BACKGROUND
infection with mycobacterium bovis  can result in tuberculosis in many mammalian species including humans.
the risk of zoonotic transmission of m. bovis from animals to humans has led to the development of the bovine tuberculosis eradication program in the united states  <cit> .
discovery of m. bovis infected livestock and/or captive wildlife is reportable in the united states and results in quarantine of animals and animal products costing livestock producers and united states government millions of dollars annually  <cit> .
antemortem diagnosis of tuberculosis in animals is based on immunological responses  to a purified protein derivative .
ppd derived from m. bovis  is produced by precipitating protein from heat-killed cultures of a laboratory-adapted strain of m. bovis, an <dig>  ppd’s are a complex mixture of proteins, carbohydrates, and lipids  <cit>  that share homology with other mycobacteria and even other bacteria .
a number of techniques have been used to evaluate an <dig> and other strains of m. bovis including m. bovis bcg to discover specific antigens to replace the complex mixture found in ppd.
several antigens have shown considerable promise in existing and emerging diagnostic assays   <cit> .
however, cross-reactivity with other mycobacterial species  <cit>  and variation in the immune response from animal to animal  <cit>  continues to hamper improved diagnostic specificity  <cit> .
a key component to development of improved diagnostic assays is to identify proteins or nucleic acid sequences that are specific to m. bovis.
ideally these are only produced by m. bovis and are significantly different from those produced by other mycobacteria.
to date most diagnostic targets have been identified using in vitro grown m. bovis and/or lab adapted strains .
an alternative approach would be to compare field strains of m. bovis to nontuberculous mycobacteria  to which cattle and captive wildlife are exposed using genomic/proteomic approaches.
identifying the ntm to use in these comparisons is critical to this approach.
early work was performed to identify mycobacteria found in the agricultural environments   <cit> .
a second approach has been to identify mycobacteria cultured from skin/γ-interferon test reactors .
these approaches have resulted in the identification of a number of potential candidates; however, a more systematic approach may be needed to identify mycobacterial infection/exposure in the united states.
regional differences may also play a role in diagnostic failure because mycobacterial exposure in the deserts of the southwest may be fundamentally different than those of the northern michigan.
the united states department of agriculture’s national veterinary services laboratories  is the primary m. bovis diagnostic laboratory in the untied states and has the most comprehensive database of mycobacterial isolates from animals in the united states.
using this database a retrospective study performed to identify mycobacteria isolated from tissues submitted for clinical evaluation that may interfere with current diagnostic tests.
RESULTS
mycobacteria were isolated from  <dig>  of  <dig>  animals or pooled samples submitted to the nvsl between  <dig> january  <dig> and  <dig> august  <dig>  these samples were collected in the united states from naturally infected animals.
the majority of the samples were from domestic cattle  and cervids  with canidae , suidae , aves , felidae , bison , non-human primates , procyonidae , cetacea , elephants , and exotic ruminants  contributing the majority of the remaining samples.
the remaining samples were from disparate species such as fish, sharks, camelids, hyrax, frogs, tortoise, horses, and other species.
no mycobacteria were isolated from 92%  of samples submitted.
fifty-five known mycobacterial species/complexes were isolated during this time period from all animal species .
the most frequently isolated mycobacteria were members of the m. avium complex.
in general m. avium species were not further characterized unless they were suspected to be m. avium ssp.
paratuberculosis .
samples specifically submitted to nvsl for map diagnostics were excluded from this study.
the second species most commonly isolated was m. bovis followed by m. fortuitum.
mycobacteria that were unable to be speciated comprised the fourth group.
this group represents mycobacterial species that could not be confidently identified to the species level.
mycobacteria isolated from cattle
nvsl received samples from  <dig>  domestic cattle during the study period.
mycobacteria were isolated from  <dig>  .
m. bovis comprised the majority  of the isolates.
m. avium complex species were isolated from  <dig> .
m. fortuitum/m.
fortuitum complex  made up  <dig> % of the isolates recovered from cattle.
mycobacteria that could not be speciated accounted for  <dig> % .
m. smegmatis was isolated from  <dig> % of the samples.
the remaining non-m. bovis mycobacteria were isolated from 2% or less of the animals .
mycobacteria isolated from abattoir and field surveillance
the primary goal of this study was to identify mycobacteria that may be interfering with current diagnostic tests.
