Mycobacterium avium subspecies paratuberculosis (Map) causes paratuberculosis or Johne's disease, a fatal chronic granulomatous enteritis. The disease occurs worldwide and is responsible for significant economic losses to livestock and associated industries [1, 2]. It is endemic in Europe with only Sweden maintaining paratuberculosis-free status. The epidemiology is poorly understood and there are important questions still to resolve, particularly with respect to interspecies transmission. Map infects principally ruminants but over the past decade it has become apparent that the organism has a much broader host range including monogastric species [3–5]. The infection of humans with Map and possible association with Crohn's disease remains a controversial issue and requires more study [6, 7]. The strain types involved and the extent to which interspecies transmission occurs have still to be elucidated. Evidence also is accumulating regarding the existence of potential wildlife reservoirs, for example, infected rabbits appear to be a particular problem in some areas of Scotland [3] but the role of such wildlife reservoirs in the epidemiology of the disease has still to be clarified.

Our knowledge and understanding of the epidemiology of Map has been hindered for many years by our inability to discriminate Map from the environmental species of Mycobacterium avium (M. avium) and to differentiate between Map isolates from different host species and different geographic locations. Recent advances in molecular biology have led to the refinement and development of molecular typing methods with sufficient discriminatory power to differentiate between M. avium subspecies and different Map isolates [8]. Genome analyses have revealed two major strain groups designated 'Type I', or 'sheep or S type' and 'Type II' or 'cattle or C type'. A sub-type of Type I strains designated 'Type III' or 'intermediate or I type' is found in sheep and goats. All three of these strain types can be differentiated by restriction fragment length polymorphism coupled with hybridization to IS900 (IS900-RFLP) [9, 10] or pulsed-field gel electrophoresis (PFGE) analyses [11, 12] and by a PCR assay based on single nucleotide polymorphisms in the gyrA and gyrB genes [13]. Single nucleotide polymorphisms in the IS1311 element also distinguish three types designated 'S' (sheep), 'C' (cattle) and 'B' (bison) [14, 15]. In this case the assay cannot distinguish between Types I and III and the 'B' type is a sub-type of Type II strains. In silico genome comparisons and techniques such as representational difference analysis and microarray analysis have identified sequence polymorphisms unique to either Type I or II strains and these have been used to develop PCRs for discriminating these strain groups [16–21]. The purpose of this study was to investigate the molecular diversity of Map isolates from a variety of hosts across Europe to enhance our understanding of the host range and distribution of the organisms and assess the potential for interspecies transmission. Previous studies have revealed limited genetic diversity; therefore, to maximise strain differentiation we evaluated several different molecular typing techniques in isolation and in combination; IS900-RFLP, PFGE and PCR-based techniques including amplified fragment length polymorphisms (AFLP) and mycobacterial interspersed repeat unit-variable number tandem repeat (MIRU-VNTR).

AFLP typing was performed at the Central Institute of Wageningen University, Lelystad, The Netherlands and MIRU-VNTR at INRA, Nouzilly, France. For PFGE and IS900-RFLP typing, the field isolates were split between two labs. PFGE typing was undertaken at the Moredun Research Institute, Scotland, UK and VISAVET, Madrid, Spain. IS900-RFLP typing was carried out at the Veterinary Research Institute in Brno, Czech Republic and VISAVET. Published standardized typing procedures were used as described in Materials and Methods. The only difference in procedures between laboratories was that at VISAVET the IS900-RFLP analysis was performed using the agarose plugs prepared for PFGE to avoid having to perform two separate DNA preparations for the different typing techniques. The correct profiles were reported by all laboratories for the duplicate isolates included to check reproducibility. All typing techniques correctly reported that the Mycobacterium phlei (M. phlei), Mycobacterium bovis BCG (M. bovis BCG) and IS901 positive M. avium were not Map. One field isolate, EU112 was found to be IS901 positive M. avium (it is not known if the isolate is M. avium subsp. avium or M. avium subsp. silvaticum) and not Map as was originally suspected. Another isolate, EU169 was found to be a mixed culture. Isolates one to 50 were typed at Institut für Mikrobiologie Stiftung Tierärztliche Hochschule Hannover, Hannover, Germany using the Type I/Type II PCR as described by Dohmann et al. [17]. EU25 and EU30 were identified as Type I and all other field isolates as Type II. These results correlated with the strain type as determined by PFGE. This PCR [17] cannot discriminate between Type I and Type III and as strain types could be discerned from the PFGE profiles, it was not considered necessary to determine the strain type of the remaining isolates by PCR. It was not possible to type all of the isolates with all typing methods as some laboratories had difficulties in subculturing some isolates to prepare sufficient cells for analyses. A total of 123 Map isolates were typed by IS900-RFLP, PFGE and MIRU-VNTR.

