BACKGROUND
rabbits have been found to be naturally susceptible to mycobacterium avium subsp.
paratuberculosis  infection in the wild , to mycobacterium avium subspecies infection in natural conditions, specifically mycobacterium avium subsp.
avium  in pigmy rabbits  <cit>  and moderately susceptible to map in laboratory conditions  <cit> .
map and maa are subspecies of mycobacterium avium, as well as mycobacterium avium subsp.
silvaticum  and mycobacterium avium subsp.
hominissuis   <cit> .
from an epidemiologic point of view, maa is typically virulent for birds and small terrestrial mammals causing a range of lesions that go from characteristic tuberculous lesions in parenchymatous organs, to lymphadenitis and disseminated infection .
mah has the human  <cit>  and the pig  <cit>  as its primary hosts and it is frequently found in soil  <cit> .
map is the causal agent of paratuberculosis in ruminants and it has been controversially associated with human inflammatory bowel disease, more precisely with crohn’s disease  and also diabetes mellitus  <cit> .
its prevalence in slaughtered cattle in europe has been estimated to be up to  <dig> %  <cit> .
mas has been isolated from wood pigeons  <cit> , roe deer  <cit>  and horses causing tuberculous-like lesions in these animals  <cit> .
however, since there is a controversy about the real existence of mas as a unique subspecies independent of maa  <cit> , for the present work we will consider mas as part of maa, and jointly refer to them as maa/mas.
mycobacterium avium subspecies members are widely spread in the environment and often enter in contact with animals and humans.
transmission from animals to humans can occur either through the consumption of contaminated foods or via direct contact with an infected animal.
apparently healthy animals thereby may represent a reservoir for mycobacterium avium subspecies and these pathogens may enter the food chain during slaughter.
most work focused on the detection of m. avium subspecies in animal species for human consumption has been performed on meat products.
map has been detected in beef, pork and chicken  <cit> , whereas mah has been detected in beef, pork and lamb  <cit> .
detection of non-tuberculous mycobacteria or mycobacterium avium subspecies at slaughter has been described in lymph nodes of pigs  and detection of map has been reported in lymph nodes, muscle and faeces of both dairy and beef cattle  <cit> .
mycobacterium avium subspecies presence in rabbits in wild conditions led us to hypothesize that these mycobacteria could also be present in commercial rabbits that go to slaughter and thus represent a route of exposure for humans.
the aim of the present study was to carry out a small survey on the frequency of mycobacterial microorganisms detected by solid and liquid culture and by a tetraplex real-time pcr for mycobacterium genus, m. avium subspecies and m. tuberculosis complex in gut associated lymphoid tissue  and caecal contents of apparently healthy rabbits at slaughter.
methods
ethics statement
animals used in this study did not undergo any manipulation prior to stunning for standard industrial slaughter according to the pertinent legislation.
for this reason, no specific ethical approval was required.
animals and sampling
we contacted the official veterinarian of the nearest rabbit slaughterhouse to set-up a sampling schedule.
this study was based on samplings that took place from may to october of  <dig>  and during january and february of  <dig>  in a rabbit slaughterhouse  processing 1300000– <dig> rabbits annually.
rabbits are typically slaughtered with an average age of two months and average live weight around  <dig> - <dig>  kg .
breeding rabbits are slaughtered at about 2– <dig>  years of age.
production rabbits are often outbred in order to maximize meat production  <cit> .
this slaughterhouse was operated by an agriculture cooperative company and complied with the pertinent basque , spanish  and european  no 1099/2009) legislation on animal welfare under the supervision of official veterinarians and the samples obtained were authorized by the slaughterhouse managers.
a total of  <dig> animals  and  <dig> animals  from  <dig> farms scattered in  <dig> provinces of north-east spain were sampled.
samples were drawn from two young animals from each batch of sacrifice and from a third additional breeding animal, if the batch included breeding rabbits.
samples were kept in refrigeration for 6-12h before being processed.
decontamination procedure
in order to determine the optimum naoh concentration for decontamination initial set-up experiments were performed.
mucosa from sacculus rotundus and vermiform appendix from  <dig> rabbits were used to test decontamination with  <dig> %,  <dig> %,  <dig> % and  <dig> % naoh.
decontamination procedures were run for  <dig> and  <dig> min.
contamination was observed in both solid and mgit culture at naoh concentrations lower than  <dig> % for both  <dig> and  <dig> min.
<dig> % naoh for  <dig> min was the lowest concentration that resulted in no contaminated samples.
for this reason, the final decontamination procedure was performed with these conditions.
also manufacturer’s instructions do not recommend running decontamination for more than  <dig> min or naoh concentrations higher than  <dig> % .
<dig> ml of sterile water were added to  <dig> g of sample tissue.
homogenization was then performed on a stomacher blender for  <dig> min at medium speed.
afterwards, the homogenized solution was transferred to a tube and  <dig> ml of 6% naoh were added.
after a vortex mix, the suspension was incubated for  <dig> min at room temperature before neutralization with  <dig> ml of phosphate buffer.
the suspension was mixed well and centrifuged for  <dig> min at  <dig>  × g and the supernatant was discarded.
pellets were suspended in  <dig> ml of sterile water.
culture
solid culture
four drops /per tube of the decontaminated suspension were seeded on solid media: herrold’s egg yolk medium ; middlebrook 7h <dig> with penicillin, amphotericin b, and chloramphenicol; lowenstein-jensen with penicillin, amphotericin b and supplemented with mycobactin j and tsukamura minimal media with cycloheximide  <cit> .
all seeded tubes were incubated at 37+/− <dig> °c and checked for growth at  <dig>   <dig>   <dig> and  <dig> weeks.
after seeding on solid culture, the remaining inoculum was used for liquid culture.
liquid culture
pellets destined to liquid culture were seeded on bbl mycobacteria growth indicator tubes  supplemented with bactec mgit growth supplement and bbl mgit panta .
tubes were incubated for  <dig> days in a bactec mgit  <dig> system and time to detection  values were recorded.
pcr evaluation
pcr performance on spiked mucosa was evaluated since this specimen is known to be tricky because of the presence of pcr inhibitors.
inocula preparation for tissue spiking
mah strain  <dig> was grown in middlebrook 7h <dig>  broth supplemented  with  <dig> % middlebrook oadc enrichment ,  <dig>  % glycerol and  <dig>  % tween  <dig> .
mycobacteria were harvested by centrifugation at  <dig>  × g. pellets were washed twice in phosphate buffered saline .
mycobacteria were resuspended in pbs containing  <dig>  % glycerol and  <dig>  % tween  <dig> .
bacterial suspensions were adjusted to  <dig> mcfarland unit after turbidity measurements were performed with a densicheck densitometer .
five ten-fold dilutions of the bacterial suspensions were prepared in pbs-gt and the highest, medium and lowest, with a  <dig> log difference between each of them were used to inoculate negative tissues.
to verify the viable number of cfu, the inocula were plated onto appropriately supplemented agar-solidified m7h <dig> flasks.
mucosa spiking
mucosa was scraped from vermiform appendix tissue from  <dig> apparently healthy rabbits previously confirmed to be negative for the presence of mycobacteria by pcr, culture and histopathology and it was thoroughly mixed and homogenized.
the blended tissues were divided in batches of  <dig> g of mixture per stomacher bag.
the stomacher bags were inoculated with  <dig> μl of one of each of the previously adjusted bacterial suspensions .
