Definitive Differentiation between Single and Mixed Mycobacterial Infections in Red Deer (Cervus elaphus) by a Combination of Duplex Amplifi
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微生物临床杂志 2005年第9期
Veterinary & Agrochemical Research Center, Brussels, Belgium
Laboratory of Applied Molecular Technology, Universite Catholique de Louvain, Brussels, Belgium
Defence Laboratories Department, Belgian Armed Forces, Brussels, Belgium
ABSTRACT
Severe emaciation and mortalities suggestive of mycobacterial infections were recently reported for both adult and young wild red deer (Cervus elaphus) in the southeastern part of Belgium. In deer, tuberculous lesions are not pathognomonic of Mycobacterium bovis infection due to gross and microscopic similarities with lesions caused by Mycobacterium avium subsp. paratuberculosis or M. avium subsp. avium. The aim of this study was to improve molecular methods for the species-specific identification of M. bovis, M. avium subsp. avium, and M. avium subsp. paratuberculosis in mycobacterial infections of deer. DNA banding patterns were assessed prior to and after Hpy188I restriction of f57-upstream (us)-p34 duplex amplicons. The duplex f57-us-p34 PCR differentiated M. bovis from M. avium subsp. paratuberculosis and M. avium subsp. avium infections, whereas the restriction step differentiated single M. avium subsp. paratuberculosis or M. avium subsp. avium infections from mixed M. avium subsp. paratuberculosis/M. avium subsp. avium infections. The endonuclease Hpy188I cleaves DNA between nucleotides N and G in the unique TCNGA sequence. This restriction site was found at position 168 upstream of the us-p34 initiation codon in all M. avium subsp. avium strains tested, regardless of their origin and the results of IS901 PCR. In contrast, the restriction site was abrogated in all M. avium subsp. paratuberculosis strains tested, independent of their origin, Mycobactin J dependency, and IS900 PCR results. Consequently, a two-step strategy, i.e., duplex us-p34-f57 PCR and Hpy188I restriction, allowed us to exclude M. bovis infection and to identify single (M. avium subsp. paratuberculosis or M. avium subsp. avium) or mixed (M. avium subsp. paratuberculosis/M. avium subsp. avium) infections in wild red deer in Belgium. Accordingly, we propose to integrate, in a functional molecular definition of M. avium subsp. paratuberculosis, the absence of the Hpy188I restriction site from the us-p34 amplicon.
INTRODUCTION
The most significant mycobacterial diseases of free-living, captive, and farmed red deer (Cervus elaphus) are bovine tuberculosis, caused by Mycobacterium bovis, Johne's disease (paratuberculosis), caused by Mycobacterium avium subsp. paratuberculosis, and avian tuberculosis, caused principally by M. avium subsp. avium (20).
Differential diagnoses of infections by M. bovis, M. avium subsp. paratuberculosis, and M. avium subsp. avium are essential because the epidemiology, zoonotic concern, and prevention and control methods are different for each of these mycobacterial infections (20). In deer, tuberculous lesions are not pathognomonic of M. bovis infection and are actually indistinguishable from lesions due to M. avium subsp. paratuberculosis or M. avium subsp. avium infection because of their gross and microscopic similarities. Moreover, the actual differential diagnosis of these mycobacterial infections is difficult due to similarities in the clinical expression and pathology associated with these infections. Antigenic similarity between M. avium subsp. avium, M. avium subsp. paratuberculosis, and M. bovis is also responsible for the lack of sensitivity and specificity of diagnostic tests (i.e., current serological and laboratory cell-mediated diagnostic tests as well as skin tests) (21).
The isolation of mycobacteria requires a decontamination treatment of the sample. Depending on the nature and the time of the decontamination treatment, mycobacteria present in the sample may also be killed. As recently demonstrated for M. avium subsp. paratuberculosis, routine protocols in the laboratory significantly decrease the number of organisms isolated per sample (for fecal, tissue, and milk samples), stressing the importance of the interpretation of negative culture results (12, 32). Conversely, atypical mycobacteria may still be grown from treated specimens (10).
Mycobacterial species are traditionally differentiated on the basis of their phenotypic characteristics. However, since these do not allow the precise identification of all species, recent molecular biology methods offer new opportunities to differentiate, identify, and type mycobacterial species (31). Several molecular targets have been used to allow species-specific identification, including IS6110 and 16S rRNA for the Mycobacterium tuberculosis complex (i.e., M. bovis, M. tuberculosis, Mycobacterium africanum, and Mycobacterium microti), IS900 and the f57 sequence for M. avium subsp. paratuberculosis (3, 4, 24, 34, 36), and IS901 for M. avium subsp. avium (14, 29). A duplex PCR has been developed to allow the differential identification of M. avium subsp. paratuberculosis, M. avium subsp. avium, and members of the Mycobacterium tuberculosis complex (4). However, the assay does not allow the differentiation of single from mixed infections.
The aim of this study was to further improve molecular methods for the species-specific identification of M. bovis, M. avium subsp. avium, and M. avium subsp. paratuberculosis in order to diagnose and differentiate single from mixed mycobacterial infections in red deer. During the period of 1999-2002, severe emaciation and mortalities, presumably related to mycobacterial infections, were reported for both adult and young wild red deer in the southeastern part of Belgium. Thirty-three carcasses (or parts of them, after scavenging had occurred) of deer that were found dead were brought to the local veterinary investigation center, and samples (mainly fecal material, as well as mesenteric lymph nodes) were shipped to our laboratories for bacteriological and molecular analysis.
MATERIALS AND METHODS
Bacterial isolates. Reference strains included two M. avium subsp. avium strains (ATCC 25291 and DSMZ 44156), three M. avium subsp. paratuberculosis strains (ATCC 19698, ATCC 43015, known as strain Linda [2], and the vaccinal strain 2E [33]), and one M. bovis strain (ATCC 19210). As controls, M. avium subsp. paratuberculosis strains (n = 12) isolated from bovine specimens during routine paratuberculosis diagnostic work between 2000 and 2003 were incorporated into this study. The controls also included M. bovis isolates from animal sources (1 from a chimpanzee and 2 from cattle) as well as M. avium subsp. paratuberculosis and M. avium subsp. avium isolates from human sources (3 M. avium subsp. paratuberculosis isolates from patients with Crohn's ileal disease and 32 M. avium subsp. avium isolates from AIDS patients with underlying lung disease).
Deer samples. Feces and/or tissue and organs known as sites of active infection, i.e., mesenteric lymph nodes and ileocecal junctions, were taken from slaughtered farmed red deer (3 animals) and from wild deer that were found dead (33 animals). In the latter case, the age, body condition (mainly emaciation and obvious soiling with green material around the tail, hindquarters, and hocks), and a description of gross lesions were recorded whenever possible. For some of these animals, organs were not available due to scavenging.
Bacteriology. All samples were subjected to diagnostic laboratory protocols for the isolation of M. avium subsp. avium, M. avium subsp. paratuberculosis, and M. bovis. Briefly, after a decontamination treatment with 0.75% hexadecylpyridinium chloride overnight, fecal samples were inoculated and tested for Mycobactin J (BioMerieux, France) dependency on Herrold’s egg yolk medium (HEYM) slants with and without Mycobactin J as previously described (11). Tissue samples were inoculated on HEYM slants with Mycobactin J and on Coletsos slants (Sanofi Diagnostics Pasteur, France) after a decontamination treatment with 3% oxalic acid for 30 min (38). Samples were incubated at 37°C for 4 to 6 months. The primary identification of M. avium subsp. paratuberculosis was based on its Mycobactin J dependency, whereas the primary identification of M. bovis was based on its susceptibility to thiophen-2-carboxylic acid hydrazide (TCH).
