Application of different methods for the diagnosis of paratuberculosis in a dairy cattle herd in Argentina.
ABSTRACT Paratuberculosis (Ptbc) has a high prevalence in Argentina, that affects dairy and beef cattle. The culture is the gold standard to the diagnosis of the disease. Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis), the aetiological agent, is difficult to isolate and grow in culture. In this study, 24 randomly selected cows of the Fresian breed from a dairy herd with a history of Ptbc were used to evaluate the performance of different diagnostic techniques. These animals did not show clinical signs of the disease. However, another animal from this herd presented evidence of clinical disease at the moment of the present study. This animal was necropsied and one strain of M. paratuberculosis was isolated from faeces, lymph nodes and intestine. Serum for indirect absorbed enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) tests and whole blood samples to perform gamma interferon (gammaIFN) release assays were obtained from each animal. Faeces and milk samples to carry out bacteriological cultures, PCR identification of M. paratuberculosis, and direct examinations of smears with Ziehl-Neelsen's (ZN) stain were also collected. Tuberculin test with bovine purified protein derivative (PPD) in the caudal fold was performed. The results showed that 10 out of 24 animals (41.6%) were positive to ELISA. Eight strains of M. paratuberculosis were isolated, six from faeces, two from milk. Five of the animals that excreted the bacteria through faeces were ELISA-positive, whereas the excreters through milk were negative to ELISA. No positive samples by AGID were obtained in clinical asymptomatic animals. Seven samples gave positive gammaIFN results with avian PPD, but only two of these animals were confirmed with culture. Direct PCR, to detect IS900 (M. paratuberculosis) in faeces and milk samples, was negative, but PCR using material taken from faecal and milk cultures gave positive results before visualizing the colonies. No sample was positive by PCR directed to IS6110 (M. tuberculosis complex). There was not always agreement between isolations and ZN in the studied samples. In conclusion, the absorbed ELISA was useful to detect positive animals and excreters through faeces but not through milk. PCR applied to cultures with incipient development before the visualization of colonies was effective to specifically determine the presence of M. paratuberculosis. The gammaIFN test was not able to detect the most positive animals confirmed by culture. The importance of using ELISA and cultures is emphasized by this study but it is necessary to continue with the gammaIFN test development for early detection of the disease.
- SourceAvailable from: Kuldeep DhamaAdvances in Animal and Veterinary Sciences 01/2014; 1(1S):1-11.
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ABSTRACT: This study aimed to evaluate the immune response in bovines following immunization with a mycobaterial Lipoarabinomannan extract (LAMe) and the effect of Map challenge. LAMe vaccine induced specific antibody levels that diminished after the challenge and affected Map excretion at least for 100 days thereafter.Brazilian Journal of Microbiology 01/2013; 44(2):511-4. · 0.76 Impact Factor
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ABSTRACT: To study the deficiency of minerals and its relationship with Paratuberculosis, blood, serum, and fecal samples were obtained from 75 adult bovines without clinical symptoms of the disease and from two bovines with clinical symptoms of the disease, from two beef herds with a previous history of Paratuberculosis in the Province of Buenos Aires, Argentina. Serum samples were processed by ELISA and feces were cultured in Herrolds medium. Copper, zinc and iron in serum were quantified by spectrophotometry and selenium was measured by the activity of glutathione peroxidase. We also determined copper, zinc, iron and molybdenum concentrations in pastures and the concentration of sulfate in water. Mycobacterium avium subsp paratuberculosis (Map) was isolated from 17.3% of fecal samples of asymptomatic animals and from the fecal samples from the two animals with clinical symptoms. All the Map-positive animals were also ELISA-positive or suspect, and among them, 84.6% presented low or marginal values of selenium and 69.2% presented low or marginal values of copper. The two animals with clinical symptoms, and isolation of Map from feces and organs were selenium-deficient and had the lowest activity of glutathione peroxidase of all the animals from both herds. All the animals negative to Map in feces and negative to ELISA had normal values of Se, while 13.8% of animals with positive ELISA or suspect and culture negative presented low levels of Se. Half of the animals that were negative both for ELISA and culture in feces were deficient in copper but none of them presented low values of selenium. The content of molybdenum and iron in pasture was high, 2.5 ppm and 1.13 ppm in one herd and 2.5 ppm and 2.02 ppm in the other, respectively, whereas the copper:molybdenum ratio was 1.5 and 5.2, respectively. These results do not confirm an interaction between imbalances of the micronutrients and clinical Paratuberculosis, but show evidence of the relationship between selenium deficiencies in animals with Map infection and ELISA positive results.Brazilian Journal of Microbiology 01/2013; 44(1):153-60. · 0.76 Impact Factor