<dig> mg of the homogenate spiked tissues were extracted as described in the dna extraction section and mycmavmtc pcr  <cit> .
in cases where pcr inhibition took place ½ and ¼ dilutions of the extracted dna were performed previous to pcr reaction and this showed to be enough to prevent inhibition.
slaughterhouse sample study
a total of  <dig> animals  were sampled and different steps that are summarized on fig.
<dig> were taken to investigate the specimens.fig.
<dig> experimental scheme showing methods followed at each stage
briefly, sacculus rotundus and vermiform appendix were scraped for mucosa and caecal contents were collected.
part of each sample was used for direct molecular detection and part for decontamination and subsequent culture.
based on set-up results, the final decontamination procedure was performed with  <dig> % naoh as described in the decontamination procedure section because it was the lowest concentration found to inhibit contamination.
both solid and liquid cultures were performed followed by pcr confirmation and sequencing of the 16sr rna gene when non-mycobacterial colonies were isolated.
histopathological analysis was performed on samples of sacculus rotundus and vermiform appendix of a total of  <dig> rabbits  including animals with and without macroscopic lesions.
dna extraction
spiked tissues and fresh rabbit slaughterhouse samples
dna extraction was performed on spiked mucosa , sacculus rotundus, vermiform appendix and caecal content , using dna extract-vk  following manufacturer’s instructions with modifications.
briefly,  <dig> mg of tissue or caecal content were weighed in a microtube.
<dig> μl of sterile distilled water, plus  <dig> μl of vk-lysis buffer and  <dig> mg of vk-extraction beads were added to the tissue and homogenized at 30hz during  <dig> min on tissuelyser ii .
following homogenization, samples were centrifuged at  <dig>  × g for  <dig> min and  <dig> μl of the supernatant transferred to a new vial.
<dig> μl of proteinase k were added to the previous solution and tubes were incubated at  <dig> °c for  <dig> min.
lysis was performed adding  <dig> μl of lysis buffer vl-lb <dig> to the previous solution and mixing thoroughly.
the solution was incubated at  <dig> °c for  <dig> min.
then,  <dig> μl of ethanol  were added and mixed thoroughly.
the final mixtures were loaded on vk-dna binding columns and centrifuged at  <dig>  × g for  <dig> min.
pass-through liquid was discarded.
washing steps were performed with  <dig> μl of wash buffer vk-wb <dig> and  <dig> μl of wash buffer vk-wb <dig> with a  <dig>  × g  <dig> min centrifuging step in between.
after washes were done,  <dig> μl of elution buffer were added to the column and the column left for  <dig> min.
dna was recovered by centrifuging at  <dig>  × g for  <dig> min.
extracted dna was stored at − <dig> °c until pcrs were performed.
negative dna extraction controls , no template and positive pcr controls  were included in all downstream pcr assays.
solid medium isolated colonies
a loopful of cells from each colony growing on solid medium was placed in  <dig> μl of sterile water.
vials were incubated at  <dig> °c for  <dig> min and centrifuged at  <dig>  × g for  <dig> min.
the supernatant was used as template for further pcr assays.
positive mgit liquid medium
one millilitre of vortexed liquid culture from the mgit tubes was collected and centrifuged at  <dig>  × g for  <dig> min, the supernatant was discarded and after a washing with  <dig> ml of sterile distilled water, the pellet was suspended in  <dig> μl of sterile water.
then  <dig> mg of zirconium beads were added and homogenized at 30hz for  <dig> min on tissuelyser ii .
following homogenization, samples were centrifuged at 7500 × g for  <dig> min and the supernatant was collected.
molecular detection
detection of mycobacteria by tetraplex real-time pcr
for initial screening purposes to test the presence of mycobacterium sp., a fourplex real-time pcr described by sevilla et al.
<cit>  was performed.
this pcr is able to detect the genus mycobacterium and then distinguish between the m. tuberculosis complex and m. avium subspecies.
the target genes were the internal transcribed spacer  between 16s and 23srrna genes, insertion sequence is <dig> and the devr genr for mycobacterium genus, m. avium subspecies and m. tuberculosis complex members respectively.
reactions were carried out in a final volume of  <dig> μl containing  <dig> μl of template dna, and 1x taqman universal pcr mastermix without amperase uracil-dna glycosylase ,  <dig>  μm of each primer,  <dig>  μm of each probe.
an internal amplification control  was included in the reaction to rule out false negative results due to inhibitors.
rox  dye was used as passive reference reporter.
amplification was carried out in a  <dig> real-time pcr instrument  and consisted of one denaturation and polymerase activation cycle of  <dig> min at 95°c, and  <dig> cycles of denaturation at  <dig> °c for  <dig> s and annealing/extension at 60°c for  <dig> min.
results were analyzed using the  <dig> system sds software v.  <dig>  .
threshold cycle  and baseline were automatically determined by the software and verified by visual examination of the threshold line in amplification plots.
m. avium subsp.
paratuberculosis detection by is <dig> & ismap <dig> real-time pcr
to identify map positive samples, a real time multiplex pcr described by sevilla et al.
<cit>  was performed.
this pcr detects is <dig> and ismap <dig> dna sequences of m. avium subsp.
paratuberculosis.
briefly, the reaction mixture contained 1× taqman universal pcr master mix , 300– <dig> nm of each primer,  <dig> nm of each probe, and 5μl of dna extract in a final volume of  <dig> μl.
an iac is also included in the pcr reaction.
amplification and real time measurement were performed in the same system and conditions as described in the previous pcr protocol.
m. avium subsp.
hominisuis and m. avium subsp.
avium/m.
avium subsp.
silvaticum detection by is <dig> & is <dig> pcr
in order to identify other m. avium subspecies, a previously published real time pcr was carried out, based on the detection of is <dig> and is <dig> insertion sequences  <cit> .
maa/mas organisms are positive for is <dig> and is <dig>  while mah is only positive for is <dig>  the pcr did not include dna concentration standards for quantification purposes because the objective was just to detect and identify mah and maa/mas.
the final reaction mixture contained the same pcr ingredients at the same concentration indicated above for map pcr but primers and probes were those previously described by slana et al.
<cit> .
amplification and real time measurement were performed in the same system and conditions as described in the previous pcr setting.