samples submitted to nvsl are primarily submitted through two mechanisms:  abattoir surveillance; or  field collected tissue samples.
in the united states, federal and state animal health inspectors examine carcasses at the abattoir for the presence of a number of diseases including tuberculosis.
when gross lesions are identified that are compatible with tuberculosis, tissues are collected and sent to nvsl for diagnosis  <cit> .
mycobacteria isolated from these lesions will allow identification of mycobacteria that may produce pathology similar to m. bovis; however the test status of these animals is not known.
additional laboratory work would be needed to determine if these mycobacteria interfere with the diagnostic tests.
the reason for each field sample submission is often not known nor routinely recorded at the laboratory; however, based on broad summary data from the laboratory, most field samples submitted were from test positive cattle.
unlike abattoir surveillance where visible granulomas trigger sample collection; non-lesioned tissues are routinely submitted for mycobacterial culture from field collected tissue samples.
examination of the mycobacteria isolated from field-collected samples may provide the most promising data since these samples come from test positive animals.
examining these two mechanisms independently may provide insight into the effect of environmental mycobacteria on diagnostics.
we hypothesize that ntm recovered from field-collected samples are more likely to interfere with current antemortem diagnostic tests.
mycobacteria were more likely to be isolated from field samples than abattoir submissions .
mycobacteria were isolated from  <dig> of  <dig>  animals  from abattoir surveillance compared to  <dig> of  <dig>   animals from field surveillance.
the top isolates from field surveillance cases in descending order were; m. bovis , m. avium complex , m. fortuitum/fortuitum complex , mycobacteria that could not be speciated , m. smegmatis , m. kansasii , and mycobacterium pulveris  .
the remaining isolates comprised less than 2% of the species isolated.
mycobacteria isolated from abattoir surveillance were: m. avium complex , m. bovis , m. fortuitum/fortuitum complex , mycobacteria that could not be speciated , m. kansasii , m. terrae complex  and m. porcinum .
the remaining isolates comprised <1% of the mycobacterial species isolated from abattoir surveillance cases .
mycobacteria isolated from tissues with mycobacterium bovis compatible lesions
histopathological examination was performed on field samples with gross lesions.
mycobacteria other than m. bovis were cultured from  <dig> field samples that contained microscopic lesions compatible with m. bovis infection but from which no m. bovis was cultured.
the ntm isolated from these samples were: m. avium complex , unable to speciate , m. smegmatis , m. kansasii , and m. lentiflavum .
ntm were cultured from  <dig> samples submitted from abattoir surveillance that contained lesions that were histopathologically compatible with m. bovis infection.
these were primarily members of the m. avium complex .
m. intermedium and m. kansasii were cultured from two abattoir submissions each.
seasonal isolation of mycobacteria
samples were submitted to nvsl year around.
there was no apparent difference in the mycobacterial species isolated at different times of the year or by submission type.
m. bovis was isolated at a similar rate regardless of the time of the year or type of submission .
isolation of mycobacteria from different regions in the united states
anecdotal data from interferon-γ release assays and serum based diagnostic test validation has suggested that cross-reactivity to ntm may be different between regions of the united states.
isolates were tabulated by the region of the united states from which they originated and by submission type.
overall, m. avium complex was most frequently isolated from abattoir surveillance whereas m. bovis was the predominate isolate from field surveillance cases.
mycobacteria isolated from samples submitted for abattoir surveillance primarily resulted in isolation of m. avium complex followed by m. bovis, m. fortuitum, uncharacterized mycobacteria, and m. kansasii.
isolations from abattoir surveillance from the south west region were predominantly m. bovis followed by m. avium complex.
in the mid-atlantic and southeast regions uncharacterized species played a more significant role .
isolation of mycobacteria from field surveillance submissions resulted in a more complex pattern.
in general, m. bovis was most frequently isolated.
there were several notable exceptions.
in the pacific region more m. avium complex and uncharacterized mycobacteria were isolated than m. bovis.
in addition, more m. avium complex and m. smegmatis were isolated than m. bovis in the southeast region.
there were two few samples to make definitive conclusions from the pacific north, northeast, and mid-atlantic regions .
isolation of multiple mycobacteria from the same animal
each submitted sample was cultured on at least one liquid medium and three solid media.