Preparation of mycobacterial DNA. DNA extraction was performed on colonies grown for 4 to 6 months. Colonies were suspended in 200 μl of lysing solution (0.1 M NaOH, 1 M NaCl, and 5% sodium dodecyl sulfate) and heated (100°C) for 20 min. The suspension was then cooled, neutralized with 3 volumes of 0.1 M Tris-HCl (pH 7.4) buffer, and centrifuged (5,000 x g, 5 min). Supernatants were extracted with phenol-chloroform, and DNAs were precipitated with ethanol, collected by centrifugation, dissolved in 50 μl of H2O, and stored at –20°C. DNAs were quantified by UV spectroscopy and/or fluorometry using Picogreen (Molecular Probes, Leiden, The Netherlands).
PCR amplification. The IS900 amplification protocol was a modified version of a previously described method (34). IS900 and IS901 primer sequences and their respective annealing temperatures are listed in Table 1. The amplification protocol was the same for the IS900 and IS901 PCRs and was performed as follows. The PCR mixture (50 μl) consisted of sample DNA (20 ng), 10 mM Tris-HCl, pH 8.3 (at 25°C), 50 mM KCl, 1.5 mM MgCl2, a 0.25 mM concentration of each deoxynucleoside triphosphate, 165 nM of each primer, and 0.75 U of AmpliTaq DNA polymerase (Perkin-Elmer Applied Biosystems, Foster City, Calif.). After an initial denaturation step (3 min at 94°C), 40 cycles of amplification were performed as follows: denaturation at 94°C for 1 min, annealing at the optimal temperature for each primer set (Table 1) for 1 min, and DNA extension at 72°C for 1 min 30 s. A final extension was performed at 72°C for 15 min.
The duplex us-p34-f57 amplification protocol was a modified version of a previously described protocol, with the original myc3 primer being degenerated and named myc32 (4). The myc1-myc32 (Fig. 1) and f57a-f57b (Fig. 2) primer sets, specific for the us-p34 and f57 target genes, respectively, were used as listed (Table 1). Briefly, the expected size of us-p34 amplicons was 257 bp for M. avium subsp. paratuberculosis and M. avium subsp. avium and 178 bp for M. bovis and other members of the M. tuberculosis complex, whereas the expected size of M. avium subsp. paratuberculosis-specific f57 amplicons was 439 bp (Fig. 3 and 4). Accordingly, the banding patterns allowed us to discriminate between these three mycobacterial species as previously described (4). Each PCR mixture (50 μl) consisted of a DNA sample (20 ng), 10 mM Tris-HCl, pH 8.3 (at 25°C), 50 mM KCl, 1.5 mM MgCl2, a 0.25 mM concentration of each deoxynucleoside triphosphate, the myc1 and myc32 primers, the f57a and f57b primers, and 0.75 U of AmpliTaq DNA polymerase (Perkin-Elmer Applied Biosystems). The amplification protocol was the same as that used for IS900 and IS901 PCR.
Identification of M. bovis by 16S rRNA PCR was performed with an Amplicor Mycobacterium tuberculosis detection kit (Amplicor MTB; Roche Diagnostic Systems, Somerville, N.J.) according to the manufacturer's instructions.
IS900, IS901, 16S rRNA, and duplex us-p34-f57 amplifications were carried out in a DNA 2400 thermocycler (Perkin-Elmer Applied Biosystems). Except for 16S rRNA amplicons, which were detected by hybridization according to the manufacturer's instructions, all of the amplified DNA fragments were run in a 2% agarose gel, stained with ethidium bromide, and visualized on a UV transilluminator.
PCR controls. A strict procedure was followed to avoid cross-contamination between samples or carryover of PCR products. For any series of reactions, contamination at the DNA level was ruled out by performing PCR analysis without a DNA template and on a negative control for DNA extraction. For positive controls, DNA samples from reference and control strains of M. avium subsp. avium, M. avium subsp. paratuberculosis, and M. bovis were included in each test. For each field sample, PCR inhibition was assessed. Briefly, 104 copies of a pCR-XL-TOPO plasmid carrying the 16S rRNA gene of Staphylococcus aureus was amplified alone and mixed with the sample. Amplification was carried out as described previously (15). PCR inhibition was considered relevant when no amplification or markedly decreased amplification of 104 plasmid copies of S. aureus from the mixture was observed.
Hpy188I restriction. Duplex us-p34-f57 amplification generates a banding pattern that does not allow discrimination between single M. avium subsp. paratuberculosis and mixed M. avium subsp. paratuberculosis/M. avium subsp. avium infections (Fig. 3). Recently, a novel endonuclease, Hpy188I, which recognizes the unique TCNGA sequence and cleaves the DNA between nucleotides N and G, was purified from Helicobacter pylori (37). In our previous work (4), the TCNGA sequence was found in M. avium subsp. avium, whereas a C-to-G single-nucleotide substitution (underlined hereafter) was identified in M. avium subsp. paratuberculosis within the TCNGA sequence, at position 168 upstream of the us-p34 initiation codon (Fig. 1). In a prevalidation step, 10 M. avium subsp. paratuberculosis and 10 M. avium subsp. avium strains were subjected to sequence analysis. Amplicons were sequenced in both orientations with a Big Dye Terminator kit (ABI PRISM; Applied Biosystems, Nieuwerkerk, The Netherlands), using an automated ABI 3100 sequencer (Applied Biosystems). The sequences were compared against those in GenBank for identity-based species identification using the BLAST program of the National Center for Biotechnology Information database. The C-to-G single-nucleotide substitution was found in all of the M. avium subsp. paratuberculosis and none of the M. avium subsp. avium isolates. In a next step, the validity of Hpy188I restriction was assessed with DNA samples from reference strains (three M. avium subsp. paratuberculosis, two M. avium subsp. avium, and one M. bovis strain) and with one reconstituted mixed M. avium subsp. avium/M. avium subsp. paratuberculosis DNA sample. Finally, Hpy188I restriction analysis was performed on control and field samples according to the manufacturer's protocol, except that amplicons were digested for 2 h (Table 2).
RESULTS
Ziehl-Neelsen staining and bacteriology of deer samples. The results of Ziehl-Neelsen staining and bacteriology of deer samples are depicted in Table 3. A large number of acid-fast bacilli were seen after staining of feces or tissue smears for all but six samples. However, mycobacteria were grown from the six Ziehl-Neelsen stain-negative samples. A total of 36 strains (33 from wild deer and 3 from farmed deer) were isolated and further characterized. Cultures on HEYM with Mycobactin J were obtained for all but one sample. Interestingly, 12 isolates assigned to the M. avium subsp. paratuberculosis infection group based on molecular techniques were classified as Mycobactin J independent, 4 were classified as Mycobactin J dependent, and 11 were not actually tested for their Mycobactin J dependency. All isolates tested for Mycobactin J dependency and assigned to the M. avium subsp. avium infection group or the mixed M. avium subsp. paratuberculosis/M. avium subsp. avium infection group were classified as Mycobactin J independent. M. bovis infection was excluded based on negative TCH test results (data not shown).
