AJVR—09-05-0189R—Thakur—0fig—3tab—VLS—BGM
Prevalence of antimicrobial resistance and association with toxin genes in Clostridium difficile in commercial swine
Siddhartha Thakur, BVSc & AH, MVSc, PhD; Michelle Putnam, MS; Pamela R. Fry, DVM;
Melanie Abley, MS; Wondwossen A. Gebreyes, DVM, PhD
Objective—To estimate prevalence and determine association between antimicrobial
resistance and toxin gene profile of Clostridium difficile in commercial pigs at the preharvest
food-safety level. Animals—68 sows and 251 young pigs from 5 farms in North Carolina and 3 in Ohio. Procedures—Fecal samples were collected from sows (8/farm) and matched young pigs
(32/farm) at farrowing. Cohorts were sampled again at nursery and finishing stages. Clos-tridium difficile isolates were tested for susceptibility to 6 antimicrobials. A PCR assay was
used to detect genes coding for enterotoxin A (tcdA), cytotoxin B (tcdB), and binary toxin
(cdtB). Results—C difficile prevalence in young pigs at farrowing was 73% (n = 183) with sig-
nificantly higher prevalence in Ohio (87.5%) than North Carolina (64%). Clostridium difficile
was isolated from 32 (47%) sows with no significant difference between the 2 regions. A
single pig had a positive test result at the nursery, and no isolate was recovered at the fin-
ishing farms. Resistance to ciprofloxacin was predominant in young pigs (91.3% of isolates)
and sows (94%). The antimicrobial resistance profile ciprofloxacin-erythromycin-tetracycline
was detected in 21.4% and 11.7% of isolates from young pigs and sows, respectively. Most
isolates had positive results for tcdA (65%), tcdB (84%), and the binary toxin cdtB (77%)
genes. Erythromycin resistance and tetracycline resistance were significantly associated
with toxin gene profiles. Conclusions and Clinical Relevance—The common occurrence of antimicrobial-resistant C difficile and the significant association of toxigenic strains with antimicrobial resistance
could contribute to high morbidity in farms with farrowing pigs. (Am J Vet Res 2010;71:
Clostridium difficile is a common nosocomial in-
fection and has been known for many decades to
bbreviAtions
cause CDAD in patients. A recent increase in the deaths
Clostridium difficile–associated diarrhea
among hospital patients has been attributed to the
pathogenic strain NAP1/027, which is hypervirulent
and has resistance to fluoroquinolones.1–4 Clostridium difficile is also an important pathogen in food animals
and is responsible for causing colitis in neonatal pigs,
enterocolitis in foals, typhocolitis in adult horses, and
enteritis in calves.5–8 Our understanding of the epide-
miologic and microbiological features of C difficile in
humans has tremendously improved over the past 2
decades. It has recently been suggested that pigs and
with identical ribotypes and toxinotypes detected in
other food animals might serve as a source of the patho-
food animals and humans.2,9–14 It is also possible that
gen for humans and cause community-acquired CDAD,
humans are responsible for pathogen transmission to
pigs; however, this has not been reported. Few studies
have been conducted to determine the epidemiologic
features and potential importance of toxigenic and an-
timicrobial-resistant strains of C difficile in pigs.
From the Department of Population Health and Pathobiology, College
In pigs, C difficile is an important cause of neonatal
of Veterinary Medicine, North Carolina State University, Raleigh,
enteritis, particularly from 1 to 7 days of age in pigs
NC 27606 (Thakur, Putnam); and Department of Veterinary Pre-
that develop CDAD characterized by colonic edema.7,15
ventive Medicine, College of Veterinary Medicine, The Ohio State
Loss of productivity is common, and affected pigs typi-
University, Columbus, OH 43210 (Fry, Abley, Gebreyes).