culture on solid media provided the opportunity to potentially identify co-infections.
there were  <dig> confirmed instances where two mycobacterial species were isolated from the same animal.
m. bovis was isolated with m. smegmatis , m. lentiflavum , and m. avium complex .
m. neoaurum was isolated with m. fortuitum  and m. smegmatis .
the remaining co-isolates were: m. simiae and m. wolinskyi; m. smegmatis and unable to speciate; m. terrae and m. thermoresistible; m. asiaticum and m. nonchromogenicum; m. fortuitum and m. pulveris.
this not an exhaustive list since ntm were generally not further identified when m. bovis had already been isolated from the animal.
most of these were recovered from the same tissue but from different media types, such as 7h <dig> agar and l-j.
occasionally culturing multiple tissues from the same animal would recover more than one mycobacteria.
these data suggest that animals can be co-infected with at least two mycobacteria at the same time.
mycobacteria isolated from cervids
nvsl received samples from  <dig>  cervids in the united states between  <dig> january  <dig> and  <dig> october  <dig>  samples were submitted from both farmed and free-ranging cervids.
submission usually consisted of lymph nodes of the head, especially the retropharyngeal lymph node, and lesions from the lung, when present.
samples submitted from farmed cervids were cultured individually.
submissions from free-ranging cervids were tested individually if lesioned, and in areas of active surveillance such as around positive cattle herds.
if deer were non-lesioned and outside of an active surveillance zone retropharyngeal lymph nodes from up to  <dig> deer were pooled.
pooled samples were treated as an individual for the purposes of this study.
mycobacteria were isolated from  <dig> of the  <dig>  samples submitted.
m. bovis  was the most commonly isolated mycobacterium, followed by m. avium complex .
uncharacterized mycobacteria comprised the third group .
the next two were m. terrae/terrae complex  and m. fortuitum/fortuitum complex  .
the remaining isolates were isolated  <dig> or fewer times during this time period.
samples from axis deer, caribou, fallow deer, mule deer, red deer, sika deer, white-tailed deer, elk, moose, muntjac and reindeer were submitted to nvsl for diagnosis .
mycobacteria isolated from individual host species was similar where significant numbers of samples were submitted.
only  <dig> samples were submitted from abattoir surveillance.
of these, m. bovis was isolated from  <dig> of the samples ,  <dig> m. kansasii  and  <dig> m. avium complex .
mycobacteria were not isolated from the remainder of the submissions from abattoir surveillance.
a includes white-tailed and mule deer.
b includes red and sika deer.
the majority of cervid samples submitted were from field surveillance.
most of the isolates  from deer were not characterized beyond confirmation they were not m. bovis.
m. bovis was isolated from  <dig> % of the animals sampled, followed by m. avium complex , m. terrae/terrae complex , uncharacterized mycobacteria , and m. fortuitum/fortuitum complex  .
mycobacteria that were characterized in elk  were m. bovis , m. avium complex  and m. nonchromogenicum  .
mycobacteria isolated from tissues with mycobacterium bovis compatible lesions
lesions that were compatible with tuberculosis were identified histologically in cervids; however, m. bovis was the only mycobacterium that was isolated from these tissues.
seasonal isolation of mycobacteria from cervids
cervid samples were primarily submitted during the fall  and winter months .
the remaining samples were submitted during the spring  and summer  months.
all of the summer samples were received over the course of two years.
this seasonal submission is expected since survey of hunter killed deer plays a substantial role in field surveillance of cervids in the united states.
there was no evidence that isolation of mycobacteria from tissues was seasonal .
adecember, january, february bmarch, april, may cjune, july, august dseptember, october, november.
isolation of multiple mycobacteria from the same animal
multiple mycobacteria were isolated from the same animal in  <dig> instances in cervids.
three mycobacteria, m. fortuitum complex, m. holsaticum, and smegmatis were isolated from one fallow deer.
other dual isolates from fallow deer included: m. nebraskense and m. simiae; and m. avium complex with m. simiae.
multiple isolations from elk included m. bovis with m. thermoresistible  and m. nonchromogenicum .
m. fortuitum and m. smegmatis were isolated from a white-tailed deer.