IS900, IS901, and 16S rRNA PCR. The IS900 and IS901 PCR results obtained with reference and control strains from different origins are depicted in Table 2. The IS901 and IS900 PCR results were negative for the four M. bovis strains. The 2 M. avium subsp. avium reference strains of avian origin were positive for IS901, whereas the 31 human and 1 bovine M. avium subsp. avium strain were consistently negative, as were the 18 M. avium subsp. paratuberculosis strains. The bovine and human M. avium subsp. paratuberculosis reference strains as well as bovine and human M. avium subsp. paratuberculosis strains isolated from clinical samples were all IS900 positive. The IS900/IS901 amplification results obtained for deer isolates are depicted in Table 3. Three patterns of IS900/IS901 results were observed: IS900 negative and IS901 positive (n = 4), IS900 positive and IS901 negative (n = 27), and IS900 positive and IS901 positive (n = 5). These results are compatible with three patterns of infection, namely, infection with M. avium subsp. avium, M. avium subsp. paratuberculosis, or a combination of M. avium subsp. paratuberculosis and M. avium subsp. avium, respectively. 16S rRNA PCR yielded positive results with the four M. bovis strains and negative results with all the other strains (data not shown).
Duplex us-p34-f57 PCR and Hpy188I restriction of duplex us-p34-f57 amplicons (PCR-restriction fragment length polymorphism [PCR-RFLP] analysis). (i) Duplex us-p34-f57 PCR. As illustrated with M. avium subsp. avium (n = 2), M. avium subsp. paratuberculosis (n = 3), and M. bovis (n = 1) reference strains, the banding pattern generated by duplex amplification can distinctly identify each species. However, duplex amplification cannot discriminate M. avium subsp. paratuberculosis alone from a mixture of M. avium subsp. paratuberculosis and M. avium subsp. avium (Fig. 3). Likewise, a mixture of M. avium subsp. paratuberculosis and M. bovis cannot be distinguished from a mixture of M. avium subsp. paratuberculosis, M. avium subsp. avium, and M. bovis.
(ii) Hpy188I restriction. Hpy188I restriction of duplex us-p34-f57 PCR amplicons obtained from the M. avium subsp. avium (n = 2), M. avium subsp. paratuberculosis (n = 3), and M. bovis (n = 1) reference strains as well as from a reconstituted mixed M. avium subsp. avium/M. avium subsp. paratuberculosis DNA sample gave the expected banding patterns. Sequence analysis of the amplicons prior to digestion confirmed the restriction patterns. Digestion of the TCCGA site in the 257-bp amplicons from M. avium subsp. avium yielded 79-bp and 178-bp fragments, whereas the C-to-G substitution in M. avium subsp. paratuberculosis suppressed the restriction site (Fig. 4). No restriction site was present in the 178-bp us-p34 amplicons from M. bovis. The digestion of M. avium subsp. paratuberculosis-specific f57 amplicons generated several fragments, of which only two (255 bp and 144 bp) were seen on the gel (Fig. 4). Accordingly, the digestion of duplex us-p34-f57 amplicons generated specific banding patterns for M. avium subsp. paratuberculosis, M. avium subsp. avium, and M. bovis alone as well as for any combination of two of these species (Fig. 4). The band size similarity (178 bp) between M. bovis us-p34 amplicons and a digested fragment of M. avium subsp. avium us-p34 amplicons could hamper the detection of mixed M. bovis, M. avium subsp. avium, and M. avium subsp. paratuberculosis infections. However, the presence of M. bovis would have been assessed by the duplex PCR in the first step of the PCR-RFLP analysis, so the hypothetical mixed M. bovis, M. avium subsp. avium, and M. avium subsp. paratuberculosis infection would also have been identified (Fig. 3).
(iii) Validation with control strains. In the following step, the PCR-RFLP method was tested on a series of strains isolated from human and animal clinical cases, including 32 M. avium subsp. avium, 15 M. avium subsp. paratuberculosis, and 3 M. bovis isolates. All of the DNA samples and the reconstituted mixture displayed the expected banding patterns according to their species. The molecular results were also in accordance with the IS900 PCR results, but no validation based on the IS901 PCR results could be made because all of the M. avium subsp. avium control strains were negative for IS901 (Table 2).
(iv) Analysis of mycobacterial colonies from deer samples. In a final step, the PCR-RFLP method was used to test 36 strains isolated from wild red deer found dead in the field (n = 33) and from slaughtered farmed deer (n = 3) (Table 3). Prior to enzymatic digestion, the duplex us-p34-f57 PCR generated only two patterns, with a us-p34-positive result for every sample (n = 36) and an f57 result that was either negative (n = 4) or positive (n = 32). No pattern consistent with the presence of M. bovis was seen, confirming the bacteriological identification results and the negative 16S rRNA PCR results. The molecular results were also in accordance with the IS900 and IS901 PCR results. For these last tests, it is worth noting that all of the M. avium subsp. avium deer isolates were IS901 positive. No PCR inhibition was observed.
Multigenotypic molecular identification of deer strains. Taken together, the results of IS900, IS901, and 16S rRNA amplification, the duplex us-p34-f57 PCR, and the duplex us-p34-f57 PCR-RFLP method identified three patterns of mycobacterial infections in deer field samples: 4 (11%) samples were identified as M. avium subsp. avium, 27 (75%) were identified as M. avium subsp. paratuberculosis, and 5 (14%) were identified as a mixture of M. avium subsp. paratuberculosis and M. avium subsp. avium, suggesting a coinfection with both mycobacteria.
DISCUSSION
Over 3 million reindeer (Rangifer tarandus) and 5 million deer belonging to other gregarious species are currently herded, held in fences, or farmed worldwide. M. avium subsp. paratuberculosis, M. bovis, and M. avium subsp. avium infections appear to be the most significant mycobacterial infections in free-living, captive, and farmed deer (18, 19). They have been reported in New Zealand (7, 20), North America (6, 22, 28, 30, 35), and Europe (11, 16, 17, 23, 27). Bacterial isolation is considered the "gold standard" and the most sensitive method for the diagnosis of mycobacterial diseases (19, 20). However, it is a very slow and labor-intensive procedure, and results are not available for several weeks (if not months) after inoculation, especially for samples containing small numbers of mycobacteria. Moreover, due to decontamination procedures, false-negative results may be obtained (12, 32). The identification of M. avium subsp. paratuberculosis is characterized by the organism's requirement for the iron-chelating growth factor Mycobactin J for in vitro growth. Recently, it was stressed that the diagnosis of paratuberculosis relying on Mycobactin J dependency also needed to be interpreted with caution (26). Therefore, as also confirmed in our study, subspecies differentiation between M. avium subsp. paratuberculosis and M. avium subsp. avium cannot rely solely on this criterion.
Likewise, indirect tests also have limitations. Although M. avium subsp. paratuberculosis and M. avium subsp. avium differ markedly in their virulence, particularly for cattle, they are antigenically and genetically very similar organisms (1). Serological tests and skin tests used for the diagnosis of paratuberculosis therefore have a low specificity. Moreover, cross reactions occur between M. avium subsp. avium, M. avium subsp. paratuberculosis, and M. bovis (21). Thus, the differential diagnosis of mycobacterial infections remains difficult with currently available tests (20).