PFGE typing

PFGE typing data were obtained for 145 Map isolates (Table 1 and Figure 1). Twenty four different SnaBI profiles were detected in this panel of isolates: 2 (n = 91); 1 (n = 15); 15 (n = 9); 29 (n = 4); 34 (n = 4); 3 (n = 3); 38 (n = 2) and 5, 9, 16, 18, 20, 26, 27, 30, 31, 32, 33, 36, 37, 39, 40, 41, 58 (n = 1 each); and 23 distinct SpeI profiles: 1 (n = 102); 25 (n = 8); 2, 15, 22 (n = 4 each); 17, 19, 21, 30, 32 (n = 2 each) and 7, 10, 11, 16, 18, 20, 23, 24, 27, 28, 29, 31, 64 (n = 1 each). The combination of both enzyme profiles gave 31 different multiplex profiles: [2-1] (n = 83); [1-1] (n = 15); [15-25] (n = 8); [29-15],[34-22] (n = 4 each); [3-2] (n = 3); [2-19],[2-30],[38-32] (n = 2 each) and [2-10], [2-17], [2-21], [2-31], [5-2], [9-7], [15-16], [16-11], [18-1], [20-1], [26-1], [27-18], [30-21], [31-17], [32-29], [33-20], [36-27], [37-23], [39-24], [40-28], [41-1],[58-64] (n = 1 each). By far the most widely distributed PFGE type was [2-1], which was found in the Czech Republic, Finland, The Netherlands, Norway, Scotland and Spain (Table 1 and see supplementary dataset in Additional file 1 and Additional file 2: Table S1). PFGE type [1-1] was the next most common occurring in the Czech Republic, Finland, The Netherlands and Spain (Table 1 and see supplementary dataset in Additional file 1 and Additional file 2: Table S1). Profile [2-30] was found in The Netherlands and Scotland and the other profiles were found in only one country (Table 1 and see supplementary dataset in Additional file 1 and Additional file 2: Table S1). The numbers of isolates detected with these profiles are too small to determine if these multiplex profiles truly are restricted in their geographical location.

The results of this study improve our knowledge of the epidemiology of paratuberculosis in Europe regarding the genetic diversity and distribution of Map isolates with respect to geographic location and host species of origin. The study has also permitted a comprehensive comparison of three standardized typing procedures, the results of which will inform future epidemiological studies as to the most appropriate and discriminative methods to employ.

This is the first study to compare the discriminatory power of IS900-RFLP, PFGE, AFLP and MIRU-VNTR for the molecular characterization of Map isolates. AFLP could not effectively discriminate between Map isolates and therefore is not suitable for epidemiological studies on paratuberculosis. A major problem with the technique was reproducibility. This was probably due in part to the variable quality of the mycobacterial DNA, which is highly dependent on growth phase and difficult to extract from Map isolates that are particularly resilient to lysis. Reproducibility could also have been affected by small variations in the experimental procedure such as shifts in electrophoretic mobility during capillary electrophoresis. Despite several attempts using alternative analytical procedures, no decrease in this variation could be obtained.

The most widely used measure of diversity is Simpson's Index of Diversity (SID), which we have employed here to estimate the discriminatory power of the various molecular typing techniques utilised in this study. When all Map isolates were considered irrespective of host or geographic origin, the SID was not significantly different between each of the individual typing techniques (IS900-RFLP, multiplex PFGE and MIRU-VNTR) and was low at a value between 0.636 and 0.664 in accordance with previous reports [23, 24]. The SID value is strongly influenced by the distribution of types rather than the number of types detected. This is clearly demonstrated by comparing the two methods with the largest difference in the number of patterns detected i.e. IS900-RFLP, which identified 15 profiles and multiplex PFGE, which detected 26 profiles. Despite the number of profiles detected, both methods have almost the same SID point estimate and 95% confidence interval. The SID for IS900-RFLP could have been improved further had it been possible to obtain PstI profiles for the isolates. The discriminatory power of the individual techniques is too low for epidemiological surveys since a SID of around 0.9 is generally considered the minimum. For isolates from mainland Europe, SID for the combination of multiplex PFGE and MIRU-VNTR, with or without IS900-RFLP, exceeded the threshold value of 0.9. The increase in SID is not surprising since the different typing techniques target different sources of genetic variation and have different limitations and will therefore complement each other when used in combination. Due to limited heterogeneity among Scottish isolates, combining all three typing techniques increased SID to 0.879 for the dataset as a whole, providing discriminatory power close to the minimum but not quite reaching the target value.