discussion
sequencing the genomes of the environmental mycobacteria that are encountered by livestock may provide researchers the ability to identify bacterial proteins or nucleic acid targets that will not cross react with m. bovis; thus enabling the development of assays with increased specificity and sensitivity.
identifying the mycobacteria that infect livestock and wildlife may enable the rational prioritization of the mycobacterial genomes to sequence to aid diagnostic test development.
a number of mycobacteria reported here have been isolated from the environment associated with cattle feed or water.
many of these have been reported to elicit reactions to ppdb based skin tests including isolates belonging to runyan group iii  <cit> , m. flavescens, m. terrae complex, m. gordonae, m. intracellulare <cit> , m. fortuitum, m. smegmatis, m. senegalense, m. scrofulaceum,  and m. kansasii <cit> .
in addition to cattle, m. fortuitum has been reported to interfere with the skin and γ-interferon test in african buffalo  <cit> .
immunological cross-reaction between these mycobacteria and ppda and ppdb suggest that immunologically similar proteins exist between mycobacteria.
cross-reaction reduces specificity and sensitivity of diagnostic tests.
m. bovis and m. avium complex species are the most common mycobacteria isolated from cervids in the united states.
these results are consistent with those reported for new zealand  <cit> .
in contrast to cattle, more m. terrae/terrae complex were isolated from cervids than m. fortuitum/fortuitum complex.
cervids may be more susceptible to mycobacterial infections.
in this study, mycobacteria were isolated at a rate of  <dig> per thousand cases compared to cattle with a rate of  <dig>  per thousand cases.
if a herd is infected with a greater range of environmental mycobacteria there is a greater probability that the immune response to the environmental mycobacteria may cross-react with ppda and/or ppdb thus reducing the specificity and/or sensitivity of the diagnostic test.
there are two caveats to the current data.
first, not all isolates were identified to the species level.
these totals may be different if all samples were speciated as far as possible.
this may be most prominent in the m. avium complex isolates.
hughes et al.
report that when improve molecular techniques were applied members of the m. avium group resulted in identification of a larger number of mycobacterial species  <cit> .
we reason that the major isolates would be similar, but some of the minor species may play a larger role than currently indicated by the data.
not all isolates were speciated due to the priority of the diagnostic laboratory to identify and report m. bovis infections.
some isolates were not speciated because they could not be confidently assigned to the species level .
this inability to speciate each mycobacterial isolate was also reported by hues et al.
<cit>  in northern ireland, suggesting that not all mycobacterial species have been identified.
a second caveat is that not all mycobacteria are cultivatable.
the possibility exists that the mycobacteria that cause significant interference with current diagnostic tests are unculturable.
a prime example of this scenario is the infrequent yet persistent finding of cct positive cattle with lesions only found in the skin.
histopathological examination reveals the presence of acid-fast bacteria, but culture attempts are always unsuccessful.
with the advent of next generation sequencing, the possibility exists that mycobacterial species could be sequenced with little capital investment.
regions or proteins unique to m. bovis could be derived by comparing the m. bovis genome with environmental mycobacteria.
these unique regions could then be tested for use in protein or nucleic acid based tests.
the problem with this approach has been the difficulty in selecting the environmental mycobacteria to sequence.
here we report the mycobacteria isolated from cattle and cervids in the united states.
these data provide a rational for selection of mycobacteria for genome sequencing to aid in the development of improved diagnostic tests.
CONCLUSIONS
between  <dig> january  <dig> and  <dig> october  <dig>   <dig>  nontuberculous mycobacteria were isolated from samples submitted for clinical diagnosis of m. bovis.
in cattle, m. avium complex, m. fortuitum/fortuitum complex, m. smegmatis, and m. kansasii were the predominate species.
un-speciated mycobacteria were the most commonly isolated category of mycobacteria isolated from deer followed by m. bovis, m. avium complex, m. terrae/terrae complex then m. fortuitum/fortuitum complex.