16s rrna gene sequencing of colonies on solid medium
the identification of colonies grown on solid media that were not mycobacteria as assessed by previously described pcr methods was performed by 16s rrna gene sequencing.
amplification of target dna was performed by pcr using bacterial universal primers paf  and 530r .
amplification was carried out in a  <dig> geneamp pcr instrument  and consisted of one denaturation and polymerase activation cycle of  <dig> min at  <dig> °c, and  <dig> cycles of denaturation at  <dig> °c for  <dig> s, annealing at  <dig> °c for  <dig> s and extension at  <dig> °c for  <dig> s. the pcr product was cleaned with illustra exoprostar 1-step  following manufacturer’s instructions.
the sequencing reaction was carried out with bigdye® terminator v <dig>  cycle sequencing kit  and resolved with a  <dig> abi  capillary sequencer.
sequencing conditions consisted of  <dig> cycles of denaturation at  <dig> °c for  <dig> s, annealing at  <dig> °c for  <dig> s and extension at  <dig> °c for  <dig> min.
sequence alignment was performed using blast  and identification with  <dig> % of coverage and 99- <dig> % of identity was made.
gross pathology
the whole digestive system of the rabbits was inspected for gross anatomical change.
samples from sacculus rotundus and vermiform appendix were collected for solid and/or liquid culture and histopathological analysis.
samples from sacculus rotundus, vermiform appendix and caecal contents were collected for detection of mycobacteria by pcr.
histopathology
histopathological analysis was performed on samples of sacculus rotundus and vermiform appendix and on other samples that presented macroscopic lesions.
the tissues were fixed in  <dig> % neutral buffered formalin for a minimum of  <dig> h, trimmed, dehydrated through graded alcohols, embedded in paraffin wax and sectioned at 5μm.
these sections were mounted on glass slides stained with haematoxylin and eosin  or by the ziehl-neelsen  method for acid fast bacteria  according to standard procedures.
slides were examined under the microscope for granuloma formation and afb presence.
statistical analysis
association of mycobacteria detection and age was tested with the chi square or fisher exact probability  tests for the frequencies of pcr results according to age using the proc freq of the sas statistical package .
for all analyses, a p value of < <dig>  was considered to be statistically significant.
RESULTS
pcr evaluation
mycmavmtc pcr results of the spiked rabbit mucosa are shown in table  <dig>  inhibition was observed at all bacterial concentrations when samples were not diluted and only probe is <dig> was detected in the highest concentration.
after dilution steps were performed, inhibitory effects were mitigated.
in the medium concentration bacterial suspension a ½ dilution was enough to detect m. avium sp.
dna by the is <dig> probe.
however, in the low concentration inoculum after ½ and ¼ dilutions the is <dig> probe was not positive and only the internal amplification control gave significant ct values.table  <dig> m. avium subsp hominissuis spiked rabbit mucosa cts of mycmavmtc pcr
is1311
is1311
is1311
mah: m. avium subps.
hominissuis, itsdenak: detects mycobacterium genus, is1311: detects mycobacterium avium, iac: internal amplification control, ud: undetermined no threshold-crossing fluorescence, devr detecting mtb complex members was ud for all assayed conditions
slaughterhouse sample study
gross pathology and digestive system parameters
of the  <dig> necropsied animals,  <dig>  showed visible pathological lesions consistent with mycobacterial infection.
they were characterized by the presence of focal thickenings of the intestinal wall in the sacculus rotundus and vermiform appendix together with the presence of pale-whitish spots consistent with lymphoid follicle hyperplasia.
jejunal and ileal wall thickening was observed in one and three animals respectively, affecting some areas of the intestine in these regions.
representative photographs of lesions observed in three different animals are shown in fig.
<dig> fig.
<dig> macroscopic lesions found in slaughtered rabbits.
a sacculus rotundus with pale white spots and thickened wall , b vermiform appendix with white spots and thickened wall , c ileum with thickened walls and a reactive peyer’s patch
molecular detection
of the  <dig> analyzed rabbits,  <dig> were positive for m. avium in galt .
of these, six animals  were positive in vermiform appendix only,  <dig>  in sacculus rotundus only, and  <dig>  in both tissue types.
cts for is <dig> probe were  <dig> +/− <dig>  for undiluted samples and  <dig> +/− <dig>  for diluted samples.
attending to tissue type, cts were  <dig> +/− <dig>  for vermiform appendix and  <dig> +/− <dig>  for sacculus rotundus.
mycobacteria were not detected by pcr in caecal contents.
the  <dig> m. avium positive samples were further analyzed by is <dig> & ismap <dig> pcr for map detection and is <dig> & is <dig> pcr for differentiation between maa/mas and mah.
all these samples were negative for the first real-time pcr, meaning that map was not detected in these rabbits.
in contrast  <dig> of the m. avium positive samples were identified as maa/mas and  <dig> was identified as mah according to the results obtained with is <dig> & is <dig> pcr .table  <dig> gross pathology and molecular detection of sacculus rotundus and vermiform appendix from analyzed slaughtered rabbits
young rabbits
breeding rabbits
n: number of tissues or animals, %: percentage of positive results, sr: sacculus rotundus, va: vermiform appendix, maa: m. avium subsp.
avium, mas: m. avium subsp silvaticum, mah: m. avium subsp hominissuis
alesions spread to the ileum in both animals, blesions spread to the ileum in one animal
culture
the culture of samples in bactec mgit recorded contamination of  <dig>  % of sacculus rotundus and  <dig>  % of vermiform appendix samples.
the remaining samples gave ttd readouts compatible with mycobacterial growth patterns on an average of  <dig>  days for samples of sacculus rotundus and  <dig>  days for samples of vermiform appendix but with a negative result in the mycmavmtc pcr.
as a consequence, all mgit cultures were finally recorded as contaminated.
no contamination on solid media was observed.
four samples cultured on lowenstein-jensen solid medium yielded colonies that were negative in the mycmavmtc.
sequencing of 16s rrna gene identified them as thermoactinomycetaceae bacterium w <dig> in three cases and thermoactinomyces sanguinis in one case .table  <dig> molecular analysis, gross pathology and histopathology results of animals with at least one positive result in any of the methods
thermoactinomyceteae
a
thermoactinomyceteae
b
thermoactinomyceteae
a
thermoactinomyces sanguinis
b
sr: sacculus rotundus, va: vermiform appendix, il: ileum, je: jejunum, ni: no isolation.
aidentified from a colony isolated from sacculus rotundus, bidentified from a colony isolated from vermiform appendix.
cintestinal wall thickening, na: not analyzed.
: without lesions, : reactive hyperplasia,  unspecific granulomatous lesions : granulomatous lesions,d: ziehl neelsen positive
histopathology
most significant lesions were found in the sacculus rotundus and vermiform appendix lymphoid tissue.
in some cases, they were formed by well demarcated granulomas compatible with mycobacterial infection, composed of between 10– <dig> macrophages with occasional multinucleated langhan’s type giant cells, located in the interfollicular areas of the lymphoid tissue.
in other animals, granulomatous lesions were categorized as unspecific and were formed by small granulomas, also seen in the interfollicular areas of lymphoid tissue, with less than  <dig> macrophages, harbouring variable amounts of a brown pigment in their cytoplasm.
microscopical examination of tissues revealed that wall thickening observed on ileum and jejunum corresponded to reactive lymphoid follicles in the ileal and jejunal peyer’s patches.
remaining samples were classified as reactive hyperplasic or absence of lesion when tissues presented a normal form.
afb were detected on two samples that were classified as absence of lesion and were also pcr positive.
complete results for all animals are shown in table  <dig>
association analysis
analysis of age and gross pathology results showed that  <dig> out of  <dig>  of the young rabbits and  <dig> out of  <dig>  breeding rabbits showed some gross changes.
however, these frequencies were not statistically significant .
microbiological results showed that  <dig> of  <dig>  young rabbits and  <dig> of  <dig>  breeding animals were m. avium subspecies positive in the pcr tests, but again, these frequencies were not statistically significant .
worth mentioning is that  <dig> %  of the samples that presented focal lesions were pcr positive.
the geographical origin of sampled rabbits is shown on fig.
3a.
all geographical areas except for f presented at least one positive farm as shown on fig.
3b.
<dig> out of  <dig> farms were positive , when positive was considered as having at least one m. avium subspecies pcr positive animal.
noticeable, is geographical area e where only  <dig> animals were sampled and one was positive for maa and the other for mah.fig.
