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SHORT COMMUNICATION |
1 Department of Microbiology and Immunology, University of British Colombia, 6174 University Blvd., Vancouver, British Columbia V6T 1Z3, Canada;
2 Department of Large Animal Clinical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, 52 Campus Drive, Saskatoon, Saskatchewan S7N 5B4, Canada;
3 Provincial Laboratory for Public Health, 3030 Hospital Drive N.W., Calgary, Alberta T2N 4W4, Canada;
4 Wildlife Division, Northwest Territories Environment and Natural Resources, 5102 50th Ave., Yellowknife, Northwest Territory X1A 3S8, Canada
5 Corresponding author (email: murray.woodbury{at}usask.ca)
ABSTRACT:
Nested polymerase chain reaction (PCR) using the Mycobacterium avium subspecies paratuberculosis (Map)–specific region, locus 251, was used as a screening tool for the detection of Map DNA in fecal samples from northern Canadian bison herds. Further characterization of positive samples (26/835) was performed because Map DNA was found without signs of disease. Strain typing, using PCR-Restriction endonucleas assay (REA), was limited to two samples but revealed that the samples corresponded to a cattle-related strain and a sheep-related strain. Sequencing of part of the IS1311 region from the two samples revealed a unique three base-pair region, which is only found within the northern Canadian bison isolates.
Key words: Bison, Mycobacterium paratuberculosis, PCR, PCR-REA.
Bison (Bison bison athabascae) in Wood Buffalo National Park (WBNP) are one of the last genetic resources of wild wood buffalo in Canada. At one point, the population of wood bison in North America was well over 100,000, but in the 1900s, due to unregulated hunting, wood bison numbers decreased to just 250 animals (Government of the Northwest Territories [NWT], 2004). From 1923 to 1928, the Canadian government moved 6,600 plains bison (Bison bison bison) from Alberta to WBNP to increase the general bison population. Unfortunately, the translocation of plains bison was associated with the introduction of bovine brucellosis (Brucella abortus) and tuberculosis (Mycobacterium bovis) to the area. Creating brucella- and tuberculosis-free bison herds is now a high priority for bison conservation in Canada. Monitoring the status of other possible pathogenic infections, such as those from Mycobacterium avium subsp. paratuberculosis (Map), has been included in the preservation plan. Infection with this organism has been previously demonstrated in farmed bison (Buergelt and Ginn, 2000), but never in the free-ranging bison from the WBNP area.
There is an extended preclinical phase of Map infection and intermittent fecal shedding of Map organisms from infected animals. A noninvasive, economical assay is needed to test asymptomatic animals for evidence of infection. Blood tests are available, but aside from the difficulty in obtaining samples from free-ranging bison, these assays suffer from low sensitivity and specificity. An easier way to sample a wild population is to collect freshly deposited fecal samples. In the recent past, fecal samples have often been tested immediately after collection by culture, despite the high costs and long incubation times. However, using polymerase chain reaction (PCR) technology, it is more economical to look for the presence of Map DNA in fecal samples and subsequently culture only the positive samples. Recently, methods for the purification of DNA directly from fecal samples have been described (Rudi et al., 2004). These procedures allow for the rapid purification of DNA directly from fecal matter, which permits further characterization or strain typing without the time and cost associated with culture. This communication describes the use of nested PCR for detection and strain typing of Map DNA directly from bison fecal samples to determine if Map is present in northern Canadian bison herds and reports the finding of Map DNA in wild, northern Canadian bison.
Biologists from the Northwest Territory (NWT) Department of Natural Resources opportunistically collected 835 fecal samples from six bison herds at various locations across northern British Columbia, and the WBNP (59°21'N, 112°17'W), Nahanni Butte (61°05'N, 123°23'W), and Fort Liard (60°13'N, 123°22'W) areas in the NWT. There is no domestic cattle or sheep production in these areas.
The method for direct isolation of bacteria from bison feces was described by Chui et al. (2004). A positive extraction control was performed by spiking 10 µl of a McFarland standard 3 suspension of Map (ATCC #19698) (Becton, Dickenson and Co., Sparks, Maryland, USA) into a 4 g sample of known Map-negative feces. This positive control was carried through all assays described in this communication.