 infection with mycobacterium bovis can result in tuberculosis in many mammalian species including humans. the risk of zoonotic transmission of m. bovis from animals to humans has led to the development of the bovine tuberculosis eradication program in the united states. discovery of m. bovis infected livestock and/or captive wildlife is reportable in the united states and results in quarantine of animals and animal products costing livestock producers and united states government millions of dollars annually. antemortem diagnosis of tuberculosis in animals is based on immunological responses to a purified protein derivative. ppd derived from m. bovis is produced by precipitating protein from heat-killed cultures of a laboratory-adapted strain of m. bovis, an ppd’s are a complex mixture of proteins, carbohydrates, and lipids that share homology with other mycobacteria and even other bacteria. a number of techniques have been used to evaluate an and other strains of m. bovis including m. bovis bcg to discover specific antigens to replace the complex mixture found in ppd. several antigens have shown considerable promise in existing and emerging diagnostic assays. however, cross-reactivity with other mycobacterial species and variation in the immune response from animal to animal continues to hamper improved diagnostic specificity. a key component to development of improved diagnostic assays is to identify proteins or nucleic acid sequences that are specific to m. bovis. ideally these are only produced by m. bovis and are significantly different from those produced by other mycobacteria. to date most diagnostic targets have been identified using in vitro grown m. bovis and/or lab adapted strains. an alternative approach would be to compare field strains of m. bovis to nontuberculous mycobacteria to which cattle and captive wildlife are exposed using genomic/proteomic approaches. identifying the ntm to use in these comparisons is critical to this approach. early work was performed to identify mycobacteria found in the agricultural environments. a second approach has been to identify mycobacteria cultured from skin/γ-interferon test reactors. these approaches have resulted in the identification of a number of potential candidates; however, a more systematic approach may be needed to identify mycobacterial infection/exposure in the united states. regional differences may also play a role in diagnostic failure because mycobacterial exposure in the deserts of the southwest may be fundamentally different than those of the northern michigan. the united states department of agriculture’s national veterinary services laboratories is the primary m. bovis diagnostic laboratory in the untied states and has the most comprehensive database of mycobacterial isolates from animals in the united states. using this database a retrospective study performed to identify mycobacteria isolated from tissues submitted for clinical evaluation that may interfere with current diagnostic tests.  mycobacteria were isolated from of animals or pooled samples submitted to the nvsl between january and august these samples were collected in the united states from naturally infected animals. the majority of the samples were from domestic cattle and cervids with canidae, suidae, aves, felidae, bison, non-human primates, procyonidae, cetacea, elephants, and exotic ruminants contributing the majority of the remaining samples. the remaining samples were from disparate species such as fish, sharks, camelids, hyrax, frogs, tortoise, horses, and other species. no mycobacteria were isolated from 92% of samples submitted. fifty-five known mycobacterial species/complexes were isolated during this time period from all animal species. the most frequently isolated mycobacteria were members of the m. avium complex. in general m. avium species were not further characterized unless they were suspected to be m. avium ssp. paratuberculosis. samples specifically submitted to nvsl for map diagnostics were excluded from this study. the second species most commonly isolated was m. bovis followed by m. fortuitum. mycobacteria that were unable to be speciated comprised the fourth group. this group represents mycobacterial species that could not be confidently identified to the species level. mycobacteria isolated from cattle nvsl received samples from domestic cattle during the study period. mycobacteria were isolated from. m. bovis comprised the majority of the isolates. m. avium complex species were isolated from. m. fortuitum/m. fortuitum complex made up % of the isolates recovered from cattle. mycobacteria that could not be speciated accounted for %. m. smegmatis was isolated from % of the samples. the remaining non-m. bovis mycobacteria were isolated from 2% or less of the animals. mycobacteria isolated from abattoir and field surveillance the primary goal of this study was to identify mycobacteria that may be interfering with current diagnostic tests. samples submitted to nvsl are primarily submitted through two mechanisms: abattoir surveillance; or field collected tissue samples. in the united states, federal and state animal health inspectors examine carcasses at the abattoir for the presence of a number of diseases including tuberculosis. when gross lesions are identified that are compatible with tuberculosis, tissues are collected and sent to nvsl for diagnosis. mycobacteria