Accordingly, a wide panel of molecular tests have therefore been proposed to improve the identification of mycobacteria. Among these, IS900- and IS901-based PCR assays have largely been used for the detection of M. avium subsp. paratuberculosis and M. avium subsp. avium, respectively (14, 24, 29, 36). Both markers, however, have limitations. Indeed, current data confirm the lack of the IS901 insertion sequence in human and bovine clinical M. avium subsp. avium strains, as reported previously for M. avium subsp. avium strains of various origins, including deer (14, 29). Interestingly, in our study, all of the M. avium subsp. avium strains isolated from deer were IS901 positive. Regarding IS900, recent data suggest that IS900-like elements are present in mycobacterial species other than M. avium subsp. paratuberculosis, generating false-positive results (5, 8, 13, 26, 34). Accordingly, false-negative and false-positive results cannot be ruled out with both insertion sequences, and IS900 or IS901 PCR results alone should therefore be interpreted with caution and compared to those obtained with other specific DNA markers. Moreover, as recently highlighted, because DNA is often not extracted from single colonies, it is possible that the extracted DNA contains a low level of DNA from another mycobacterial strain that is masked by the differential growth of the predominant mycobacteria in the sample. This was the most likely explanation for IS901-positive results obtained with DNA extracted from a sample containing M. avium subsp. paratuberculosis, as recently reported (26).
In our study, IS900-positive results were obtained with 89% (32/36) of samples from deer, whereas 25% of the samples (9/36) were IS901 positive. Among the latter, 4/9 were IS900 negative and 5/9 were IS900 positive. Accordingly, the combined IS900 and IS901 results suggested either single M. avium subsp. paratuberculosis and M. avium subsp. avium infections, or in some cases, a mixed M. avium subsp. paratuberculosis/M. avium subsp. avium infection. However, due to the fact that DNAs were not extracted from single colonies and due to the potential limitations of the IS900/901 PCR, the definitive identification of the IS900/901 pattern in deer samples was still pending.
Since the us-p34-f57 duplex PCR (4) does not allow us to discriminate between M. avium subsp. paratuberculosis infection alone and a mixed M. avium subsp. avium and M. avium subsp. paratuberculosis infection, Hpy188I restriction was carried out on duplex amplicons. This two-step strategy, i.e., integration of the pre- and postrestriction results, allowed the identification of single (M. avium subsp. paratuberculosis or M. avium subsp. avium) and mixed (M. avium subsp. paratuberculosis/M. avium subsp. avium) infections. The results of the banding patterns confirmed the IS900 and IS901 PCR results supporting the mixed infection hypothesis.
At this point, the TCNGA sequence had been found in all M. avium subsp. avium strains tested, regardless of their origin, Mycobactin J dependency, and IS901 PCR results, whereas the C-to-G single-nucleotide substitution within the TCNGA sequence, at position 168 upstream of the us-p34 initiation codon, was identified in all M. avium subsp. paratuberculosis strains tested. Consequently, all M. avium subsp. avium strains were restricted by Hpy188I, whereas none of the M. avium subsp. paratuberculosis strains were restricted. No restriction site was present in the 178-bp us-p34 amplicons from M. bovis.
These results suggest that Hpy188I restriction performed on duplex amplicons will allow a clear discrimination between M. avium subsp. avium and M. avium subsp. paratuberculosis strains, regardless of the Mycobactin J and IS900/901 PCR results. Moreover, this technique will also assess the possible contamination of M. avium subsp. paratuberculosis DNA by M. avium subsp. avium DNA and hence will help to further explain unexpected IS900/901 results, as recently documented (26), allowing validation of further molecular typing studies. We therefore propose to integrate the absence of the Hpy188I restriction site at position 168 upstream of the us-p34 initiation codon in a functional molecular definition of M. avium subsp. paratuberculosis.
Taken together, our molecular results strengthen the hypothesis that besides well-documented M. avium subsp. paratuberculosis infections, M. avium subsp. avium infections were the cause of macroscopic tuberculous lesions and eventually of death in a substantial number of wild red deer in Belgium. Moreover, we cannot rule out that mixed M. avium subsp. paratuberculosis/M. avium subsp. avium infections may result in more aggressive acute clinical outbreaks, particularly in young animals.
As recently reemphasized, there are very limited data, if any, on the proportions of M. avium subsp. paratuberculosis and M. avium subsp. avium subclinically infected deer that show no macroscopic evidence of infection, so that background information is missing (20). In our opinion, because of its ubiquitous nature, the issue becomes the identification of factors contributing to the implication of M. avium subsp. avium, alone or in association with M. avium subsp. paratuberculosis, in infection and eventually in clinical diseases in wild red deer. Factors likely to affect the susceptibility of deer to mycobacterial infection include age, environment, population density, exposure, and genetics (9, 18, 20).
Clinical outbreaks due to M. avium subsp. avium were reported for groups of deer kept in feedlots or indoors in winter, where they are exposed to food or water contaminated by bird feces (19). The hypothesis of a great exposition of these animals to M. avium subsp. avium is also strengthened, for farmed red deer, by the widespread reactivity to M. avium subsp. avium in skin tests and lymphocyte transformation assays (20). As reported for M. bovis infections of white-tailed deer (Odocoileus virginianus) (25) and M. avium subsp. paratuberculosis and M. avium subsp. avium infections of red deer (20), supplemental feeding or winter feeding may combine several risk factors, such as population density, exposure, and cross-contamination by infected vectors and contaminated water, soil, or food. High-density and winter feeding practices could at least partly explain the unusual pattern of mycobacterial infections in Belgian wild deer by enhancing exposure to M. avium subsp. avium so that a "clinical threshold" is reached and pathology is seen.
In conclusion, M. avium subsp. avium, besides or in association with M. avium subsp. paratuberculosis, causes clinical disease and eventually death in wild red deer in Belgium. Hence, the diagnosis of mycobacteriosis in deer is difficult, and current immunological tests do not allow a clear discrimination between both infections. Mixed infections may further complicate the picture and even challenge the definite bacteriological diagnosis of mycobacterial infections in deer. Therefore, the identification of mycobacterial infections requires a combination of phenotypic and molecular markers.
Our studies suggest that a combination of classical bacteriological isolation and identification methods, IS900/IS901- and 16S rRNA-based PCRs, and duplex us-p34-f57 PCR followed by Hpy188I restriction is a useful tool for the identification of mycobacterial infection in deer, particularly to discriminate between single M. avium subsp. paratuberculosis or M. avium subsp. avium infections, mixed M. avium subsp. paratuberculosis/M. avium subsp. avium infections, and M. bovis infections.
ACKNOWLEDGMENTS
This project was funded by the ACOS-Strat, Staff section of the Belgian Armed Forces supporting research and development (grant MED-03), by the Walloon Region (grants 981/3902 and 011/4853), and by the Ministry of Health, Food Chain Safety and Environment (grant S5992).
We thank J. Bughin from the regional veterinary investigation center (ARSIA) for providing us with deer samples. We also thank P.-A. Fonteyn (Scientific Institute of Public Health) for stimulating discussions on the microbiological results and F. Portaels (Institute of Tropical Medicine) and M. Fauville-Dufaux (Pasteur Institute of Brussels) for providing some human and/or bovine mycobacterial strains. The expert technical assistance of P. Vannoorenberghe is acknowledged.
These authors contributed equally to this work.
