 3 loci missing from type II (C type) strains. The 233bp locus identified as pig-RDA10 (AY266300) by Dohmann et al. (2003), forms part of the 16-kb region called as LSPA 18 by Semret et al. (2006). The 197-bp locus identified as pig-RDA20 (AY266301) by Dohmann et al. (2003), is located within the 26-kb segment called as LSPA 4-II by Semret et al. (2006).
Map unique IS elements Genome sequencing of Map K10 had identified three more Map unique IS elements; ISMav2, ISMAP02 and ISMAP04 present in 3, 6 and 4 copies, respectively, in Map K10 genome in addition to previously known IS 900 (Li et al., 2005). Small-scale studies using these newly identified Map unique IS elements have provided encouraging results. Strommenger et al. (2001) had shown that ISMav2 has potential to act as Map diagnostic Marker, but has not yet been adequately evaluated at large scale. Stabel and Bannantine (2005) evaluated the sensitivity and specificity of the ISMAP02 element in both conventional and realtime PCR tests for the detection of Map in bovine fecal samples in comparison to the sensitivity and specificity of the IS900 element target. They found that ISMAP02 is 100% specific for Map. The sensitivity of detection for the ISMAP02 element in either conventional or real-time PCR format was less than 100 fg DNA or 102 CFU/ml. The results were comparable to those obtained for the IS900 element. PCR analyses of ISMAP02 by Paustian et al. (2004), also demonstrated that the sequence was present in all 39 isolates of Map examined but was absent from all other mycobacterial species and subspecies tested. This analysis further substantiates the specificity of this sequence. To date, there is no data available on the use of ISMAP04 as specific diagnostic marker for Map.
Variable number tandem repeat (VNTR) analysis Tandem repeat sequences often represent polymorphic structures in the genomes of microbes.
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VNTR typing offers a promising PCR-based study for typing of mycobacteria based on variation in repeat numbers. Overduin et al. (2004) explored the strain typing ability of VNTRs by multilocus variable number tandem repeat analysis (MLVA) and compared with IS 900 RFLP. By testing 20 VNTR loci across 49 Map isolates, they identified 5 polymorphic VNTRs. These 5 polymorphic VNTR loci yielded 6 VNTR types for 49 Map isolates and were able to subdivide the most predominant IS 900 RFLP type, R01, into six subtypes. Interesting finding was all human isolates revealed the same MLVA type as found for the majority of the cattle strains. Biet et al. (2005) identified 8 polymorphic MIRU-VNTR loci by screening 72 Map isolates; they found 12 different MIRU-VNTR types for these 8 polymorphic repeat elements. In total, MIRU-VNTR grouped 69 isolates into 9 clusters whereas 3 MIRU-VNTR patterns were unique. MIRU-VNTR typing sub-divided the major RFLP type into 11 different MIRU-VNTR types, thus providing an additional method to study the diversity of Map.
Short sequence repeats (SSRs) and Map typing SSRs in bacterial DNA have been used as markers for the differentiation and sub-typing strains of several bacterial species, including M. tuberculosis (Gascoyne-Binzi et al., 2001; Kremer et al., 1999). It has been recognized that regions of mono-, di-, and tri-nucleotide tandem repeats are often the most diverse in a bacterial genome, while complex longer repeats generally have lower levels of diversity (Keim et al., 2000). Results of multi locus short sequence repeat (MLSSR) sequencing by Amonsin et al. (2004), enabled facile and reproducible high-resolution sub-typing of Map isolates for molecular epidemiologic and population genetic analyses. They identified 11 poly-morphic short sequence repeats (SSRs), with an average of 3.2 alleles per locus. This analysis differentiated the 33 Map isolates into 20 distinct MLSSR types. MLSSR analysis easily and reproducibly differentiated strains representing the predominant MPIL genotype and AFLP genotypes. Further analysis of Map isolates using SSR analysis by Motiwala et al. (2004), enabled the differentiation of the Map isolates in clade A18 (by MPIL analysis) into seven distinct alleles. The analysis revealed eight subtypes among the 33 species of animals, suggesting the interspecies transmission of specific strains. Also SSR analysis of Map isolates by Ghadiali et al. (2004), from CD patients identified two alleles,
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J.S. Sohal et al.
both of which clustered with strains derived from animals with JD (Fig. 1).
Single nucleotide polymorphisms (SNPs) and Map typing SNPs are the most abundant form of DNA polymorphism. Compared with other molecular markers, SNPs are slow molecular clocks, making them valuable for phylogenetic and evolutionary analysis. The large-scale studies on the SNP markers for Map isolates are lacking that has been used for studying evolution, pathogenesis and epidemiology of in clinical isolates of M. tuberculosis (Alland et al., 2003). Marsh et al. (2005) studied SNPs using PCR and sequencing-based comparison of S and C Map strains at 30 loci across 29 Map genes. They found 8 (Table 3) contained a total of 11 SNPs in S strain compared to C strain. Sequences from both the strains were used to query the Map K10 genome. The C strain was identical and 11 SNPs were confirmed in S strain. The efforts of
Marsh et al. (2005) can be extended on Map isolates from different host species and different geographical regions to identify more SNP markers to type the Map strains.