isolated from these lesions will allow identification of mycobacteria that may produce pathology similar to m. bovis; however the test status of these animals is not known. additional laboratory work would be needed to determine if these mycobacteria interfere with the diagnostic tests. the reason for each field sample submission is often not known nor routinely recorded at the laboratory; however, based on broad summary data from the laboratory, most field samples submitted were from test positive cattle. unlike abattoir surveillance where visible granulomas trigger sample collection; non-lesioned tissues are routinely submitted for mycobacterial culture from field collected tissue samples. examination of the mycobacteria isolated from field-collected samples may provide the most promising data since these samples come from test positive animals. examining these two mechanisms independently may provide insight into the effect of environmental mycobacteria on diagnostics. we hypothesize that ntm recovered from field-collected samples are more likely to interfere with current antemortem diagnostic tests. mycobacteria were more likely to be isolated from field samples than abattoir submissions. mycobacteria were isolated from of animals from abattoir surveillance compared to of animals from field surveillance. the top isolates from field surveillance cases in descending order were; m. bovis, m. avium complex, m. fortuitum/fortuitum complex, mycobacteria that could not be speciated, m. smegmatis, m. kansasii, and mycobacterium pulveris. the remaining isolates comprised less than 2% of the species isolated. mycobacteria isolated from abattoir surveillance were: m. avium complex, m. bovis, m. fortuitum/fortuitum complex, mycobacteria that could not be speciated, m. kansasii, m. terrae complex and m. porcinum. the remaining isolates comprised <1% of the mycobacterial species isolated from abattoir surveillance cases. mycobacteria isolated from tissues with mycobacterium bovis compatible lesions histopathological examination was performed on field samples with gross lesions. mycobacteria other than m. bovis were cultured from field samples that contained microscopic lesions compatible with m. bovis infection but from which no m. bovis was cultured. the ntm isolated from these samples were: m. avium complex, unable to speciate, m. smegmatis, m. kansasii, and m. lentiflavum. ntm were cultured from samples submitted from abattoir surveillance that contained lesions that were histopathologically compatible with m. bovis infection. these were primarily members of the m. avium complex. m. intermedium and m. kansasii were cultured from two abattoir submissions each. seasonal isolation of mycobacteria samples were submitted to nvsl year around. there was no apparent difference in the mycobacterial species isolated at different times of the year or by submission type. m. bovis was isolated at a similar rate regardless of the time of the year or type of submission. isolation of mycobacteria from different regions in the united states anecdotal data from interferon-γ release assays and serum based diagnostic test validation has suggested that cross-reactivity to ntm may be different between regions of the united states. isolates were tabulated by the region of the united states from which they originated and by submission type. overall, m. avium complex was most frequently isolated from abattoir surveillance whereas m. bovis was the predominate isolate from field surveillance cases. mycobacteria isolated from samples submitted for abattoir surveillance primarily resulted in isolation of m. avium complex followed by m. bovis, m. fortuitum, uncharacterized mycobacteria, and m. kansasii. isolations from abattoir surveillance from the south west region were predominantly m. bovis followed by m. avium complex. in the mid-atlantic and southeast regions uncharacterized species played a more significant role. isolation of mycobacteria from field surveillance submissions resulted in a more complex pattern. in general, m. bovis was most frequently isolated. there were several notable exceptions. in the pacific region more m. avium complex and uncharacterized mycobacteria were isolated than m. bovis. in addition, more m. avium complex and m. smegmatis were isolated than m. bovis in the southeast region. there were two few samples to make definitive conclusions from the pacific north, northeast, and mid-atlantic regions. isolation of multiple mycobacteria from the same animal each submitted sample was cultured on at least one liquid medium and three solid media. culture on solid media provided the opportunity to potentially identify co-infections. there were confirmed instances where two mycobacterial species were isolated from the same animal. m. bovis was isolated with m. smegmatis, m. lentiflavum, and m. avium complex. m. neoaurum was isolated with m. fortuitum and m. smegmatis. the remaining co-isolates were: m. simiae and m. wolinskyi; m. smegmatis and unable to speciate; m. terrae and m. thermoresistible; m. asiaticum and m. nonchromogenicum; m. fortuitum and m. pulveris. this not an exhaustive list since ntm were generally not further identified when m. bovis had already been isolated from the animal. most of these were recovered from the same tissue but from different media types, such as 7h agar and l-j. occasionally culturing multiple tissues from