Present address: University of Pretoria, Faculty of Veterinary Science, Department of Veterinary Tropical Diseases, Private Bag X04, Onderstepoort 0110, South Africa.
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Laboratory of Applied Molecular Technology, Universite Catholique de Louvain, Brussels, Belgium
Defence Laboratories Department, Belgian Armed Forces, Brussels, Belgium
ABSTRACT
Severe emaciation and mortalities suggestive of mycobacterial infections were recently reported for both adult and young wild red deer (Cervus elaphus) in the southeastern part of Belgium. In deer, tuberculous lesions are not pathognomonic of Mycobacterium bovis infection due to gross and microscopic similarities with lesions caused by Mycobacterium avium subsp. paratuberculosis or M. avium subsp. avium. The aim of this study was to improve molecular methods for the species-specific identification of M. bovis, M. avium subsp. avium, and M. avium subsp. paratuberculosis in mycobacterial infections of deer. DNA banding patterns were assessed prior to and after Hpy188I restriction of f57-upstream (us)-p34 duplex amplicons. The duplex f57-us-p34 PCR differentiated M. bovis from M. avium subsp. paratuberculosis and M. avium subsp. avium infections, whereas the restriction step differentiated single M. avium subsp. paratuberculosis or M. avium subsp. avium infections from mixed M. avium subsp. paratuberculosis/M. avium subsp. avium infections. The endonuclease Hpy188I cleaves DNA between nucleotides N and G in the unique TCNGA sequence. This restriction site was found at position 168 upstream of the us-p34 initiation codon in all M. avium subsp. avium strains tested, regardless of their origin and the results of IS901 PCR. In contrast, the restriction site was abrogated in all M. avium subsp. paratuberculosis strains tested, independent of their origin, Mycobactin J dependency, and IS900 PCR results. Consequently, a two-step strategy, i.e., duplex us-p34-f57 PCR and Hpy188I restriction, allowed us to exclude M. bovis infection and to identify single (M. avium subsp. paratuberculosis or M. avium subsp. avium) or mixed (M. avium subsp. paratuberculosis/M. avium subsp. avium) infections in wild red deer in Belgium. Accordingly, we propose to integrate, in a functional molecular definition of M. avium subsp. paratuberculosis, the absence of the Hpy188I restriction site from the us-p34 amplicon.
INTRODUCTION
The most significant mycobacterial diseases of free-living, captive, and farmed red deer (Cervus elaphus) are bovine tuberculosis, caused by Mycobacterium bovis, Johne's disease (paratuberculosis), caused by Mycobacterium avium subsp. paratuberculosis, and avian tuberculosis, caused principally by M. avium subsp. avium (20).
Differential diagnoses of infections by M. bovis, M. avium subsp. paratuberculosis, and M. avium subsp. avium are essential because the epidemiology, zoonotic concern, and prevention and control methods are different for each of these mycobacterial infections (20). In deer, tuberculous lesions are not pathognomonic of M. bovis infection and are actually indistinguishable from lesions due to M. avium subsp. paratuberculosis or M. avium subsp. avium infection because of their gross and microscopic similarities. Moreover, the actual differential diagnosis of these mycobacterial infections is difficult due to similarities in the clinical expression and pathology associated with these infections. Antigenic similarity between M. avium subsp. avium, M. avium subsp. paratuberculosis, and M. bovis is also responsible for the lack of sensitivity and specificity of diagnostic tests (i.e., current serological and laboratory cell-mediated diagnostic tests as well as skin tests) (21).
The isolation of mycobacteria requires a decontamination treatment of the sample. Depending on the nature and the time of the decontamination treatment, mycobacteria present in the sample may also be killed. As recently demonstrated for M. avium subsp. paratuberculosis, routine protocols in the laboratory significantly decrease the number of organisms isolated per sample (for fecal, tissue, and milk samples), stressing the importance of the interpretation of negative culture results (12, 32). Conversely, atypical mycobacteria may still be grown from treated specimens (10).
Mycobacterial species are traditionally differentiated on the basis of their phenotypic characteristics. However, since these do not allow the precise identification of all species, recent molecular biology methods offer new opportunities to differentiate, identify, and type mycobacterial species (31). Several molecular targets have been used to allow species-specific identification, including IS6110 and 16S rRNA for the Mycobacterium tuberculosis complex (i.e., M. bovis, M. tuberculosis, Mycobacterium africanum, and Mycobacterium microti), IS900 and the f57 sequence for M. avium subsp. paratuberculosis (3, 4, 24, 34, 36), and IS901 for M. avium subsp. avium (14, 29). A duplex PCR has been developed to allow the differential identification of M. avium subsp. paratuberculosis, M. avium subsp. avium, and members of the Mycobacterium tuberculosis complex (4). However, the assay does not allow the differentiation of single from mixed infections.
The aim of this study was to further improve molecular methods for the species-specific identification of M. bovis, M. avium subsp. avium, and M. avium subsp. paratuberculosis in order to diagnose and differentiate single from mixed mycobacterial infections in red deer. During the period of 1999-2002, severe emaciation and mortalities, presumably related to mycobacterial infections, were reported for both adult and young wild red deer in the southeastern part of Belgium. Thirty-three carcasses (or parts of them, after scavenging had occurred) of deer that were found dead were brought to the local veterinary investigation center, and samples (mainly fecal material, as well as mesenteric lymph nodes) were shipped to our laboratories for bacteriological and molecular analysis.
MATERIALS AND METHODS
Bacterial isolates. Reference strains included two M. avium subsp. avium strains (ATCC 25291 and DSMZ 44156), three M. avium subsp. paratuberculosis strains (ATCC 19698, ATCC 43015, known as strain Linda [2], and the vaccinal strain 2E [33]), and one M. bovis strain (ATCC 19210). As controls, M. avium subsp. paratuberculosis strains (n = 12) isolated from bovine specimens during routine paratuberculosis diagnostic work between 2000 and 2003 were incorporated into this study. The controls also included M. bovis isolates from animal sources (1 from a chimpanzee and 2 from cattle) as well as M. avium subsp. paratuberculosis and M. avium subsp. avium isolates from human sources (3 M. avium subsp. paratuberculosis isolates from patients with Crohn's ileal disease and 32 M. avium subsp. avium isolates from AIDS patients with underlying lung disease).
Deer samples. Feces and/or tissue and organs known as sites of active infection, i.e., mesenteric lymph nodes and ileocecal junctions, were taken from slaughtered farmed red deer (3 animals) and from wild deer that were found dead (33 animals). In the latter case, the age, body condition (mainly emaciation and obvious soiling with green material around the tail, hindquarters, and hocks), and a description of gross lesions were recorded whenever possible. For some of these animals, organs were not available due to scavenging.
Bacteriology. All samples were subjected to diagnostic laboratory protocols for the isolation of M. avium subsp. avium, M. avium subsp. paratuberculosis, and M. bovis. Briefly, after a decontamination treatment with 0.75% hexadecylpyridinium chloride overnight, fecal samples were inoculated and tested for Mycobactin J (BioMerieux, France) dependency on Herrold’s egg yolk medium (HEYM) slants with and without Mycobactin J as previously described (11). Tissue samples were inoculated on HEYM slants with Mycobactin J and on Coletsos slants (Sanofi Diagnostics Pasteur, France) after a decontamination treatment with 3% oxalic acid for 30 min (38). Samples were incubated at 37°C for 4 to 6 months. The primary identification of M. avium subsp. paratuberculosis was based on its Mycobactin J dependency, whereas the primary identification of M. bovis was based on its susceptibility to thiophen-2-carboxylic acid hydrazide (TCH).