Supported by National Pork Board grant ID NPB No. 07-044.
cally weigh 10% to 15% less at weaning than those in
Address correspondence to Dr. Thakur (sthakur@ncsu.edu).
unaffected litters.2 An important concern of swine pro-
duction medicine is the development of resistance in
um taurocholate, and cefoxitin were added and tubes
pathogens against important classes of antimicrobials.
were incubated under anaerobic conditions at 35°C for
This is particularly important in toxigenic C difficile
7 days. Following incubation, the culture broths were
strains that can damage the intestines and prolong treat-
centrifuged (7,800 X g for 5 minutes), treated with 96%
ment, leading to high morbidity and mortality rates. Al-
ethanol for 50 minutes to select for spores, and plated
though previous studies2,6,7,11,15 have reported C difficile
on cycloserine cefoxitin fructose agara with C difficile
in swine, no study has been conducted to determine
selective supplementb supplemented with 7% laked
and compare its prevalence in the same pigs sampled at
horse blood.c The presumptive C difficile isolates were
3 stages of production and from 2 distinct geographic
biochemically tested via detection of l-proline amino-
regions in the United States. In addition, to our knowl-
peptidase activity on discs.d The identity of the isolates
edge, no reports concerning a statistical association be-
was confirmed via PCR amplification of the specific
tween antimicrobial resistance and the virulence pro-
marker housekeeping gene, tpi, which encodes for tri-
file in the C difficile population have been published.
Prompted by the lack of such data, the main objective
of the study reported here was to determine the preva-
Antimicrobial susceptibility testing—The MIC
lence of C difficile in pigs at the preharvest food-safety
was determined for a panel of 6 antimicrobials by use
level. We also aimed to assess the occurrence of MDR
of strips with exponential gradients of antimicrobial
phenotypes and determine the statistical association
concentrations, which correspond to specific MIC dilu-
between toxin genes and antimicrobial resistance of
tions.e Susceptibility testing was conducted on Muller
C difficile in commercial pigs at different stages of pro-
Hinton plates supplemented with 5% sheep bloodf fol-
lowing the manufacturer’s instructions.e The antimicro-
bials tested, abbreviations, dilution range, and number
Materials and Methods
of breakpoints used included ampicillin (Amp; dilu-
tion range, 0.016 to 256 µg/mL; 2 breakpoints), cip-
Sample collection and bacterial isolation—Pig
rofloxacin (Cip; dilution range, 0.002 to 32 µg/mL; 8
fecal samples were collected from 8 farms, including
5 from North Carolina and 3 from Ohio. Thirty-two
breakpoints), erythromycin (Ery; dilution range, 0.016
young pigs (1 to 7 days old) and 8 sows were sampled
to 256 µg/mL; 2 breakpoints), metronidazole (Met;
at every farm. Fecal samples were collected from 4
dilution range, 0.016 to 256 µg/mL; 16 breakpoints),
healthy young pigs/sow if available. The probability of
tetracycline (Tet; dilution range, 0.016 to 256 µg/mL;
a diseased young pig surviving to the time of slaughter
4 breakpoints), and vancomycin (Van; dilution range,
is low; therefore, healthy-appearing young pigs were
0.016 to 256 µg/mL; 4 breakpoints). Superscript let-
selected to determine the profile of C difficile in each
ter R was used with an abbreviation to indicate resis-
pig as it moved from farrowing, nursery, and finishing
tance to that drug (eg, VanR). The breakpoint used for
farms to slaughter. Every pig was sampled 3 times; the
the fluoroquinolone ciprofloxacin was 8 µg/mL.17 The
first sample was taken at the farrowing unit from 8 to
breakpoint values used in a previous study18 were used
10 days of birth, with subsequent sampling at nursery
for the remaining antimicrobials. The MIC and MIC
and finishing farms. The pigs were part of an all-in and
all-out production flow and therefore were ear tagged
Toxin gene detection—The DNA was extracted
for identification and subsequent sample collections
from the C difficile colonies by a resin-based DNA ex-
during each production phase. Samples were randomly
traction kit following manufacturer’s instructions.g Am-
collected from more than 8 sows and associated young
plification of the housekeeping gene tpi and the toxin
pigs at a few farms. This was done primarily to avoid
genes including tcdA, tcdB, and cdtB coding for TcdA
a decrease in sample size because of pig deaths at the