<dig>
a geographical origin of sampled slaughtered rabbits.
a: asturias, b: bizkaia, c: cantabria, d: gipuzkoa, e: navarra, f: burgos and number of analyzed animals per area.
b
m. avium subspecies positivity of each farm identified as letters  representing each geographical area and a number  identifying the farm.
black bars represent prevalence , numbers on bars represent total number of analyzed animals from each farm
discussion
there is not much information in the veterinary literature concerning microbiological findings from rabbits at slaughter.
a few studies have assessed listeria, salmonella, and e.coli among others  <cit> .
it is not surprising then, that data from mycobacteria and more specifically m. avium subspecies is scarce and mainly limited to other animal species such as cattle  <cit>  or pigs  <cit> .
therefore, to the best of our knowledge, this is the first study reporting m. avium subspecies detection in slaughtered rabbits.
in rabbits, apart from mesenteric lymph nodes and peyer’s patches, the galt which accounts for  <dig> % of the lymphoid tissue includes two specific structures: the sacculus rotundus, and the vermiform appendix.
both tissue types present m cells  <cit>  which actively uptake and present particular antigens and microorganisms to the immune cells of the lymphoid follicle to induce an effective immune response  <cit> .
map has been reported to enter the domes of the peyer’s patches through m cells  <cit>  so orally ingested mycobacteria should end up in these sites and/or in caecal content and therefore we chose these specimens for our analysis.
regarding pcr evaluation, the possibility of inhibition was the main concern.
mycmavmtc pcr performed well at 104- <dig> cfu/g of tissue.
according to our results if we had +/− <dig> cfu/g of mycobacteria we would probably not detect them at least in our tissue type because we would necessarily have to perform dilution steps.
the detection limit for this pcr for m. avium hominissuis in a mixture of bovine tissues has been reported as  <dig> × <dig> cfu/g  <cit>  which is in near agreement with our findings.
we must add that the dna extraction methods of both studies are not coincident and that the bovine tissue mixture may present a more favorable chemistry for the dna extraction and pcr reaction compared to rabbit mucosa.
in any case, for the slaughterhouse study we decided to dilute template dna if inhibition took place, maybe missing positive samples in return.
in the slaughterhouse study, although gross pathology compatible with mycobacterial infection was seen, histological analysis of galt did not reveal presence of granulomas in all gross pathology positive animals and afb detected by zn were only found in two cases.
the presence of granulomas in galt associated to mah have been reported before in sheep  <cit> .
the unspecific granulomas may not be due to infectious agents and they may be “garbage” granulomas or on the other hand they can be ancient granulomas that accumulate waxy pigments that have been attributed to mycobacteria in previous studies  <cit> .
dna of m. avium was present in  <dig>  % of the analyzed galt samples.
if we compare ct values in sampled tissues with the pcr of artificially spiked tissues we can estimate that the bacterial load is probably below  <dig> cfu/g.
in an experimental infection in rabbits with map, isolation was variable, since some sacculus rotundus and vermiform appendix samples that had bacterial loads of 102- <dig> genomic equivalents/g estimated by qpcr gave positive solid culture results, whereas the same tissue site samples from other animals with 101- <dig> genomic equivalents/g gave negative culture results .
of course, isolation variability can be affected by the irregular distribution of afb in the analyzed tissues among other factors.
as for, liquid cultures, they were eventually contaminated and other bacteria could have used up the nutrients necessary for mycobacterial growth.
mycmavmtc pcr of mgit cultures was negative meaning that mycobacterial load was possibly under  <dig> × <dig> cfu/g, since this was the reported detection limit for mgit culture in a bovine tissue mixture using  <dig> % naoh  <cit> .
if more than  <dig> % naoh were to be used as in the present study , it would probably affect mycobacterial viability and interfere with isolation in both solid and liquid media.
in solid media, only thermoactinomyces, bacteria were isolated.
thermoactinomyces species have been implicated as causal agents of farmer’s lung diseases  <cit>  since the disease appears in farmers that are in direct contact with mouldy hay and cereal grains where these bacterial species are known to be abundant.
also they are found in soil, rivers and dairy products  <cit> .
we are not sure about the meaning of this finding but it could be that these bacteria are present in the grains that the rabbits are fed or in the straw litter that maybe used for nesting before kindling.
in any case, it is worth mentioning that these bacteria survive decontamination techniques used for mycobacterial culture and that no macro or microscopic signs of pathology in the digestive system were detected in the animals these bacteria were isolated from.
association analysis revealed that neither gross pathology nor mycobacterial presence was affected by age.
maa has been detected before in rabbits that had been housed with infected pigeons  <cit>  or with doves, ducks and chickens  <cit>  suggesting in both cases that transmission had probably occurred from infected fowl to rabbits.
in our case, although not specifically examined, given the standard production housing characteristics in the studied region, it is most probable that rabbits did not have direct contact with fowl.
in any case, young rabbits do present maa implicating an early contact in their life that might point to water or feeding.
the results from this study show that m. avium subspecies are widely spread and in contact with farmed rabbits since only 2– <dig> animals analyzed from each batch of  <dig> farms has shown  <dig>  % positivity among animals and  <dig> % positivity among farms.
detection of mycobacterial dna was achieved in  <dig> % of the tissues with focal lesions, slightly higher than what has been reported for map in cattle  <cit> .
at this point, we are not sure about the real significance of detecting m. avium subspecies in galt tissue of slaughtered rabbits either from an animal pathological and epidemiological or public health perspective.
although detected granulomas suggest the generation of lesions, the focal nature of these granulomas could be compatible with a latent and controlled infection.
rabbit galt does not enter the food chain, but if it occurred, properly cooked affected parts should not pose a risk to healthy or immunocompromised individuals.
scarcity of previous reports indicates that the problem probably does not compromise rabbit production to a big extent.
lack of other information on the infected animals such as clinical status or weight at slaughter impedes drawing conclusions.
it is noteworthy to state that no map was detected or isolated, therefore indicating that its presence in wild rabbits might be related to environmental factors that do not occur in more controlled farm conditions or that its prevalence is lower and a higher number of animals should be tested.
CONCLUSIONS
this study should be considered as a preliminary survey that might draw attention to a hitherto undetected potential problem.
future studies should consider including other intestinal sites, associated lymph nodes and respiratory tissues as well.
the detection mac bacteria other than map indicates that a niche for this group of mycobacteria does exist in farmed rabbits.
abbreviations
mapmycobacterium avium subsp.
paratuberculosis
maamycobacterium avium subsp.
avium
mahmycobacterium avium subsp.
hominissuis
macmycobacterium avium complex
afbacid fast bacilli
galtgut associated lymphoid tissue
hehaematoxylin and eosin
znziehl-neelsen
heymherrold’s egg yolk medium
pbsphosphate buffered saline
pbs-gtpbs  <dig>  % glycerol  <dig>  % tween 80
mgitmycobacteria growth indicator tubes
ttdtime to detection
fetfisher exact probability
cfucolony forming unit
pcrpolymerase chain reaction
ctthreshold cycle
iacinternal amplification control
rox6-carboxyl-x-rhodamine
udundetermined
nino isolation
nanot analyzed
srsacculus rotundus
vavermiform appendix
ilileum
jejejunum
competing interests
the authors declare that they have no competing interests.
authors’ contribution
ra collected the samples, participated in all the microbiological and pathological studies and collaborated in the interpretation of the data and writing of the paper, is participated in the analysis of the data, interpretation of the results and writing of the paper, em contributed to the sample collection and microbiological studies, vp collaborated in the histopathological interpretation and paper drafting, jg collaborated in the interpretation of the results and the paper drafting, rj conceived and designed the experiment, performed data analysis and drafting of the paper, ne conceived and designed the experiment, participated in sample collection, pathological studies, interpretation of the results and performed data analysis and writing of the paper.
all the authors read and approved the final manuscript.
authors’ information
rakel arrazuria, dvmd; iker a sevilla, bsc, phd; elena molina, bsc; valentín pérez, dvmd, phd, joseba m garrido, dvmd, phd; ramón a juste, dvmd, phd; natalia elguezabal, bsc, phd.