Preparation of mycobacterial DNA. DNA extraction was performed on colonies grown for 4 to 6 months. Colonies were suspended in 200 μl of lysing solution (0.1 M NaOH, 1 M NaCl, and 5% sodium dodecyl sulfate) and heated (100°C) for 20 min. The suspension was then cooled, neutralized with 3 volumes of 0.1 M Tris-HCl (pH 7.4) buffer, and centrifuged (5,000 x g, 5 min). Supernatants were extracted with phenol-chloroform, and DNAs were precipitated with ethanol, collected by centrifugation, dissolved in 50 μl of H2O, and stored at –20°C. DNAs were quantified by UV spectroscopy and/or fluorometry using Picogreen (Molecular Probes, Leiden, The Netherlands).
PCR amplification. The IS900 amplification protocol was a modified version of a previously described method (34). IS900 and IS901 primer sequences and their respective annealing temperatures are listed in Table 1. The amplification protocol was the same for the IS900 and IS901 PCRs and was performed as follows. The PCR mixture (50 μl) consisted of sample DNA (20 ng), 10 mM Tris-HCl, pH 8.3 (at 25°C), 50 mM KCl, 1.5 mM MgCl2, a 0.25 mM concentration of each deoxynucleoside triphosphate, 165 nM of each primer, and 0.75 U of AmpliTaq DNA polymerase (Perkin-Elmer Applied Biosystems, Foster City, Calif.). After an initial denaturation step (3 min at 94°C), 40 cycles of amplification were performed as follows: denaturation at 94°C for 1 min, annealing at the optimal temperature for each primer set (Table 1) for 1 min, and DNA extension at 72°C for 1 min 30 s. A final extension was performed at 72°C for 15 min.
The duplex us-p34-f57 amplification protocol was a modified version of a previously described protocol, with the original myc3 primer being degenerated and named myc32 (4). The myc1-myc32 (Fig. 1) and f57a-f57b (Fig. 2) primer sets, specific for the us-p34 and f57 target genes, respectively, were used as listed (Table 1). Briefly, the expected size of us-p34 amplicons was 257 bp for M. avium subsp. paratuberculosis and M. avium subsp. avium and 178 bp for M. bovis and other members of the M. tuberculosis complex, whereas the expected size of M. avium subsp. paratuberculosis-specific f57 amplicons was 439 bp (Fig. 3 and 4). Accordingly, the banding patterns allowed us to discriminate between these three mycobacterial species as previously described (4). Each PCR mixture (50 μl) consisted of a DNA sample (20 ng), 10 mM Tris-HCl, pH 8.3 (at 25°C), 50 mM KCl, 1.5 mM MgCl2, a 0.25 mM concentration of each deoxynucleoside triphosphate, the myc1 and myc32 primers, the f57a and f57b primers, and 0.75 U of AmpliTaq DNA polymerase (Perkin-Elmer Applied Biosystems). The amplification protocol was the same as that used for IS900 and IS901 PCR.
Identification of M. bovis by 16S rRNA PCR was performed with an Amplicor Mycobacterium tuberculosis detection kit (Amplicor MTB; Roche Diagnostic Systems, Somerville, N.J.) according to the manufacturer's instructions.
IS900, IS901, 16S rRNA, and duplex us-p34-f57 amplifications were carried out in a DNA 2400 thermocycler (Perkin-Elmer Applied Biosystems). Except for 16S rRNA amplicons, which were detected by hybridization according to the manufacturer's instructions, all of the amplified DNA fragments were run in a 2% agarose gel, stained with ethidium bromide, and visualized on a UV transilluminator.
PCR controls. A strict procedure was followed to avoid cross-contamination between samples or carryover of PCR products. For any series of reactions, contamination at the DNA level was ruled out by performing PCR analysis without a DNA template and on a negative control for DNA extraction. For positive controls, DNA samples from reference and control strains of M. avium subsp. avium, M. avium subsp. paratuberculosis, and M. bovis were included in each test. For each field sample, PCR inhibition was assessed. Briefly, 104 copies of a pCR-XL-TOPO plasmid carrying the 16S rRNA gene of Staphylococcus aureus was amplified alone and mixed with the sample. Amplification was carried out as described previously (15). PCR inhibition was considered relevant when no amplification or markedly decreased amplification of 104 plasmid copies of S. aureus from the mixture was observed.
Hpy188I restriction. Duplex us-p34-f57 amplification generates a banding pattern that does not allow discrimination between single M. avium subsp. paratuberculosis and mixed M. avium subsp. paratuberculosis/M. avium subsp. avium infections (Fig. 3). Recently, a novel endonuclease, Hpy188I, which recognizes the unique TCNGA sequence and cleaves the DNA between nucleotides N and G, was purified from Helicobacter pylori (37). In our previous work (4), the TCNGA sequence was found in M. avium subsp. avium, whereas a C-to-G single-nucleotide substitution (underlined hereafter) was identified in M. avium subsp. paratuberculosis within the TCNGA sequence, at position 168 upstream of the us-p34 initiation codon (Fig. 1). In a prevalidation step, 10 M. avium subsp. paratuberculosis and 10 M. avium subsp. avium strains were subjected to sequence analysis. Amplicons were sequenced in both orientations with a Big Dye Terminator kit (ABI PRISM; Applied Biosystems, Nieuwerkerk, The Netherlands), using an automated ABI 3100 sequencer (Applied Biosystems). The sequences were compared against those in GenBank for identity-based species identification using the BLAST program of the National Center for Biotechnology Information database. The C-to-G single-nucleotide substitution was found in all of the M. avium subsp. paratuberculosis and none of the M. avium subsp. avium isolates. In a next step, the validity of Hpy188I restriction was assessed with DNA samples from reference strains (three M. avium subsp. paratuberculosis, two M. avium subsp. avium, and one M. bovis strain) and with one reconstituted mixed M. avium subsp. avium/M. avium subsp. paratuberculosis DNA sample. Finally, Hpy188I restriction analysis was performed on control and field samples according to the manufacturer's protocol, except that amplicons were digested for 2 h (Table 2).
RESULTS
Ziehl-Neelsen staining and bacteriology of deer samples. The results of Ziehl-Neelsen staining and bacteriology of deer samples are depicted in Table 3. A large number of acid-fast bacilli were seen after staining of feces or tissue smears for all but six samples. However, mycobacteria were grown from the six Ziehl-Neelsen stain-negative samples. A total of 36 strains (33 from wild deer and 3 from farmed deer) were isolated and further characterized. Cultures on HEYM with Mycobactin J were obtained for all but one sample. Interestingly, 12 isolates assigned to the M. avium subsp. paratuberculosis infection group based on molecular techniques were classified as Mycobactin J independent, 4 were classified as Mycobactin J dependent, and 11 were not actually tested for their Mycobactin J dependency. All isolates tested for Mycobactin J dependency and assigned to the M. avium subsp. avium infection group or the mixed M. avium subsp. paratuberculosis/M. avium subsp. avium infection group were classified as Mycobactin J independent. M. bovis infection was excluded based on negative TCH test results (data not shown).