(toxin A), TcdB (toxin B), and CDT (binary) toxins,
nursery and finishing farms, and these 4 pigs were in-
respectively, was done by use of specific primers as de-
cluded in the analysis only when used to replace miss-
scribed.16,19 The PCR running conditions included an
ing samples caused by pig deaths. Fecal samples from
initial denaturing step at 95°C for 5 minutes, followed
the young pigs in the farrowing barns were collected
by 30 cycles of denaturation at 95°C for 1 minute, an-
with sterile loops. All samples from nursery and fin-
nealing at 54°C for 1 minute, and an extension-elonga-
ishing-age pigs and sows in the farrowing barns were
tion step of 72°C for 1 minute. This was followed by a
collected with gloved hands directly from the rectum
final extension step at 72°C for 7 minutes. Amplified
and transported to the laboratory in a sterilized cup at
products were run on 2% agarose gel containing ethid-
4°C. Pathogen isolation for the entire study was con-
ium bromide in 0.5X Tris-acetate EDTA buffer.h
ducted in North Carolina, and samples originating in
Ohio were shipped overnight under refrigerated con-
Statistical analysis—The frequency of antimicro-
ditions. The study was reviewed and approved by the
bial resistance profiles and comparison of MIC values
North Carolina State University Institutional Animal
between isolates from different production phases and
region were evaluated by use of the χ2 test and Fisher
Clostridium difficile was isolated by transferring
exact 2-tailed test, when applicable, by use of a statis-
2 g of fecal sample in 10 mL of C difficile broth con-
tical software package.i Significant association of anti-
sisting of proteose peptone (4%), disodium hydrogen
microbial resistance and virulence gene profiles for the
phosphate (0.5%), potassium dihydrogen phosphate
C difficile isolates was determined by use of the OR test
(0.1%), magnesium sulfate (0.01%), sodium chloride
with 95% CIs by use of commercially available software.j
(0.2%), and fructose (0.6%). Cycloserine, 0.1% sodi-
Values of P ≤ 0.05 were considered significant.
systems, than in North Carolina (14% in young pigs
and 9% in sows). The MIC for the C difficile isolates
C difficile prevalence—Two hundred fifty-one
from both sources to erythromycin was > 256 µg/mL
young pigs and 68 sows were sampled from 5 farms in
(the highest concentration tested). The MIC to tetra-
North Carolina (155 young pigs and 44 sows) and 3
cycline varied from 64 µg/mL in isolates from sows to
farms in Ohio (96 young pigs and 24 sows). In North
16 µg/mL in isolates from young pigs. The MIC and
Carolina, 13 sows only had 3 young pigs/sow available
MIC for isolates to ciprofloxacin were > 32 µg/mL for
for sampling. In addition, we could not locate 8 pigs
representing 2 finishing farms in North Carolina dur-
ing sampling. It was assumed that these pigs died dur-
Phenotyping based on antimicrobial resistance
ing their stay at the nursery farm or finishing farm or
profile—Nine distinct antimicrobial resistance pro-
during transport. The overall C difficile prevalence in
files were detected. Ciprofloxacin resistance was rep-
young pigs was 73% (n = 183) with significantly (P <
resented in 5 profiles. The antimicrobial resistance
0.001) higher prevalence in Ohio (87.5% [n = 84]) than
profile CipR-EryR-TetR was the predominant pattern
in North Carolina (64% [99]). The overall C difficile
and was represented by 19.5% (n = 42) of the iso-
prevalence in sows was 47% (n = 32) with no significant
lates. Specific resistance phenotypes were found to
geographic difference between North Carolina (50%)
be associated with the region of sample collection.
and Ohio (41.7%). All the C difficile–positive pigs were
For example, the CipR-TetR pattern was found in iso-
detected at the farrowing stage, except a single nursery-
lates from young pigs (n = 6) and sows (34) in North
age pig from North Carolina. No deaths were observed
Carolina, but none of the isolates from Ohio had this
when pigs were sampled at the nursery farms, and none
pattern. This clear distinction of resistance pheno-
of the pigs tested positive at the finishing farms.
types in the 2 geographic regions was also observed
Antimicrobial susceptibility—Clostridium diffi-
for the antimicrobial resistance profile CipR-EryR-
cile isolates had resistance to 4 of the 6 antimicrobials
TetR, with significantly (P < 0.001) higher frequency
tested (Table 1). Overall, the frequency of CipR was sig-
among young pigs (35.7%) and sows (40%) in Ohio.