Conclusions Though the present phase is beginning of the post-genomic phase in Map research, but some initiatives taken in this field can guide in designing our future strategies to combat this devastating disease of animals and human beings. This paper dwelt extensively on the diagnostic and strain typing strategies to overcome limitations of the present methods. Studies indicate that many LSP regions are heterogeneously distributed across geographically diverse isolates, so lacked the specificity required for diagnostics. However, subset of LSPs, appear highly specific (LSPP2, LSPP4, LSPP15) and sensitive (LSPP15) for Map and may prove useful in the Clinical isolate
Cattle 8g-4ggt 9g-4ggt 13g-5ggt 14g-5ggt
Sheep
P3ggt 15g-3ggt P7g-3ggt
10g-5ggt 15g-5ggt
3 Primer based Test for LSP 8LSP LSP 8 Present
11g-5ggt 12g-5ggt Goat
7g-4ggt 7g-5ggt
LSP 8 Absent
Non-Map Isolate
Map Isolate 3 Primer Test for LSPA 4-II, LSPA18 and LSPA 20
LSPA4-IIandLSPA18Present
LSPA 20 Present
Human
Figure 1. SSR, allele distribution across various host species suggests strain sharing and interspecies transmission among Map strains. Table 3.
Map S type
Map C type
Figure 2. Diagnostic algorithm for detecting and typing Map isolate based on the presence/absence of LSPs.
Eleven SNPs differentiating S and C Map strains
Gene
S strain to C strain
S strain amino acid
C strain amino acid
Change
Hsp65
T to C A to G C to G T to C T to G C to T C to T T to G T to C G to A G to A
Thr Glu Leu Leu Ala Arg Ser Ile Val Gln Pro
Thr Glu Val Pro Ala Trp Ser Met Ala Gln Pro
Conserved Conserved Neutral to neutral Neutral to neutral Conserved Basic to neutral Conserved Neutral to polar Neutral to neutral Conserved Conserved
SodA DnaA DnaN RecF GyrB
InhA pks8
ARTICLE IN PRESS Mycobacterium avium subspecies paratuberculosis diagnosis
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Forward Primer 1 LSP
Flanking Region
Reverse Primer 1
Forward Primer 2
Reverse Primer 2
Flanking Region
Figure 3. Four primers-based strategy for testing the presence/absence of LSP. The Forward Primer (green) and Reverse Primer 2 (yellow) are in genomic regions flanking the LSP and Forward Primer 2 Reverse Primer 1 (red) is an internal primer within LSP. A positive PCR with both pairs of Forward Primer and Reverse Primer will indicate the presence of LSP and positive PCR with Forward Primer 1 and Reverse Primer 2 will indicate the absence of the LSP region.
development of effective diagnostics for JD. LSPA8 have immediate applicability in the diagnostics of Map. The simple PCR strategy to diagnose and characterize Map isolates based on LSPs proposed by Semret et al. (2006) (Fig. 2) is valuable. The newly discovered IS elements (ISMav2, ISMAP02 and ISMAP04) are of particular interest for their use as specific potential diagnostic targets due to their absence in other mycobacteria and in recent future these elements can offer an alternative or additional PCR-based method for the rapid detection of Map in biological samples. Since IS elements are mobile and many of the LSPPs have characteristics of mobile genetic elements and include genes annotated as encoding transposes and phage-like integrases (Semret et al., 2005). Consistent with their mobile nature, they may be variably present in Map. Thus a diagnostic test targeting different markers (LSPs and IS elements) can be designed either in multiplex form or singly to assure that the isolate is not Map. Preliminary studies using tandem repeat DNA elements (VNTRs, SSRs) provide a promising molecular sub-typing approach. The analysis using these elements enables the genetic characterization of Map isolates from different host species and provide evidence for the host specificity of some Map strains as well as sharing of strains between wild, domesticated animal species and human beings. But the studies conducted above, do not involve large number of Map isolates. Further analysis on extended panel of Map isolates may yield an epidemiological tool with an higher resolution, enabling a clear interpretation of comparative DNA fingerprinting of Map isolates from animal and human sources. Studies have shown that 2 LSPs can be employed to type Map isolates (LSPA4 and LSPP20). More number of such sequences can be explored to type host associated variants of Map from diverse geographical regions. Based on the presence/absence of particular LSP
and VNTR/SSR type, unique molecular signatures can be assigned to different host-associated variants of Map for epidemiological studies. In our efforts to characterize Indian Map isolates, we have designed a four primer-based strategy (Fig. 3) to look at the presence/absence of particular LSP (unpublished data). This study will also identify more of such sequences specific for particular hostassociated variant of Map.
Acknowledgment Authors are grateful to Head, Institute of Molecular Medicine, New Delhi for extending necessary facilities for genomic studies of Indian Map isolates.
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