 rabbits have been found to be naturally susceptible to mycobacterium avium subsp. paratuberculosis infection in the wild, to mycobacterium avium subspecies infection in natural conditions, specifically mycobacterium avium subsp. avium in pigmy rabbits and moderately susceptible to map in laboratory conditions. map and maa are subspecies of mycobacterium avium, as well as mycobacterium avium subsp. silvaticum and mycobacterium avium subsp. hominissuis. from an epidemiologic point of view, maa is typically virulent for birds and small terrestrial mammals causing a range of lesions that go from characteristic tuberculous lesions in parenchymatous organs, to lymphadenitis and disseminated infection. mah has the human and the pig as its primary hosts and it is frequently found in soil. map is the causal agent of paratuberculosis in ruminants and it has been controversially associated with human inflammatory bowel disease, more precisely with crohn’s disease and also diabetes mellitus. its prevalence in slaughtered cattle in europe has been estimated to be up to %. mas has been isolated from wood pigeons, roe deer and horses causing tuberculous-like lesions in these animals. however, since there is a controversy about the real existence of mas as a unique subspecies independent of maa, for the present work we will consider mas as part of maa, and jointly refer to them as maa/mas. mycobacterium avium subspecies members are widely spread in the environment and often enter in contact with animals and humans. transmission from animals to humans can occur either through the consumption of contaminated foods or via direct contact with an infected animal. apparently healthy animals thereby may represent a reservoir for mycobacterium avium subspecies and these pathogens may enter the food chain during slaughter. most work focused on the detection of m. avium subspecies in animal species for human consumption has been performed on meat products. map has been detected in beef, pork and chicken, whereas mah has been detected in beef, pork and lamb. detection of non-tuberculous mycobacteria or mycobacterium avium subspecies at slaughter has been described in lymph nodes of pigs and detection of map has been reported in lymph nodes, muscle and faeces of both dairy and beef cattle. mycobacterium avium subspecies presence in rabbits in wild conditions led us to hypothesize that these mycobacteria could also be present in commercial rabbits that go to slaughter and thus represent a route of exposure for humans. the aim of the present study was to carry out a small survey on the frequency of mycobacterial microorganisms detected by solid and liquid culture and by a tetraplex real-time pcr for mycobacterium genus, m. avium subspecies and m. tuberculosis complex in gut associated lymphoid tissue and caecal contents of apparently healthy rabbits at slaughter. methods ethics statement animals used in this study did not undergo any manipulation prior to stunning for standard industrial slaughter according to the pertinent legislation. for this reason, no specific ethical approval was required. animals and sampling we contacted the official veterinarian of the nearest rabbit slaughterhouse to set-up a sampling schedule. this study was based on samplings that took place from may to october of and during january and february of in a rabbit slaughterhouse processing 1300000– rabbits annually. rabbits are typically slaughtered with an average age of two months and average live weight around - kg. breeding rabbits are slaughtered at about 2– years of age. production rabbits are often outbred in order to maximize meat production. this slaughterhouse was operated by an agriculture cooperative company and complied with the pertinent basque, spanish and european no 1099/2009) legislation on animal welfare under the supervision of official veterinarians and the samples obtained were authorized by the slaughterhouse managers. a total of animals and animals from farms scattered in provinces of north-east spain were sampled. samples were drawn from two young animals from each batch of sacrifice and from a third additional breeding animal, if the batch included breeding rabbits. samples were kept in refrigeration for 6-12h before being processed. decontamination procedure in order to determine the optimum naoh concentration for decontamination initial set-up experiments were performed. mucosa from sacculus rotundus and vermiform appendix from rabbits were used to test decontamination with %, %, % and % naoh. decontamination procedures were run for and min. contamination was observed in both solid and mgit culture at naoh concentrations lower than % for both and min. % naoh for min was the lowest concentration that resulted in no contaminated samples. for this reason, the final decontamination procedure was performed with these conditions. also manufacturer’s instructions do not recommend running decontamination for more than min or naoh concentrations higher than %. ml of sterile water were added to g of sample tissue. homogenization was then performed on a stomacher blender for min at medium speed. afterwards, the homogenized solution was transferred to a tube and ml of 6% naoh were added. after a vortex mix, the suspension was incubated for min at room temperature before neutralization with ml of phosphate buffer. the suspension was mixed well and centrifuged for min at  × g and the supernatant was discarded. pellets were suspended in ml of sterile water. culture solid culture four drops /per tube of the decontaminated suspension were seeded on solid media: herrold’s egg yolk medium; middlebrook 7h with penicillin, amphotericin b, and chloramphenicol; lowenstein-jensen with penicillin, amphotericin b and supplemented with mycobactin j and tsukamura minimal media with cycloheximide. all seeded tubes were incubated at 37+/− °c and checked for growth at and weeks. after seeding on solid culture, the remaining inoculum was used for liquid culture. liquid culture pellets destined to liquid culture were seeded on bbl mycobacteria growth indicator tubes supplemented with bactec mgit growth supplement and bbl mgit panta. tubes were incubated for days in a bactec mgit system and time to detection values were recorded. pcr evaluation pcr performance on spiked mucosa was evaluated since this specimen is known to be tricky because of the presence of pcr inhibitors. inocula preparation for tissue spiking mah strain was grown in middlebrook 7h broth supplemented with % middlebrook oadc enrichment, % glycerol and % tween. mycobacteria were harvested by centrifugation at  × g. pellets were washed twice in phosphate buffered saline. mycobacteria were resuspended in pbs containing % glycerol and % tween. bacterial suspensions were adjusted to mcfarland unit after turbidity measurements were performed with a densicheck densitometer. five ten-fold dilutions of the bacterial suspensions were prepared in pbs-gt and the highest, medium and lowest, with a log difference between each of them were used to inoculate negative tissues. to verify the viable number of cfu, the inocula were plated onto appropriately supplemented agar-solidified m7h flasks. mucosa spiking mucosa was scraped from vermiform appendix tissue from apparently healthy rabbits previously confirmed to be negative for the presence of mycobacteria by pcr, culture and histopathology and it was thoroughly mixed and homogenized. the blended tissues were divided in batches of g of mixture per stomacher bag. the stomacher bags were inoculated with μl of one of each of the previously adjusted bacterial suspensions. mg of the homogenate spiked tissues were extracted as described in the dna extraction section and mycmavmtc pcr. in cases where pcr inhibition took place ½ and ¼ dilutions of the extracted dna were performed previous to pcr reaction and this showed to be enough to prevent inhibition. slaughterhouse sample study a total of animals were sampled and different steps that are summarized on fig.  were taken to investigate the specimens.fig. experimental scheme showing methods followed at each stage briefly, sacculus rotundus and vermiform appendix were scraped for mucosa and caecal contents were collected. part of each sample was used for direct molecular detection and part for decontamination and subsequent culture. based on set-up results, the final decontamination procedure was performed with % naoh as described in the decontamination procedure section because it was the lowest concentration found to inhibit contamination. both solid and liquid cultures were performed followed by pcr confirmation and sequencing of the 16sr rna gene when non-mycobacterial colonies were