the same animal would recover more than one mycobacteria. these data suggest that animals can be co-infected with at least two mycobacteria at the same time. mycobacteria isolated from cervids nvsl received samples from cervids in the united states between january and october samples were submitted from both farmed and free-ranging cervids. submission usually consisted of lymph nodes of the head, especially the retropharyngeal lymph node, and lesions from the lung, when present. samples submitted from farmed cervids were cultured individually. submissions from free-ranging cervids were tested individually if lesioned, and in areas of active surveillance such as around positive cattle herds. if deer were non-lesioned and outside of an active surveillance zone retropharyngeal lymph nodes from up to deer were pooled. pooled samples were treated as an individual for the purposes of this study. mycobacteria were isolated from of the samples submitted. m. bovis was the most commonly isolated mycobacterium, followed by m. avium complex. uncharacterized mycobacteria comprised the third group. the next two were m. terrae/terrae complex and m. fortuitum/fortuitum complex. the remaining isolates were isolated or fewer times during this time period. samples from axis deer, caribou, fallow deer, mule deer, red deer, sika deer, white-tailed deer, elk, moose, muntjac and reindeer were submitted to nvsl for diagnosis. mycobacteria isolated from individual host species was similar where significant numbers of samples were submitted. only samples were submitted from abattoir surveillance. of these, m. bovis was isolated from of the samples, m. kansasii and m. avium complex. mycobacteria were not isolated from the remainder of the submissions from abattoir surveillance. a includes white-tailed and mule deer. b includes red and sika deer. the majority of cervid samples submitted were from field surveillance. most of the isolates from deer were not characterized beyond confirmation they were not m. bovis. m. bovis was isolated from % of the animals sampled, followed by m. avium complex, m. terrae/terrae complex, uncharacterized mycobacteria, and m. fortuitum/fortuitum complex. mycobacteria that were characterized in elk were m. bovis, m. avium complex and m. nonchromogenicum. mycobacteria isolated from tissues with mycobacterium bovis compatible lesions lesions that were compatible with tuberculosis were identified histologically in cervids; however, m. bovis was the only mycobacterium that was isolated from these tissues. seasonal isolation of mycobacteria from cervids cervid samples were primarily submitted during the fall and winter months. the remaining samples were submitted during the spring and summer months. all of the summer samples were received over the course of two years. this seasonal submission is expected since survey of hunter killed deer plays a substantial role in field surveillance of cervids in the united states. there was no evidence that isolation of mycobacteria from tissues was seasonal. adecember, january, february bmarch, april, may cjune, july, august dseptember, october, november. isolation of multiple mycobacteria from the same animal multiple mycobacteria were isolated from the same animal in instances in cervids. three mycobacteria, m. fortuitum complex, m. holsaticum, and smegmatis were isolated from one fallow deer. other dual isolates from fallow deer included: m. nebraskense and m. simiae; and m. avium complex with m. simiae. multiple isolations from elk included m. bovis with m. thermoresistible and m. nonchromogenicum. m. fortuitum and m. smegmatis were isolated from a white-tailed deer. discussion sequencing the genomes of the environmental mycobacteria that are encountered by livestock may provide researchers the ability to identify bacterial proteins or nucleic acid targets that will not cross react with m. bovis; thus enabling the development of assays with increased specificity and sensitivity. identifying the mycobacteria that infect livestock and wildlife may enable the rational prioritization of the mycobacterial genomes to sequence to aid diagnostic test development. a number of mycobacteria reported here have been isolated from the environment associated with cattle feed or water. many of these have been reported to elicit reactions to ppdb based skin tests including isolates belonging to runyan group iii, m. flavescens, m. terrae complex, m. gordonae, m. intracellulare, m. fortuitum, m. smegmatis, m. senegalense, m. scrofulaceum, and m. kansasii. in addition to cattle, m. fortuitum has been reported to interfere with the skin and γ-interferon test in african buffalo. immunological cross-reaction between these mycobacteria and ppda and ppdb suggest that immunologically similar proteins exist between mycobacteria. cross-reaction reduces specificity and sensitivity of diagnostic tests. m. bovis and m. avium complex species are the most common mycobacteria isolated from cervids in the united states. these results are consistent with those reported for new zealand. in contrast to cattle, more m. terrae/terrae complex were isolated from cervids than m. fortuitum/fortuitum complex. cervids may be more susceptible to mycobacterial infections. in this study, mycobacteria were isolated at a rate of per thousand cases compared to cattle with a rate of per thousand cases. if a herd is infected with a greater