Definitive Differentiation between Single and Mixed Mycobacterial Infections in Red Deer (Cervus elaphus) by a Combination of Duplex Amplification of p34 and f57 Sequences and Hpy188I Enzymatic Restriction of Duplex Amplicons

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.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 ...

Development and validation of a triplex real-time PCR for rapid detection and specific identification of M. avium sub sp. paratuberculosis in faecal samples

“…This report highlights the main diagnostic challenges: the intermittent nature of Map shedding and/ or the lack of sensitivity of culture. Moreover, Map dependency for mycobactin J should be interpreted with caution as some Map strains may grow without mycobactin J (Motiwala et al, 2004;Godfroid et al, 2005). Culture limitations are therefore of particular concern regarding specificity and sensitivity of Map detection in cattle (Sockett et al, 1992).…”

“…However, recent reports have challenged the specificity of IS900-based assays due to the discovery of IS900-like sequences in non-Map mycobacteria (Englund et al, 2002;Cousins et al, 1999;Godfroid et al, 2005;Motiwala et al, 2004;Tasara et al, 2005). In order to improve PCR identification of Map, a triplex realtime PCR (TRT-PCR) co-amplifying in the same tube the multi-copy IS900, the single-copy Map-restricted f57 target (Coetsier et al, 2000;Godfroid et al, 2005;Poupart et al, 1993) and the new multi-copy Map-restricted ISMAP02 genetic target (Paustian et al, 2004) was designed. TRT-PCR results were compared on the same faecal samples with methods routinely used for Map identification (i.e.…”

“…relevant biological samples (Godfroid et al, 2005). Specific and sensitive diagnostic methods allowing rapid antemortem detection of Map are critically needed.…”

Paratuberculosis or avian tuberculosis in red deer with chronic diarrhea?

“…avium (Maa). Co-infection with Map and Maa, which causes clinical disease and eventually death, complicates and challenges the bacteriological diagnosis of mycobacterial infections in deer (Godfroid et al 2005). Besides, just as in the case of avian tuberculosis, lesions found at slaughter in deer subclinically infected with paratuberculosis may cause problems because of their gross and histopathological similarity to lesions of tuberculosis (Campbell 1995).…”

Comparison of Three Immunological Diagnostic Tests for the Detection of Avian Tuberculosis in Naturally Infected Red Deer (Cervus Elaphus)

“…4,14 It is generally accepted that lesions and clinical progression of both infections are indistinguishable, and immunological or etiologic probes are required to distinguish between the 2 agents. 5,14 The comparative cervical tuberculin (CCT) skin test has been applied in cervids in some cases to detect the tuberculous infection, obtaining good results. 8,11,24 However, the CCT test has 2 very important limitations: 1) it has been proved in domestic ruminants that CCT cannot detect some phases of mycobacterial infection, 10,14 and 2) wild ruminants must be captured twice, increasing the stress and the risk of accidents for the animals and for the personnel handling them.…”