IS900, IS901, and 16S rRNA PCR. The IS900 and IS901 PCR results obtained with reference and control strains from different origins are depicted in Table 2. The IS901 and IS900 PCR results were negative for the four M. bovis strains. The 2 M. avium subsp. avium reference strains of avian origin were positive for IS901, whereas the 31 human and 1 bovine M. avium subsp. avium strain were consistently negative, as were the 18 M. avium subsp. paratuberculosis strains. The bovine and human M. avium subsp. paratuberculosis reference strains as well as bovine and human M. avium subsp. paratuberculosis strains isolated from clinical samples were all IS900 positive. The IS900/IS901 amplification results obtained for deer isolates are depicted in Table 3. Three patterns of IS900/IS901 results were observed: IS900 negative and IS901 positive (n = 4), IS900 positive and IS901 negative (n = 27), and IS900 positive and IS901 positive (n = 5). These results are compatible with three patterns of infection, namely, infection with M. avium subsp. avium, M. avium subsp. paratuberculosis, or a combination of M. avium subsp. paratuberculosis and M. avium subsp. avium, respectively. 16S rRNA PCR yielded positive results with the four M. bovis strains and negative results with all the other strains (data not shown).
Duplex us-p34-f57 PCR and Hpy188I restriction of duplex us-p34-f57 amplicons (PCR-restriction fragment length polymorphism [PCR-RFLP] analysis). (i) Duplex us-p34-f57 PCR. As illustrated with M. avium subsp. avium (n = 2), M. avium subsp. paratuberculosis (n = 3), and M. bovis (n = 1) reference strains, the banding pattern generated by duplex amplification can distinctly identify each species. However, duplex amplification cannot discriminate M. avium subsp. paratuberculosis alone from a mixture of M. avium subsp. paratuberculosis and M. avium subsp. avium (Fig. 3). Likewise, a mixture of M. avium subsp. paratuberculosis and M. bovis cannot be distinguished from a mixture of M. avium subsp. paratuberculosis, M. avium subsp. avium, and M. bovis.
(ii) Hpy188I restriction. Hpy188I restriction of duplex us-p34-f57 PCR amplicons obtained from the M. avium subsp. avium (n = 2), M. avium subsp. paratuberculosis (n = 3), and M. bovis (n = 1) reference strains as well as from a reconstituted mixed M. avium subsp. avium/M. avium subsp. paratuberculosis DNA sample gave the expected banding patterns. Sequence analysis of the amplicons prior to digestion confirmed the restriction patterns. Digestion of the TCCGA site in the 257-bp amplicons from M. avium subsp. avium yielded 79-bp and 178-bp fragments, whereas the C-to-G substitution in M. avium subsp. paratuberculosis suppressed the restriction site (Fig. 4). No restriction site was present in the 178-bp us-p34 amplicons from M. bovis. The digestion of M. avium subsp. paratuberculosis-specific f57 amplicons generated several fragments, of which only two (255 bp and 144 bp) were seen on the gel (Fig. 4). Accordingly, the digestion of duplex us-p34-f57 amplicons generated specific banding patterns for M. avium subsp. paratuberculosis, M. avium subsp. avium, and M. bovis alone as well as for any combination of two of these species (Fig. 4). The band size similarity (178 bp) between M. bovis us-p34 amplicons and a digested fragment of M. avium subsp. avium us-p34 amplicons could hamper the detection of mixed M. bovis, M. avium subsp. avium, and M. avium subsp. paratuberculosis infections. However, the presence of M. bovis would have been assessed by the duplex PCR in the first step of the PCR-RFLP analysis, so the hypothetical mixed M. bovis, M. avium subsp. avium, and M. avium subsp. paratuberculosis infection would also have been identified (Fig. 3).
(iii) Validation with control strains. In the following step, the PCR-RFLP method was tested on a series of strains isolated from human and animal clinical cases, including 32 M. avium subsp. avium, 15 M. avium subsp. paratuberculosis, and 3 M. bovis isolates. All of the DNA samples and the reconstituted mixture displayed the expected banding patterns according to their species. The molecular results were also in accordance with the IS900 PCR results, but no validation based on the IS901 PCR results could be made because all of the M. avium subsp. avium control strains were negative for IS901 (Table 2).
(iv) Analysis of mycobacterial colonies from deer samples. In a final step, the PCR-RFLP method was used to test 36 strains isolated from wild red deer found dead in the field (n = 33) and from slaughtered farmed deer (n = 3) (Table 3). Prior to enzymatic digestion, the duplex us-p34-f57 PCR generated only two patterns, with a us-p34-positive result for every sample (n = 36) and an f57 result that was either negative (n = 4) or positive (n = 32). No pattern consistent with the presence of M. bovis was seen, confirming the bacteriological identification results and the negative 16S rRNA PCR results. The molecular results were also in accordance with the IS900 and IS901 PCR results. For these last tests, it is worth noting that all of the M. avium subsp. avium deer isolates were IS901 positive. No PCR inhibition was observed.
Multigenotypic molecular identification of deer strains. Taken together, the results of IS900, IS901, and 16S rRNA amplification, the duplex us-p34-f57 PCR, and the duplex us-p34-f57 PCR-RFLP method identified three patterns of mycobacterial infections in deer field samples: 4 (11%) samples were identified as M. avium subsp. avium, 27 (75%) were identified as M. avium subsp. paratuberculosis, and 5 (14%) were identified as a mixture of M. avium subsp. paratuberculosis and M. avium subsp. avium, suggesting a coinfection with both mycobacteria.
DISCUSSION
Over 3 million reindeer (Rangifer tarandus) and 5 million deer belonging to other gregarious species are currently herded, held in fences, or farmed worldwide. M. avium subsp. paratuberculosis, M. bovis, and M. avium subsp. avium infections appear to be the most significant mycobacterial infections in free-living, captive, and farmed deer (18, 19). They have been reported in New Zealand (7, 20), North America (6, 22, 28, 30, 35), and Europe (11, 16, 17, 23, 27). Bacterial isolation is considered the "gold standard" and the most sensitive method for the diagnosis of mycobacterial diseases (19, 20). However, it is a very slow and labor-intensive procedure, and results are not available for several weeks (if not months) after inoculation, especially for samples containing small numbers of mycobacteria. Moreover, due to decontamination procedures, false-negative results may be obtained (12, 32). The identification of M. avium subsp. paratuberculosis is characterized by the organism's requirement for the iron-chelating growth factor Mycobactin J for in vitro growth. Recently, it was stressed that the diagnosis of paratuberculosis relying on Mycobactin J dependency also needed to be interpreted with caution (26). Therefore, as also confirmed in our study, subspecies differentiation between M. avium subsp. paratuberculosis and M. avium subsp. avium cannot rely solely on this criterion.
Likewise, indirect tests also have limitations. Although M. avium subsp. paratuberculosis and M. avium subsp. avium differ markedly in their virulence, particularly for cattle, they are antigenically and genetically very similar organisms (1). Serological tests and skin tests used for the diagnosis of paratuberculosis therefore have a low specificity. Moreover, cross reactions occur between M. avium subsp. avium, M. avium subsp. paratuberculosis, and M. bovis (21). Thus, the differential diagnosis of mycobacterial infections remains difficult with currently available tests (20).