A volume (250 µl) of fecal wash was extracted using the MagaZorb® DNA Mini-Prep Kit (Cortex Biochem, San Leandro, California, USA). Nested PCR for the detection of Map DNA was performed using gene 254 of the Map-specific locus 251 (Bannantine et al., 2002), as the target sequence. The primers used in the primary reaction have previously been established (Bannantine et al., 2002), while the secondary primers (254-F2, 5'-TCG GGG CTG GAT TCG TAT TC-3' and 254-R2, 5'-GCC AAC TTT CCG GTG CTC AA-3') were specifically designed for this nested protocol. The secondary primers were designed by analyzing Map DNA sequences from Genbank using Lasergene software (DNASTAR Inc., Madison, Wisconsin, USA). The secondary primers were checked for specificity in silico through Genbank and in vitro by testing other bacterial species similar to Map (Table 1
). The PCR conditions are shown in Table 2. A subset (n=8) of the PCR positive fecal samples from different geographic areas was sent to the Animal Health Monitoring Lab (Abbotsford, British Columbia, Canada) for confirmation with this laboratory’s PCR assay and BACTEC culture.
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. PCR-restriction endonuclease (PCR-REA) digest of IS1311 was carried out as previously described (Whittington et al., 2001), with the exception that the digest was performed on the secondary PCR product, and therefore the digest sizes are different than previously described. The expected, modified restriction fragment sizes are shown in Table 1. The amplified region of the IS1311 element was also sequenced (Plant Bio-technology Institute, Saskatoon, Saskatchewan, Canada) using primers M42 and M545. Mycobacterium paratuberculosis DNA from five amplified products from bison, cattle, and sheep strains, and two different northern bison fecal samples were sequenced in this manner. Sequences were aligned manually with previously established M. paratuberculosis IS1311 sequences (Genbank AJ 223975 and AJ223974) using MegAlign software (DNASTAR, Inc).
Twenty-six of 835 fecal samples were positive for the presence of the Map-specific gene 254 from locus 251, but no positive results were obtained when other Mycobacterium or other bacterial species were similarly tested (Table 1). Map DNA positive fecal samples were collected from the Sweet Grass (58°83' N, 111°96'W) (n=2), Pine Lake (59°67'N, 112°19'W) (n=2), Hook Lake (60°72'N, 112°76'W) (n=1), and Grand Detour (60°36'N, 112°80'W) (n=21) herds in the NWT. Samples from Nahanni (61°05'N, 123° 23'W) and Salt Plains (59°96'N, 112° 34'W) herds were negative. Of the eight samples sent to the Animal Health Monitoring Laboratory, three were positive for Map DNA by this laboratory’s diagnostic PCR assay, but no Map colonies were isolated using BACTEC culture. Therefore, further molecular analysis was performed at the Western College of Veterinary Medicine (Saskatoon, Saskatchewan, Canada) to characterize Map directly from bison fecal samples.
Of the 26 fecal samples positive for the presence of Map DNA, two yielded sufficient Map DNA for strain typing. Strain typing revealed the presence of a sheep-related strain in the Sweet Grass herd and a cattle-related strain in the Grand Detour herd (Table 1). The amplified IS1311 region from the Sweet Grass isolate and Grand Detour isolate was sequenced and revealed a unique 3 base-pair (bp) region (CA-T, located at position 32 of IS1311) not found in other previously sequenced isolates in Genbank. Furthermore, a previously identified Map bison isolate from the United States did not demonstrate the unique nucleotide sequence found in samples from the northern Canadian herds.
This report demonstrates the existence of Map DNA in wild, northern Canadian bison. Isolation of bacteria from fecal samples and the DNA extraction protocol were chosen after comparison to other commonly used extraction methods in which amplification was not observed. The presence of Map DNA was detected with the use of a nested Map-specific PCR assay and a nested Map strain-typing protocol. It was important to modify the PCR assay and the PCR-REA from previous publications to improve the detection limit from less than satisfactory sample specimens.
The unsuccessful cultivation of Map organisms from the fecal samples may have resulted from the use of unsuitable laboratory growth conditions for these particular isolates. Previous evidence of toxicity associated with growth elements (sodium pyruvate) in Map culture media has been described (Juste et al., 1991). PCR may be more sensitive than culture for the detection of mycobacteria (Saboor et al., 1992); therefore, low levels of viable Map shed in the feces may be undetected by culture. Reduced viability of organisms at culture due to frozen storage or repeated freeze-thawing of samples is also possible, and therefore the percentage of northern Canadian bison positive for Map DNA could potentially be higher than fecal culture would indicate.