isolated. histopathological analysis was performed on samples of sacculus rotundus and vermiform appendix of a total of rabbits including animals with and without macroscopic lesions. dna extraction spiked tissues and fresh rabbit slaughterhouse samples dna extraction was performed on spiked mucosa, sacculus rotundus, vermiform appendix and caecal content, using dna extract-vk following manufacturer’s instructions with modifications. briefly, mg of tissue or caecal content were weighed in a microtube. μl of sterile distilled water, plus μl of vk-lysis buffer and mg of vk-extraction beads were added to the tissue and homogenized at 30hz during min on tissuelyser ii. following homogenization, samples were centrifuged at  × g for min and μl of the supernatant transferred to a new vial. μl of proteinase k were added to the previous solution and tubes were incubated at °c for min. lysis was performed adding μl of lysis buffer vl-lb to the previous solution and mixing thoroughly. the solution was incubated at °c for min. then, μl of ethanol were added and mixed thoroughly. the final mixtures were loaded on vk-dna binding columns and centrifuged at  × g for min. pass-through liquid was discarded. washing steps were performed with μl of wash buffer vk-wb and μl of wash buffer vk-wb with a  × g min centrifuging step in between. after washes were done, μl of elution buffer were added to the column and the column left for min. dna was recovered by centrifuging at  × g for min. extracted dna was stored at − °c until pcrs were performed. negative dna extraction controls, no template and positive pcr controls were included in all downstream pcr assays. solid medium isolated colonies a loopful of cells from each colony growing on solid medium was placed in μl of sterile water. vials were incubated at °c for min and centrifuged at  × g for min. the supernatant was used as template for further pcr assays. positive mgit liquid medium one millilitre of vortexed liquid culture from the mgit tubes was collected and centrifuged at  × g for min, the supernatant was discarded and after a washing with ml of sterile distilled water, the pellet was suspended in μl of sterile water. then mg of zirconium beads were added and homogenized at 30hz for min on tissuelyser ii. following homogenization, samples were centrifuged at 7500 × g for min and the supernatant was collected. molecular detection detection of mycobacteria by tetraplex real-time pcr for initial screening purposes to test the presence of mycobacterium sp., a fourplex real-time pcr described by sevilla et al. was performed. this pcr is able to detect the genus mycobacterium and then distinguish between the m. tuberculosis complex and m. avium subspecies. the target genes were the internal transcribed spacer between 16s and 23srrna genes, insertion sequence is and the devr genr for mycobacterium genus, m. avium subspecies and m. tuberculosis complex members respectively. reactions were carried out in a final volume of μl containing μl of template dna, and 1x taqman universal pcr mastermix without amperase uracil-dna glycosylase, μm of each primer, μm of each probe. an internal amplification control was included in the reaction to rule out false negative results due to inhibitors. rox dye was used as passive reference reporter. amplification was carried out in a real-time pcr instrument and consisted of one denaturation and polymerase activation cycle of min at 95°c, and cycles of denaturation at °c for s and annealing/extension at 60°c for min. results were analyzed using the system sds software v.. threshold cycle and baseline were automatically determined by the software and verified by visual examination of the threshold line in amplification plots. m. avium subsp. paratuberculosis detection by is & ismap real-time pcr to identify map positive samples, a real time multiplex pcr described by sevilla et al. was performed. this pcr detects is and ismap dna sequences of m. avium subsp. paratuberculosis. briefly, the reaction mixture contained 1× taqman universal pcr master mix, 300– nm of each primer, nm of each probe, and 5μl of dna extract in a final volume of μl. an iac is also included in the pcr reaction. amplification and real time measurement were performed in the same system and conditions as described in the previous pcr protocol. m. avium subsp. hominisuis and m. avium subsp. avium/m. avium subsp. silvaticum detection by is & is pcr in order to identify other m. avium subspecies, a previously published real time pcr was carried out, based on the detection of is and is insertion sequences. maa/mas organisms are positive for is and is while mah is only positive for is the pcr did not include dna concentration standards for quantification purposes because the objective was just to detect and identify mah and maa/mas. the final reaction mixture contained the same pcr ingredients at the same concentration indicated above for map pcr but primers and probes were those previously described by slana et al.. amplification and real time measurement were performed in the same system and conditions as described in the previous pcr setting. 16s rrna gene sequencing of colonies on solid medium the identification of colonies grown on solid media that were not mycobacteria as assessed by previously described pcr methods was performed by 16s rrna gene sequencing. amplification of target dna was performed by pcr using bacterial universal primers paf and 530r. amplification was carried out in a geneamp pcr instrument and consisted of one denaturation and polymerase activation cycle of min at °c, and cycles of denaturation at °c for s, annealing at °c for s and extension at °c for s. the pcr product was cleaned with illustra exoprostar 1-step following manufacturer’s instructions. the sequencing reaction was carried out with bigdye® terminator v cycle sequencing kit and resolved with a abi capillary sequencer. sequencing conditions consisted of cycles of denaturation at °c for s, annealing at °c for s and extension at °c for min. sequence alignment was performed using blast and identification with % of coverage and 99- % of identity was made. gross pathology the whole digestive system of the rabbits was inspected for gross anatomical change. samples from sacculus rotundus and vermiform appendix were collected for solid and/or liquid culture and histopathological analysis. samples from sacculus rotundus, vermiform appendix and caecal contents were collected for detection of mycobacteria by pcr. histopathology histopathological analysis was performed on samples of sacculus rotundus and vermiform appendix and on other samples that presented macroscopic lesions. the tissues were fixed in % neutral buffered formalin for a minimum of h, trimmed, dehydrated through graded alcohols, embedded in paraffin wax and sectioned at 5μm. these sections were mounted on glass slides stained with haematoxylin and eosin or by the ziehl-neelsen method for acid fast bacteria according to standard procedures. slides were examined under the microscope for granuloma formation and afb presence. statistical analysis association of mycobacteria detection and age was tested with the chi square or fisher exact probability tests for the frequencies of pcr results according to age using the proc freq of the sas statistical package. for all analyses, a p value of < was considered to be statistically significant.  