Accordingly, a wide panel of molecular tests have therefore been proposed to improve the identification of mycobacteria. Among these, IS900- and IS901-based PCR assays have largely been used for the detection of M. avium subsp. paratuberculosis and M. avium subsp. avium, respectively (14, 24, 29, 36). Both markers, however, have limitations. Indeed, current data confirm the lack of the IS901 insertion sequence in human and bovine clinical M. avium subsp. avium strains, as reported previously for M. avium subsp. avium strains of various origins, including deer (14, 29). Interestingly, in our study, all of the M. avium subsp. avium strains isolated from deer were IS901 positive. Regarding IS900, recent data suggest that IS900-like elements are present in mycobacterial species other than M. avium subsp. paratuberculosis, generating false-positive results (5, 8, 13, 26, 34). Accordingly, false-negative and false-positive results cannot be ruled out with both insertion sequences, and IS900 or IS901 PCR results alone should therefore be interpreted with caution and compared to those obtained with other specific DNA markers. Moreover, as recently highlighted, because DNA is often not extracted from single colonies, it is possible that the extracted DNA contains a low level of DNA from another mycobacterial strain that is masked by the differential growth of the predominant mycobacteria in the sample. This was the most likely explanation for IS901-positive results obtained with DNA extracted from a sample containing M. avium subsp. paratuberculosis, as recently reported (26).
In our study, IS900-positive results were obtained with 89% (32/36) of samples from deer, whereas 25% of the samples (9/36) were IS901 positive. Among the latter, 4/9 were IS900 negative and 5/9 were IS900 positive. Accordingly, the combined IS900 and IS901 results suggested either single M. avium subsp. paratuberculosis and M. avium subsp. avium infections, or in some cases, a mixed M. avium subsp. paratuberculosis/M. avium subsp. avium infection. However, due to the fact that DNAs were not extracted from single colonies and due to the potential limitations of the IS900/901 PCR, the definitive identification of the IS900/901 pattern in deer samples was still pending.
Since the us-p34-f57 duplex PCR (4) does not allow us to discriminate between M. avium subsp. paratuberculosis infection alone and a mixed M. avium subsp. avium and M. avium subsp. paratuberculosis infection, Hpy188I restriction was carried out on duplex amplicons. This two-step strategy, i.e., integration of the pre- and postrestriction results, allowed the identification of single (M. avium subsp. paratuberculosis or M. avium subsp. avium) and mixed (M. avium subsp. paratuberculosis/M. avium subsp. avium) infections. The results of the banding patterns confirmed the IS900 and IS901 PCR results supporting the mixed infection hypothesis.
At this point, the TCNGA sequence had been found in all M. avium subsp. avium strains tested, regardless of their origin, Mycobactin J dependency, and IS901 PCR results, whereas the C-to-G single-nucleotide substitution within the TCNGA sequence, at position 168 upstream of the us-p34 initiation codon, was identified in all M. avium subsp. paratuberculosis strains tested. Consequently, all M. avium subsp. avium strains were restricted by Hpy188I, whereas none of the M. avium subsp. paratuberculosis strains were restricted. No restriction site was present in the 178-bp us-p34 amplicons from M. bovis.
These results suggest that Hpy188I restriction performed on duplex amplicons will allow a clear discrimination between M. avium subsp. avium and M. avium subsp. paratuberculosis strains, regardless of the Mycobactin J and IS900/901 PCR results. Moreover, this technique will also assess the possible contamination of M. avium subsp. paratuberculosis DNA by M. avium subsp. avium DNA and hence will help to further explain unexpected IS900/901 results, as recently documented (26), allowing validation of further molecular typing studies. We therefore propose to integrate the absence of the Hpy188I restriction site at position 168 upstream of the us-p34 initiation codon in a functional molecular definition of M. avium subsp. paratuberculosis.
Taken together, our molecular results strengthen the hypothesis that besides well-documented M. avium subsp. paratuberculosis infections, M. avium subsp. avium infections were the cause of macroscopic tuberculous lesions and eventually of death in a substantial number of wild red deer in Belgium. Moreover, we cannot rule out that mixed M. avium subsp. paratuberculosis/M. avium subsp. avium infections may result in more aggressive acute clinical outbreaks, particularly in young animals.
As recently reemphasized, there are very limited data, if any, on the proportions of M. avium subsp. paratuberculosis and M. avium subsp. avium subclinically infected deer that show no macroscopic evidence of infection, so that background information is missing (20). In our opinion, because of its ubiquitous nature, the issue becomes the identification of factors contributing to the implication of M. avium subsp. avium, alone or in association with M. avium subsp. paratuberculosis, in infection and eventually in clinical diseases in wild red deer. Factors likely to affect the susceptibility of deer to mycobacterial infection include age, environment, population density, exposure, and genetics (9, 18, 20).
Clinical outbreaks due to M. avium subsp. avium were reported for groups of deer kept in feedlots or indoors in winter, where they are exposed to food or water contaminated by bird feces (19). The hypothesis of a great exposition of these animals to M. avium subsp. avium is also strengthened, for farmed red deer, by the widespread reactivity to M. avium subsp. avium in skin tests and lymphocyte transformation assays (20). As reported for M. bovis infections of white-tailed deer (Odocoileus virginianus) (25) and M. avium subsp. paratuberculosis and M. avium subsp. avium infections of red deer (20), supplemental feeding or winter feeding may combine several risk factors, such as population density, exposure, and cross-contamination by infected vectors and contaminated water, soil, or food. High-density and winter feeding practices could at least partly explain the unusual pattern of mycobacterial infections in Belgian wild deer by enhancing exposure to M. avium subsp. avium so that a "clinical threshold" is reached and pathology is seen.
In conclusion, M. avium subsp. avium, besides or in association with M. avium subsp. paratuberculosis, causes clinical disease and eventually death in wild red deer in Belgium. Hence, the diagnosis of mycobacteriosis in deer is difficult, and current immunological tests do not allow a clear discrimination between both infections. Mixed infections may further complicate the picture and even challenge the definite bacteriological diagnosis of mycobacterial infections in deer. Therefore, the identification of mycobacterial infections requires a combination of phenotypic and molecular markers.
Our studies suggest that a combination of classical bacteriological isolation and identification methods, IS900/IS901- and 16S rRNA-based PCRs, and duplex us-p34-f57 PCR followed by Hpy188I restriction is a useful tool for the identification of mycobacterial infection in deer, particularly to discriminate between single M. avium subsp. paratuberculosis or M. avium subsp. avium infections, mixed M. avium subsp. paratuberculosis/M. avium subsp. avium infections, and M. bovis infections.
ACKNOWLEDGMENTS
This project was funded by the ACOS-Strat, Staff section of the Belgian Armed Forces supporting research and development (grant MED-03), by the Walloon Region (grants 981/3902 and 011/4853), and by the Ministry of Health, Food Chain Safety and Environment (grant S5992).
We thank J. Bughin from the regional veterinary investigation center (ARSIA) for providing us with deer samples. We also thank P.-A. Fonteyn (Scientific Institute of Public Health) for stimulating discussions on the microbiological results and F. Portaels (Institute of Tropical Medicine) and M. Fauville-Dufaux (Pasteur Institute of Brussels) for providing some human and/or bovine mycobacterial strains. The expert technical assistance of P. Vannoorenberghe is acknowledged.
These authors contributed equally to this work.
Present address: University of Pretoria, Faculty of Veterinary Science, Department of Veterinary Tropical Diseases, Private Bag X04, Onderstepoort 0110, South Africa.
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