nificantly greater, compared with other antimicrobials,
Eleven young pigs from 5 sows had matching antimi-
irrespective of the source or the region, with 91.3% fre-
quency among isolates from young pigs and 94% from
Toxin gene profile—The PCR testing of the 215
sows. All the C difficile isolates in this study were sus-
C difficile isolates for the presence of important toxin
ceptible to metronidazole (MIC -to-MIC range, 0.13
genes revealed that toxigenic strains commonly oc-
to 0.25 µg/mL) and vancomycin (MIC -to-MIC range,
curred among the isolates. Genes for toxins A (65%
0.5 to > 0.5 µg/mL), 2 drugs of choice for treatment of
of isolates) and B (84%) and the binary toxin coding
C difficile in human medicine. Only 6 isolates, includ-
genes (77%) were detected. Further analysis revealed 4
ing 5 from young pigs (Ohio, n = 4; North Carolina, 1)
toxin gene profile combinations of the 3 toxin encoding
and a single isolate from a sow (North Carolina), were
genes tcdA, tcdB, and cdtB(Table 2). The predominant
susceptible to all drugs tested in the study. Resistance
toxin gene profile coding for A+B+CDT+ was found in
to ampicillin was detected in 2.7% (n = 5) of isolates
59% (n = 127) of the isolates. Forty-two (19.5%) iso-
from young pigs only. The single isolate from the nurs-
lates had the A–B+CDT+ profile, whereas 33 (15.3%)
ery farm was susceptible to all the antimicrobials except
isolates tested negative for all toxins tested. A signifi-
ciprofloxacin. Antimicrobial resistance to tetracycline
cantly (P < 0.001) higher number of C difficile isolates
was detected in isolates from sows (31.3%) and young
from young pigs in Ohio farms (n = 28) had that pro-
pigs (46%) and to erythromycin in sows (34.4%) and
file. Twenty-two young pigs representing 13 sows had
matching toxin profiles. The pansusceptible C difficile
A significantly (P < 0.001) higher number of iso-
isolate from a sow and a single young pig from North
lates from young pigs (66.7%) and sows (90%) in Ohio
Carolina had the A–B+CDT+ toxin profile. The remain-
had resistance to the macrolide, erythromycin, a class
ing 4 pansusceptible isolates from young pigs in Ohio
of antimicrobial commonly used in swine production
Table 1—Frequency of antimicrobial resistance at different MICs in Clostridium difficile isolated from 183 young pigs and 32 sows. Antimicrobial (dilution range [µg/mL]) Breakpoint (µg/mL) MIC range (µg/mL) MIC (µg/mL) Resistance
*Indicates number (%) of C difficile isolates with resistance to an antimicrobial.
Table 2—Toxin gene profiles (number [%] or proportion) in C dif-
had a prevalence of 47%, which was higher than re-
ficile isolated from the same pigs as in Table 1.
ported for sows and boars in a previous study (3.8%).k
It is important to mention that none of the pigs tested
Source Region A+B+CDT+ A–B+CDT+ A–B–CDT– A+B+CDT–
in this study had diarrhea or signs of any illness. The
Young North Carolina 59 (59.6) 25 (25.3)
young pigs in this study appeared healthy, yet the prev-
alence of C difficile in these pigs was high. However,
during stress or illness caused by another pathogen, it
42 (19.5) 33 (15.3) 13 (6)
is possible that C difficile might contribute to the dis-
A = Toxin encoded by gene TcdA. B = Toxin encoded by gene
eased state of the pig. Other than a single pig that had
positive results at the nursery stage, none of the pigs
Two isolates from young pigs and 1 from a sow in North Carolina
had positive results at the nursery and finishing stages
had the A–B+CDT– toxin profile; 1 isolate from a young pig in North
Carolina had the A–B–CDT+ profile.
of production. A previous studyk has also reported that
C difficile is primarily clustered in young pigs, and the
prevalence in nursery and finishing age pigs is signifi-
Table 3—Distribution (number [%] or proportion) of C difficile iso-
cantly lower. An important reason for lower prevalence
lates with antimicrobial resistance to tetracycline or erythromy-
among older pigs could be reduced susceptibility to the
cin by virulence genes, source, and geographic region.
pathogen. Exposure to C difficile elicits an immune re-
Antimicrobial tcdB cdtB
sponse, which could be more pronounced in adults.22,23
However, more studies are needed to determine the rea-
sons for this finding. The difference in prevalence in
young pigs between the 2 regions could be the diverse
potential sources of C difficile or a random error caused
by the limited sample size of this study.