pcr evaluation mycmavmtc pcr results of the spiked rabbit mucosa are shown in table  inhibition was observed at all bacterial concentrations when samples were not diluted and only probe is was detected in the highest concentration. after dilution steps were performed, inhibitory effects were mitigated. in the medium concentration bacterial suspension a ½ dilution was enough to detect m. avium sp. dna by the is probe. however, in the low concentration inoculum after ½ and ¼ dilutions the is probe was not positive and only the internal amplification control gave significant ct values.table m. avium subsp hominissuis spiked rabbit mucosa cts of mycmavmtc pcr is1311 is1311 is1311 mah: m. avium subps. hominissuis, itsdenak: detects mycobacterium genus, is1311: detects mycobacterium avium, iac: internal amplification control, ud: undetermined no threshold-crossing fluorescence, devr detecting mtb complex members was ud for all assayed conditions slaughterhouse sample study gross pathology and digestive system parameters of the necropsied animals, showed visible pathological lesions consistent with mycobacterial infection. they were characterized by the presence of focal thickenings of the intestinal wall in the sacculus rotundus and vermiform appendix together with the presence of pale-whitish spots consistent with lymphoid follicle hyperplasia. jejunal and ileal wall thickening was observed in one and three animals respectively, affecting some areas of the intestine in these regions. representative photographs of lesions observed in three different animals are shown in fig.  fig. macroscopic lesions found in slaughtered rabbits. a sacculus rotundus with pale white spots and thickened wall, b vermiform appendix with white spots and thickened wall, c ileum with thickened walls and a reactive peyer’s patch molecular detection of the analyzed rabbits, were positive for m. avium in galt. of these, six animals were positive in vermiform appendix only, in sacculus rotundus only, and in both tissue types. cts for is probe were +/− for undiluted samples and +/− for diluted samples. attending to tissue type, cts were +/− for vermiform appendix and +/− for sacculus rotundus. mycobacteria were not detected by pcr in caecal contents. the m. avium positive samples were further analyzed by is & ismap pcr for map detection and is & is pcr for differentiation between maa/mas and mah. all these samples were negative for the first real-time pcr, meaning that map was not detected in these rabbits. in contrast of the m. avium positive samples were identified as maa/mas and was identified as mah according to the results obtained with is & is pcr.table gross pathology and molecular detection of sacculus rotundus and vermiform appendix from analyzed slaughtered rabbits young rabbits breeding rabbits n: number of tissues or animals, %: percentage of positive results, sr: sacculus rotundus, va: vermiform appendix, maa: m. avium subsp. avium, mas: m. avium subsp silvaticum, mah: m. avium subsp hominissuis alesions spread to the ileum in both animals, blesions spread to the ileum in one animal culture the culture of samples in bactec mgit recorded contamination of % of sacculus rotundus and % of vermiform appendix samples. the remaining samples gave ttd readouts compatible with mycobacterial growth patterns on an average of days for samples of sacculus rotundus and days for samples of vermiform appendix but with a negative result in the mycmavmtc pcr. as a consequence, all mgit cultures were finally recorded as contaminated. no contamination on solid media was observed. four samples cultured on lowenstein-jensen solid medium yielded colonies that were negative in the mycmavmtc. sequencing of 16s rrna gene identified them as thermoactinomycetaceae bacterium w in three cases and thermoactinomyces sanguinis in one case.table molecular analysis, gross pathology and histopathology results of animals with at least one positive result in any of the methods thermoactinomyceteae a thermoactinomyceteae b thermoactinomyceteae a thermoactinomyces sanguinis b sr: sacculus rotundus, va: vermiform appendix, il: ileum, je: jejunum, ni: no isolation. aidentified from a colony isolated from sacculus rotundus, bidentified from a colony isolated from vermiform appendix. cintestinal wall thickening, na: not analyzed.: without lesions,: reactive hyperplasia, unspecific granulomatous lesions: granulomatous lesions,d: ziehl neelsen positive histopathology most significant lesions were found in the sacculus rotundus and vermiform appendix lymphoid tissue. in some cases, they were formed by well demarcated granulomas compatible with mycobacterial infection, composed of between 10– macrophages with occasional multinucleated langhan’s type giant cells, located in the interfollicular areas of the lymphoid tissue. in other animals, granulomatous lesions were categorized as unspecific and were formed by small granulomas, also seen in the interfollicular areas of lymphoid tissue, with less than macrophages, harbouring variable amounts of a brown pigment in their cytoplasm. microscopical examination of tissues revealed that wall thickening observed on ileum and jejunum corresponded to reactive lymphoid follicles in the ileal and jejunal peyer’s patches. remaining samples were classified as reactive hyperplasic or absence of lesion when tissues presented a normal form. afb were detected on two samples that were classified as absence of lesion and were also pcr positive. complete results for all animals are shown in table  association analysis analysis of age and gross pathology results showed that out of of the young rabbits and out of breeding rabbits showed some gross changes. however, these frequencies were not statistically significant. microbiological results showed that of young rabbits and of breeding animals were m. avium subspecies positive in the pcr tests, but again, these frequencies were not statistically significant. worth mentioning is that % of the samples that presented focal lesions were pcr positive. the geographical origin of sampled rabbits is shown on fig. 3a. all geographical areas except for f presented at least one positive farm as shown on fig. 3b. out of farms were positive, when positive was considered as having at least one m. avium subspecies pcr positive animal. noticeable, is geographical area e where only animals were sampled and one was positive for maa and the other for mah.fig. a geographical origin of sampled slaughtered rabbits. a: asturias, b: bizkaia, c: cantabria, d: gipuzkoa, e: navarra, f: burgos and number of analyzed animals per area. b m. avium subspecies positivity of each farm identified as letters representing each geographical area and a number identifying the farm. black bars represent prevalence, numbers on bars represent total number of analyzed animals from each farm discussion there is not much information in the veterinary literature concerning microbiological findings from rabbits at slaughter. a few studies have assessed listeria, salmonella, and e.coli among others. it is not surprising then, that data from mycobacteria and more specifically m. avium subspecies is scarce and mainly limited to other animal species such as cattle or pigs. therefore, to the best of our knowledge, this is the first study reporting m. avium subspecies detection in slaughtered rabbits. in rabbits, apart from mesenteric lymph nodes and peyer’s patches, the galt which accounts for % of the lymphoid tissue includes two specific structures: the sacculus rotundus, and the vermiform appendix. both tissue types present m cells which actively uptake and present particular antigens and microorganisms to the immune cells of the lymphoid follicle to induce an effective immune response. map has been reported to enter the domes of the peyer’s patches through m cells so orally ingested mycobacteria should end up in these sites and/or in caecal content and therefore we chose these specimens for our analysis. regarding pcr evaluation, the possibility of inhibition was the main concern. mycmavmtc pcr performed well at 104- cfu/g of tissue. according to our results if we had +/− cfu/g of mycobacteria we would probably not detect them at least in our tissue type because we would necessarily have to perform dilution steps. the detection limit for this pcr for m. avium hominissuis in a mixture of bovine tissues has been reported as × cfu/g which is in near agreement with our findings. we must add that the dna extraction methods of both studies are not coincident and that the bovine tissue mixture may present a more favorable chemistry for the dna extraction and pcr reaction compared to rabbit mucosa. in any case, for the slaughterhouse study we decided to dilute template dna if inhibition took place, maybe missing positive samples in return. in the slaughterhouse study, although gross pathology compatible with mycobacterial infection was seen, histological analysis of galt did not reveal presence of granulomas in all gross pathology positive animals and afb detected by zn were only found in two cases. the presence of granulomas in galt associated to mah have been reported before in sheep. the unspecific granulomas may not be due to infectious agents and they may be “garbage” granulomas or on the other hand they can be ancient granulomas that accumulate waxy pigments that have been attributed to mycobacteria in previous studies. dna of m. avium was present in % of the analyzed galt samples. if we compare ct values in sampled tissues with the pcr of artificially spiked tissues we can estimate that the bacterial load is probably below cfu/g. in an experimental infection in rabbits with map, isolation was variable, since some sacculus rotundus and vermiform appendix samples that had bacterial loads of 102- genomic equivalents/g estimated by qpcr gave positive solid culture results, whereas the same tissue site samples from other animals with 101- genomic equivalents/g gave negative culture results. of