Although the combination of all three typing techniques gives the greatest discrimination, this is generally not practical or cost effective for large national or international studies and often a compromise is sought. The choice of typing method will be influenced by the predominant isolate type in the population to be tested. This is highlighted in this study by considering the data shown in Table 4 for the isolates from Scotland versus those from mainland Europe and the combined European dataset (i.e. all isolates). The isolates from Scotland comprise a homogeneous population in which the B-C17 IS900-RFLP profile predominates and is therefore a rigorous test for the combination approach. Comparing the SIDs for the various combinations of typing techniques there was no difference between multiplex PFGE + MIRU-VNTR and the combination of all three typing techniques. Therefore, a combination of multiplex PFGE + MIRU-VNTR would be suitable for epidemiological studies in Scotland. A combination of multiplex PFGE + MIRU-VNTR would also be appropriate for mainland Europe but here a combination of IS900-RFLP and multiplex PFGE would also perform well. The best combination for the combined European dataset was all three typing techniques. The SID for the isolates from mainland Europe was often higher than that for the combined European dataset, the latter being affected by the inclusion of the less heterogeneous Scottish isolates. Based on these results a small pilot study of the population of interest is recommended before undertaking a large epidemiological survey. For further epidemiological studies in Scotland, it would be advantageous to undertake a pilot study including short sequence repeat analysis [25], which may improve the discriminatory power for this homogeneous population of isolates.

The study identified the common isolate types within the European countries examined. IS900-RFLP profile C1 was the most widespread, consistent with previous reports from individual countries [26–31]. This profile has a global distribution, being found in the United States, Australia and New Zealand [10, 30, 32]. Although IS900-RFLP profile C17 is commonly isolated in Scotland it is reported to be relatively rare in other European countries [30, 31]. It was identified in isolates from The Netherlands and Norway in this study and has been reported previously in Germany [31] and is predominant in specific regions of Argentina [30, 33]. The most common PFGE profile was [2-1] found in six of the seven countries examined, closely followed by [1-1] found in four. INVM 2 was found in six countries and INVM 1 in five. Further investigations will be required to determine if this distribution is a consequence of animal movements, increased virulence or whether these isolates have characteristics that allow them to transmit more readily. There is evidence to suggest that different mycobacterial strain types vary in their ability to cause disease. Caws et al. [34] provided evidence that M. tuberculosis genotype influences clinical disease phenotype and demonstrated a significant interaction between host and bacterial genotypes and the development of tuberculosis. Gollnick et al. [35] reported that the survival of Map in bovine monocyte-derived macrophages was not affected by host infection status but by the infecting strain type. Two recent studies suggest that different Map strain types may play a role in polarizing the host immune responses during infection [36, 37]. Also, different Map strains have been found to differ in virulence in experimental infections of deer [38] and in a mouse model (KS, unpublished data) and Verna et al. have provided data to show how the strain type may influence the pathology of ovine paratuberculosis [39].

Surprisingly, no Type I strains (corresponding to S Type strains in the literature [40]) were identified within the 27 sheep and 33 goat field isolates submitted by the partners. This may be a reflection of the difficulties encountered in isolating and growing these strains in vitro. Typically, isolates of strain Type I are slow-growing, taking longer than 16 weeks and sometimes as long as 18 months to isolate on solid medium. Cultures are often not retained this long in diagnostic laboratories. Furthermore, studies have shown that the decontamination procedures or media used for isolation can significantly affect recovery of these strains. Reddacliff et al. [41] reported the detrimental effects of various decontamination protocols on the recovery of Type I strains from tissues and faeces. The addition of egg yolk and mycobactin J to BACTEC 12B or 7H9 broth was found to be essential for the isolation of Australian sheep strains from faeces and to enhance their recovery from tissue samples [42]. Other workers have successfully isolated Type I or III strains on LJ or Middlebrook 7H11 supplemented with mycobactin J [43, 44]. The addition of antibiotics can also affect growth. Both ampicillin and vancomycin hydrochloride can retard growth of Type I strains [45]. The various laboratories participating in this study used a range of decontamination procedures and culture media but it is not possible to rule out a culture bias.