There is little published information regarding the
antimicrobial resistance profile of C difficile isolated
from pigs. Antimicrobial resistance to ciprofloxacin
Tet = Tetracycline. Ery = Erythromycin.
was observed at the highest concentration tested (32
µg/mL) in most of the isolates from young pigs (91.3%)
and sows (94%). The results are in accordance with
Association between antimicrobial resistance
other studies3,24 that have reported high frequency of
and virulence gene profile—The association between
resistance to ciprofloxacin in C difficile isolates from
antimicrobial resistance (TetR and EryR) and virulence
different sources, including pigs and humans. It should
determinants (tcdB and cdtB) in C difficile isolates re-
be noted that this class of antimicrobials had not been
covered from young pigs was determined (Table 3).
used in swine production in the United States for any
This association was tested at 2 levels—the source and
geographic location of the sample. The low number of
purpose at the time of the study. Recently, the FDA ap-
isolates recovered from sows precluded testing this as-
proved therapeutic treatment for respiratory infections
sociation in sows. A strong association was detected
in swine, but all the samples in this study were collect-
between TetR C difficile isolated from young pigs with
ed prior to that approval. Fluoroquinolone resistance
the virulence genes tcdB (OR, 9) and cdtB (OR, 4). A
in C difficile is possibly attributable to the extensive use
strong association was detected between EryR isolates
of this class of antimicrobial in hospitals, which might
from young pigs and the tcdB genes (OR, 3). However,
lead to nonsynonymous mutations in the gyrase region.
no significant association was detected between EryR
These acquired mutations in the C difficile gyrA and gyrB
and the cdtB toxin gene (OR, 1.7; 95% CI, 0.7 to 4.2)
regions are stable and have resulted in the clonal ex-
for the 2 sources. When isolates were stratified by geo-
pansion of the resistant strains. Pigs may have acquired
graphic region, North Carolina isolates had a stronger
these antimicrobial-resistant strains from humans, but
association between TetR and tcdB (OR, 73; 95% CI, 17
this was not investigated in the present study.
to 298) or cdtB (OR, 16.4; 95% CI, 5.5 to 49) genes.
There was no indication of high MICs for metro-
However, the CI for the tcdB and the cdtB genes was
nidazole or vancomycin, the 2 drugs most commonly
wide. The same pattern was observed in the EryR iso-
used for treating infections in humans. These results
lates from young pigs in North Carolina with signifi-
are in accordance with previous reports.3,18,24,25 In the
cant association with tcdB (OR, 12.5; 95% CI, 1.9 to 78)
present study, resistance was detected to erythromycin
genes. However, the EryR isolates from young pigs in
in isolates from young pigs (38.3%) and sows (34.4%)
Ohio were significantly more associated with the cdtB
with a clear distinction based on the geographic ori-
gin of isolates. The reason for this difference was not
apparent because farms in both regions used erythro-
Discussion
mycin in the feed for growth promotion purposes. It
is possible that other factors including environment,
The high prevalence (73%) of C difficile in young
type of flooring, and production flow might be associ-
pigs in this study was not surprising because CDAD
ated with the dissemination of these C difficile strains in
is a known cause of neonatal enteritis.7,15,20 Previous
specific geographic locations. The bimodal distribution
studies21,k,l have detected C difficile prevalence in young
(2 distinct clusterings of MIC values) of erythromycin
pigs from 25.9% to 49.5%, and the findings in this study
susceptibility, as indicated by the MIC and MIC ,
are in agreement with those reports. Sows in this study
among isolates from young pigs and sows in this study,
has been reported in isolates from pigs and humans.24,26
The main objective of determining a significant
This distribution has been attributed to the possible
association between antimicrobial resistance and viru-
random circulation of resistance coding determinants
lence profile was to identify and devise strategies to dif-
in the population and the use of tylosin as a growth
ferentiate, target, and control the pathogenic species of
promotor.26 The detection of specific resistance profiles
C difficile on farrowing farms, thereby reducing young
associated with geographic location of sampling in the
pig morbidity. Significant associations were detected
present study was interesting. This may indicate that
between the TetR and the EryR isolates with virulence
specific C difficile strains are circulating in specific loca-
markers that were dependent on the geographic origin
tions. But it is also important to note that we did not
of the isolates. There are conflicting reports in the liter-
observe a single antimicrobial resistance or toxin gene
ature regarding this association between antimicrobial
profile that was restricted to either North Carolina or
resistance and pathogen virulence. Although no study
Ohio. However, the differences between the 2 states
has reported on this possible association in C difficile, a
could be attributable to random error because the study
recent study36 found a positive association between re-
was conducted in a limited number of farms and may
sistance and virulence in Escherichia coli isolated from
not have external validity for making generalized infer-
healthy pigs. In contrast, a study37 conducted with En-
ences. The detection of resistance to important antimi-
terococcus faecalis isolated from retail foods revealed
crobials is concerning and should be studied in more
both positive and negative associations between antimi-
detail by use of representative samples.