course, isolation variability can be affected by the irregular distribution of afb in the analyzed tissues among other factors. as for, liquid cultures, they were eventually contaminated and other bacteria could have used up the nutrients necessary for mycobacterial growth. mycmavmtc pcr of mgit cultures was negative meaning that mycobacterial load was possibly under × cfu/g, since this was the reported detection limit for mgit culture in a bovine tissue mixture using % naoh. if more than % naoh were to be used as in the present study, it would probably affect mycobacterial viability and interfere with isolation in both solid and liquid media. in solid media, only thermoactinomyces, bacteria were isolated. thermoactinomyces species have been implicated as causal agents of farmer’s lung diseases since the disease appears in farmers that are in direct contact with mouldy hay and cereal grains where these bacterial species are known to be abundant. also they are found in soil, rivers and dairy products. we are not sure about the meaning of this finding but it could be that these bacteria are present in the grains that the rabbits are fed or in the straw litter that maybe used for nesting before kindling. in any case, it is worth mentioning that these bacteria survive decontamination techniques used for mycobacterial culture and that no macro or microscopic signs of pathology in the digestive system were detected in the animals these bacteria were isolated from. association analysis revealed that neither gross pathology nor mycobacterial presence was affected by age. maa has been detected before in rabbits that had been housed with infected pigeons or with doves, ducks and chickens suggesting in both cases that transmission had probably occurred from infected fowl to rabbits. in our case, although not specifically examined, given the standard production housing characteristics in the studied region, it is most probable that rabbits did not have direct contact with fowl. in any case, young rabbits do present maa implicating an early contact in their life that might point to water or feeding. the results from this study show that m. avium subspecies are widely spread and in contact with farmed rabbits since only 2– animals analyzed from each batch of farms has shown % positivity among animals and % positivity among farms. detection of mycobacterial dna was achieved in % of the tissues with focal lesions, slightly higher than what has been reported for map in cattle. at this point, we are not sure about the real significance of detecting m. avium subspecies in galt tissue of slaughtered rabbits either from an animal pathological and epidemiological or public health perspective. although detected granulomas suggest the generation of lesions, the focal nature of these granulomas could be compatible with a latent and controlled infection. rabbit galt does not enter the food chain, but if it occurred, properly cooked affected parts should not pose a risk to healthy or immunocompromised individuals. scarcity of previous reports indicates that the problem probably does not compromise rabbit production to a big extent. lack of other information on the infected animals such as clinical status or weight at slaughter impedes drawing conclusions. it is noteworthy to state that no map was detected or isolated, therefore indicating that its presence in wild rabbits might be related to environmental factors that do not occur in more controlled farm conditions or that its prevalence is lower and a higher number of animals should be tested.  this study should be considered as a preliminary survey that might draw attention to a hitherto undetected potential problem. future studies should consider including other intestinal sites, associated lymph nodes and respiratory tissues as well. the detection mac bacteria other than map indicates that a niche for this group of mycobacteria does exist in farmed rabbits. abbreviations mapmycobacterium avium subsp. paratuberculosis maamycobacterium avium subsp. avium mahmycobacterium avium subsp. hominissuis macmycobacterium avium complex afbacid fast bacilli galtgut associated lymphoid tissue hehaematoxylin and eosin znziehl-neelsen heymherrold’s egg yolk medium pbsphosphate buffered saline pbs-gtpbs % glycerol % tween 80 mgitmycobacteria growth indicator tubes ttdtime to detection fetfisher exact probability cfucolony forming unit pcrpolymerase chain reaction ctthreshold cycle iacinternal amplification control rox6-carboxyl-x-rhodamine udundetermined nino isolation nanot analyzed srsacculus rotundus vavermiform appendix ilileum jejejunum competing interests the authors declare that they have no competing interests. authors’ contribution ra collected the samples, participated in all the microbiological and pathological studies and collaborated in the interpretation of the data and writing of the paper, is participated in the analysis of the data, interpretation of the results and writing of the paper, em contributed to the sample collection and microbiological studies, vp collaborated in the histopathological interpretation and paper drafting, jg collaborated in the interpretation of the results and the paper drafting, rj conceived and designed the experiment, performed data analysis and drafting of the paper, ne conceived and designed the experiment, participated in sample collection, pathological studies, interpretation of the results and performed data analysis and writing of the paper. all the authors read and approved the final manuscript. authors’ information rakel arrazuria, dvmd; iker a sevilla, bsc, phd; elena molina, bsc; valentín pérez, dvmd, phd, joseba m garrido, dvmd, phd; ramón a juste, dvmd, phd; natalia elguezabal, bsc, phd. 
apparently healthy farmed rabbits that go to slaughter may carry m. avium subspecies in gut associated lymphoid tissue. % of the animals presented gross lesions suggestive of lymphoid activation, % presented granulomatous lesions and % contained acid fast bacilli. although, rabbit meat production worldwide is million tons per year, microbiological data on this source of meat is lacking and more specifically reports of mycobacterial presence in industrially reared rabbit for human consumption have not been published. rabbits are susceptible to infection by different species of the genus mycobacterium. paratuberculosis, both subspecies of the m. avium complex, has been reported in wildlife conditions. hominissuis was detected in % of gut associated lymphoid tissue of sampled animals by pcr, whereas caecal contents were negative. particularly, development of specific lesions and isolation of mycobacterium avium subsp. to this end, we sought mycobacteria by microbiological and histopathological methods paying special attention to mycobacterium avium subsp. avium was detected in % and mycobacterium avium subsp. animal pathogensepidemiologymycobacterium avium subspmycobacterium avium complexthermoactinomyces sprabbitsslaughterissue-copyright-statement© the author 2015
 rabbits are susceptible to infection by different species of the genus mycobacterium. particularly, development of specific lesions and isolation of mycobacterium avium subsp. avium and mycobacterium avium subsp. paratuberculosis, both subspecies of the m. avium complex, has been reported in wildlife conditions. although, rabbit meat production worldwide is million tons per year, microbiological data on this source of meat is lacking and more specifically reports of mycobacterial presence in industrially reared rabbit for human consumption have not been published. to this end, we sought mycobacteria by microbiological and histopathological methods paying special attention to mycobacterium avium subsp. paratuberculosis in rabbits from commercial rabbitries from the north east of spain.  m. avium subsp. paratuberculosis was not detected either by culture or pcr. however, mycobacterium avium subsp. avium was detected in % and mycobacterium avium subsp. hominissuis was detected in % of gut associated lymphoid tissue of sampled animals by pcr, whereas caecal contents were negative. % of the animals presented gross lesions suggestive of lymphoid activation, % presented granulomatous lesions and % contained acid fast bacilli. mycobacterial isolation from samples was not achieved, although colonies of thermoactinomycetes sp. were identified by 16s rrna sequencing in % of sampled animals.  apparently healthy farmed rabbits that go to slaughter may carry m. avium subspecies in gut associated lymphoid tissue. keywords animal pathogensepidemiologymycobacterium avium subspmycobacterium avium complexthermoactinomyces sprabbitsslaughterissue-copyright-statement© the author 2015 