The practical significance of finding Map DNA in northern Canadian bison herds is uncertain because the bison in and around WBNP have never and do not now manifest the clinical signs of Map infection (Johne’s disease). It is possible but unlikely that wild bison can become infected with Map and eventually clear the infection, or that northern Canadian bison are in fact dying of Johne’s disease but are not discovered, perhaps due to the remoteness of the herds and the efficient work of scavenging wildlife. Alternatively, the apparent lack of disease in northern Canadian bison could be due to the recovery of DNA from a less virulent strain of Map. Our study suggests that the strains infecting northern Canadian bison are distinct from those previously described. Perhaps these strains are not capable of creating clinical disease. Although the sequences obtained only differ by three nucleotides in a region of the gene, which appears to be variable, this is significant considering that the current strain-typing technique (PCR-REA) relies on a single nucleotide polymorphism. Whether or not this unique sequence is limited to bison, or more specifically, to bison in northern Canada, remains unknown.
The samples from northern Canadian bison appear to be unique in their IS1311 sequence and should be characterized further to shed some light on epidemiological questions related to host susceptibility, virulence, and geography. Further studies with larger sample numbers will determine if the northern Canadian bison IS1311 nucleotide sequence is unique.
The authors wish to thank M. Chirino, R. O’Brien, K. Stevenson, and E. Manning for contributing positive control cultures of M. paratuberculosis. This research was supported by grants from the government of Northwest Territories, Parks Canada, the Canada-Saskatchewan Agri-Food Innovation Fund (AFIF), and Saskatchewan Agriculture, Food and Rural Revitalization.
BANNANTINE, J. P., E. BAECHLER, Q. ZHANG, L. LI, AND V. KAPUR. 2002. Genome scale comparison of Mycobacterium avium subsp. paratuberculosis with Mycobacterium avium subsp. avium reveals potential diagnostic sequences. Journal of Clinical Microbiology 40: 1303–1310.
BUERGELT, C. D., AND P. E. GINN. 2000. The histopathologic diagnosis of subclinical Johne’s disease in North American bison (Bison bison). Veterinary Microbiology 77: 325–331.[Medline]
CHUI, L. W., R. KING, P. LU, K. MANNINEN, AND J. SIM. 2004. Evaluation of four DNA extraction methods for the detection of Mycobacterium avium subsp. paratuberculosis by polymerase chain reaction. Diagnostic Microbiology and Infectious Disease 48: 39–45.[Medline]
GOVERNMENT OF THE NORTHWEST TERRITORIES. 2004. Wildlife and Fisheries, http://www.nwtwildlife.com/Publications/speciesatriskweb/woodbison.htm. Accessed 20 July 2007.
JUSTE, R. A., J. C. MARCO, D. O. SAEZ, AND J. J. ADURIZ. 1991. Comparison of different media for the isolation of small ruminant strains of Mycobacterium paratuberculosis. Veterinary Microbiology 28: 385–390.[Medline]
MARSH, I., R. WHITTINGTON, AND D. COUSINS. 1999. PCR-restriction endonuclease analysis for identification and strain typing of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium based on polymorphisms in IS1311. Molecular and Cellular Probes 13: 115–126.[Medline]
RUDI, K., H. K. HOIDAL, T. KATLA, B. K. JOHANSEN, J. NORDAL, AND K. S. JAKOBSEN. 2004. Direct real-time PCR quantification of Campylobacter jejuni in chicken fecal and cecal samples by integrated cell concentration and DNA purification. Applied and Environmental Microbiology 70: 790–797.
SABOOR, S. A., N. M. JOHNSON, AND J. MCFADDEN. 1992. Detection of mycobacterial DNA in sarcoidosis and tuberculosis with polymerase chain reaction. Lancet 339: 1012–1015.[Medline]
WHITTINGTON, R. J., I. B. MARSH, AND R. H. WHITLOCK. 2001. Typing of IS 1311 polymorphisms confirms that bison (Bison bison) with paratuberculosis in Montana are infected with a strain of Mycobacterium avium subsp. paratuberculosis distinct from that occurring in cattle and other domesticated livestock. Molecular and Cellular Probes 15: 139–145.[Medline]
Received for publication 26 September 2006.
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