crobial resistance and virulence determinants. It is pos-
The toxin profile of the isolates characterized in this
sible that antimicrobial resistance has the potential for
study was interesting. The TcdA+B+CDT+ profile was the
selecting virulence in bacteria, which may result in high
predominant gene profile and was found in 59% of the
mortality rates in animals. Other investigators have also
isolates. Detection of toxins A and B in the feces has
reported a similar association in E coli with the concern
been used as a standard for diagnosis of C difficile infec-
that antimicrobial use may contribute to persistence of
tion in pigs and humans.6,27 However, the detection of
virulent strains.38 It is important to mention that asso-
toxin-negative C difficile strains from the feces of pigs
ciation between virulence and antimicrobial resistance
in the present study (15.3%) was an important find-
could be dependent on the bacterial population, strain,
ing and indicates the importance of pathogen isolation
source, and other important factors that must be taken
and not simply relying on toxin detection. This also has
into account before making valid interpretations.
important implications if a particular C difficile strain is
toxin negative but resistant to multiple antimicrobials.
Cycloserine-cefoxitin-fructose agar, Oxoid, Baskingstoke,
To the authors’ knowledge, the present study is the
Clostridium difficile selective supplement, Oxoid, Baskingstoke,
first to report the TcdA–B+CDT+ toxin profile in C dif-ficile isolates from pigs. Clostridium difficile strains with
Laked horse blood, Hemostat Laboratories, Dixon, Calif.
that toxin profile have been reported to cause outbreaks
and clinical cases in humans and have been isolated
Epsilometric test, AB Biodisk, Solna, Sweden.
with increasing frequency from infants and elderly hu-
5% sheep blood, BD Diagnostics, Franklin Lakes, NJ.
mans.28–32 The prevalence of binary toxin coding genes
Chelex, InstaGene Matrix, Biorad Laboratories, Hercules, Calif.
Tris-Acetate EDTA, Fisher Scientific, Fair Lawn, NJ.
was high, with 77% (n = 166) of the isolates testing
positive for the cdtB gene. Previous reports1,33 indicate
Egret, version 2.0.1, Cytel Software Corp, Cambridge, Mass.
a higher prevalence of this toxin in young pigs, rang-
Harvey RB, Norman KN, Scott HM, et al. Prevalence of Clostridium
ing from 78.4% to 83%. Recently, binary toxin has been
difficile in an integrated swine operation (abstr), in Proceedings. 9th
increasingly present in strains responsible for commu-
Bienn Cong Anaerobe Soc Am 2008;5. Available at: www.anaerobe.
nity-acquired CDAD in humans.34,35 The role of binary
org/2008/ASA 2008 Session 14.pdf. Accessed Aug 11, 2010.
Zidaric V, Rupnik M, Avbersek J, et al. Prevalence and diversity
toxin in the pathogenesis of C difficile infection and its
of Clostridium difficile in poultry, pigs and calves (abstr), in Pro-
role in conjunction with TcdA and TcdB toxins need
ceedings. 9th Bienn Cong Anaerobe Soc Am 2008;12. Available
further investigation. In the present study, the isolation
at: www.anaerobe.org/2008/ASA 2008 Session 14.pdf . Accessed
of C difficile isolates with similar antimicrobial resis-
tance and toxin gene profiles from the sows and young
pigs may imply direct transmission of C difficile from
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Author: Please verify missing info in ref 24.
1. A Revista Crítica de Ciências Sociais publica textos originais que possam contribuir para enriquecer a investigação científica transdisciplinar da realidade social, nacional e internacional, para promover de modo aprofundado a reflexão e a discussão sobre os instrumentos dessa mesma investigação e para fornecer informações e orientações a quantos, de um modo ou de outro, se
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