range of environmental mycobacteria there is a greater probability that the immune response to the environmental mycobacteria may cross-react with ppda and/or ppdb thus reducing the specificity and/or sensitivity of the diagnostic test. there are two caveats to the current data. first, not all isolates were identified to the species level. these totals may be different if all samples were speciated as far as possible. this may be most prominent in the m. avium complex isolates. hughes et al. report that when improve molecular techniques were applied members of the m. avium group resulted in identification of a larger number of mycobacterial species. we reason that the major isolates would be similar, but some of the minor species may play a larger role than currently indicated by the data. not all isolates were speciated due to the priority of the diagnostic laboratory to identify and report m. bovis infections. some isolates were not speciated because they could not be confidently assigned to the species level. this inability to speciate each mycobacterial isolate was also reported by hues et al. in northern ireland, suggesting that not all mycobacterial species have been identified. a second caveat is that not all mycobacteria are cultivatable. the possibility exists that the mycobacteria that cause significant interference with current diagnostic tests are unculturable. a prime example of this scenario is the infrequent yet persistent finding of cct positive cattle with lesions only found in the skin. histopathological examination reveals the presence of acid-fast bacteria, but culture attempts are always unsuccessful. with the advent of next generation sequencing, the possibility exists that mycobacterial species could be sequenced with little capital investment. regions or proteins unique to m. bovis could be derived by comparing the m. bovis genome with environmental mycobacteria. these unique regions could then be tested for use in protein or nucleic acid based tests. the problem with this approach has been the difficulty in selecting the environmental mycobacteria to sequence. here we report the mycobacteria isolated from cattle and cervids in the united states. these data provide a rational for selection of mycobacteria for genome sequencing to aid in the development of improved diagnostic tests.  between january and october nontuberculous mycobacteria were isolated from samples submitted for clinical diagnosis of m. bovis. in cattle, m. avium complex, m. fortuitum/fortuitum complex, m. smegmatis, and m. kansasii were the predominate species. un-speciated mycobacteria were the most commonly isolated category of mycobacteria isolated from deer followed by m. bovis, m. avium complex, m. terrae/terrae complex then m. fortuitum/fortuitum complex. 
to identify mycobacteria other than m. bovis that may be interfering with current diagnostic tests, a retrospective study was performed to identify mycobacteria isolated from clinical tissues at the national veterinary services laboratories between january and october these data provide information characterizing the species and relative prevalence of mycobacteria other than m. bovis that may interfere with current diagnostic tests. mycobacteria other than m. bovis may interfere with current bovine tuberculosis diagnostic tests resulting in false positive test results. as the prevalence of m. bovis decreases in the united states, interference from other mycobacteria play an increasingly important role in preventing the eradication of m. bovis. during the study period, mycobacteria other than m. bovis were isolated from samples submitted for clinical diagnosis of m. bovis. in cattle, m. avium complex, m. fortuitum/fortuitum complex, m. smegmatis, m. kansasii, and m. terrae complex were the predominate species other than m. bovis isolated from tissues submitted for culture. mycobacteria other than m. bovis isolated from deer were predominantly m. avium complex, m. terrae/terrae complex, and m. fortuitum/fortuitum complex. fifty-five mycobacterial species were isolated during this time period.
 mycobacteria other than m. bovis may interfere with current bovine tuberculosis diagnostic tests resulting in false positive test results. as the prevalence of m. bovis decreases in the united states, interference from other mycobacteria play an increasingly important role in preventing the eradication of m. bovis. to identify mycobacteria other than m. bovis that may be interfering with current diagnostic tests, a retrospective study was performed to identify mycobacteria isolated from clinical tissues at the national veterinary services laboratories between january and october  during the study period, mycobacteria other than m. bovis were isolated from samples submitted for clinical diagnosis of m. bovis. fifty-five mycobacterial species were isolated during this time period. in cattle, m. avium complex, m. fortuitum/fortuitum complex, m. smegmatis, m. kansasii, and m. terrae complex were the predominate species other than m. bovis isolated from tissues submitted for culture. mycobacteria other than m. bovis isolated from deer were predominantly m. avium complex, m. terrae/terrae complex, and m. fortuitum/fortuitum complex.  these data provide information characterizing the species and relative prevalence of mycobacteria other than m. bovis that may interfere with current diagnostic tests. 