The results of this survey highlight an interesting difference between the epidemiology of Map in Europe and Australia. This study shows that in Europe, Type II strains (corresponding to C Type strains in the literature [10]) are commonly isolated from sheep, goats and cattle whereas in Australia, Type II strains are rarely, if ever, isolated from sheep -the predominant type being Type I. We can only speculate as to the reasons for this difference. Management practices will affect the circulation of strains and can differ between some parts of Europe and Australia. The scale of farming operations and relative proportions of the different livestock co- or sequentially grazing may also be a factor. Paratuberculosis is more common in sheep in Australia than in cattle and the Type I strain is more virulent for sheep than cattle [39].

In this study, Map was isolated from 19 different host species, which included both ruminants and non-ruminants. This is the first report of the isolation of Map from a giraffe. The Type II strains appear to have greater propensity for infecting a broad range of host species whereas the epidemiological data available for Type I strains suggests that they have a preference for sheep and goats [23]. Since our results show that the same profiles are found in isolates from different species, it strongly suggests that strain sharing occurs. Even more convincing was the observation that the same profiles were isolated from wildlife species and domestic ruminants on the same farm. The frequency or ease with which interspecies transmission occurs are unknown entities and require further investigation. Similarly, the relative risk of transmission from domestic livestock to wildlife or vice versa remains to be determined.

All animals in contact with Map contaminated faeces on an infected property will contribute to the spread of disease through passive transmission. However, Map infects a variety of wildlife host species that potentially could be reservoirs for infection of domestic livestock and have serious implications for control of paratuberculosis. The role of wildlife reservoirs in the epidemiology of paratuberculosis will depend on a number of factors which need to be taken into consideration when undertaking a risk assessment for interspecies transmission. Although Map can infect many wildlife species, only wild ruminants and lagomorphs show evidence of disease as determined by the presence of gross or microscopic lesions with associated acid fast bacteria [46]. These wildlife species have the capacity to excrete Map and spread disease to other susceptible species primarily through further faecal contamination of the environment. Potentially, they could constitute wildlife reservoirs. By definition, to constitute a wildlife reservoir the infection would need to be sustained within the species population. Evidence is available for vertical, pseudovertical and horizontal transmission within natural rabbit populations which could contribute to the maintenance of Map infections within such populations [47, 48].

The other wildlife species in this study could be categorised into predators and scavengers that probably acquire the disease through eating contaminated prey or carrion, respectively. It has been reported previously that these animals show no clinical signs of disease and only minor histopathological changes with a few acid fast bacteria in tissues [4, 5]. Such infected predators and scavengers are probably 'dead-end hosts' and are not high risk factors for interspecies transmission.

Information pertaining to strain types can assist in designing and evaluating disease control programmes. It is beneficial to know the predominant strain type in a population or the virulence of a particular strain type particularly for developing new vaccines. Singh et al. [49] recently reported the effectiveness and advantage of using a vaccine based on a local 'bison-type' strain.

In conclusion, this survey has helped to expand our knowledge to improve our understanding of the epidemiology of paratuberculosis. It is hoped that the information provided will facilitate future surveys and research strategies to resolve the outstanding epidemiological questions regarding this disease.

The results of this study were in agreement with previous reports indicating that Map isolates comprise a relatively homogeneous population exhibiting little genetic diversity compared with other bacterial pathogens. As a result it is necessary to use multiple genotyping techniques targeting different sources of genetic variation to obtain the level of discrimination necessary to investigate transmission dynamics and trace the source of infections. Identical genotypes were obtained from Map isolated from different host species co-habiting on the same property strongly suggesting that interspecies transmission occurs. Interspecies transmission of Map between wildlife species and domestic livestock on the same farm provides further evidence to support a role for wildlife reservoirs of infection. However, in assessing the relative risk of transmission between wildlife and domestic livestock, distinction needs to be made between passive and active transmission as well as the potential for contact.