Veterinary Microbiology 50 (1996) 59-71
Comparison of different genetic markers for
molecular epidemiology of bovine tuberculosis
Maria I. Roman0 aT * , Alicia Alito b, Juan C. Fisanotti a,
Fabiana Bigi a, Isabel Kantor ‘, Maria E. Cicuta d,
Angel Cataldi a
a Instituto de Biotecnologia, Centro de Investigaciones en Ciencias Veterinarias, Institute National de
Tecrwlogia Agropecuaria, P.O. Box 77, 1708 Moron, Buenos Aires, Argentina
b Instituto de Patobiologia, Centro de Investigaciones en Ciencias Veterinarias, Institute National de
Tecnologia Agropecuaria, 1708 Moron, Buenos Aires, Argentina
’ Instituto Panamericano de Protection de Alimentos y Zoonosis COPS/ OMS), Martinez, Argentina
’ Facultad de Ciencias Veterinarias, Uniuersidad National de1 Nordeste, Corrientes, Argentina
Received 24 May 1995; accepted 24 October 1995
IS6110, the direct repeat (DR) sequence flanking IS6110, and a polymorphic
sequence (PGRS), called pMBA2. Two restriction enzymes were used, PouII for IS6110 and DR
(DR/PvuII) and AluI for DR (DR/AU) and pMBA2. DNA from 85 of M. bovis isolates was
digested with PvuII and hybridized with IS6110 and DR. IS6110 was not useful to differentiate
M. bovis because most of the isolates contain a single monomorphic
a limited degree of differentiation. DNA from 44 of these isolates was also digested with AluI and
hybridized with DR and pMBA2. In this condition these probes differentiated
different RPLP types. By combining the patterns generated with DR/AluI
possible to increase the differentiation up to obtain 30 different
isolates were differentiated because they showed a unique pattern. Six isolates of M. bovis
involved in two different outbreaks of bovine tuberculosis were correctly identified. Thus, DR and
pMBA2 could be, at the moment, the probes of choice for comparisons
different regions and for epidemiological surveillance of bovine tuberculosis.
bovis (M. bovis) isolated in Argentina. The markers were: the insertion sequence
copy. The use of DR allowed
the isolates in many
and pMBA2 it was
RPLP types and 54% of the
of M. bovis isolates in
Keywords: Myobacterium bouis; RFLP; Genetic markers; Epidemiology
* Corresponding author. Tel.: 54-1-621-1447, Fax: 54-l-481-2975, e-mail: firstname.lastname@example.org
0 1996 Elsevier Science B.V. All tights reserved
M.I. Rornuno et al. / Veterinary Microbiology 50 f 19%) 59- 71
on animal production
livestock industry in Europe and America (Kantor and Alvarez, I991 >.
Although cattle are the main host and reservoir
mammalian species, including human, are susceptible to M. bouis, the causative agent of
Argentina holds nearly 5 1 million head of cattle, with a mean prevalence of BT equal
to or higher than 4%. This infection is spread over most of the territory where 38% of
the herds have one or more cattle positive to the tuberculin
and Alvarez. 199 1).
There is scarce epidemiological information
human health in Argentina. In Santa Fe, an Argentine
breeding and dairy industry, it was found that 2.5% of human
diagnosed in a lo-year period were due to M. boLlis (Latini et al.. 1990).
A more precise understanding of the epidemiology
will contribute to achieving better control or eradication
clearly identify and differentiate strains and trace back the origin of infection
facilitate this task. For that purpose. it is necessary to have a precise correlation between
typing and epidemiological data. However, in Argentina this is hard to achieve, because
cattle management is complicated. This situation is further impaired by the high bovine
tuberculosis incidence and sometimes, uncompleted
M. boris belongs, along with Mycobacterium
and Mycobacterium africanum to the genetically
culosis complex. These are slow growing,
difficult to differentiate by microbiological phenotyping
Phage typing has been applied for strain differentiation
tuberculosis complex in a few reference laboratories.
laborious and the results are not discriminatory
powerful recently developed tool to trace infection
differentiation by DNA techniques. One of these techniques
analysis (REA). Its main limitation is the slight differences
often difficult to compare and analyze (Collins and de Lisle, 1984. Collins and de Lisle,
1985, Collins and de Lisle, 1987; Cousins
polymorphic repetitive DNA sequences in the M. tuberculosis
resulted in the application of restriction fragment length polymorphism
to the epidemiology of human tuberculosis (Hermans et al., 1990, Hermans et al., 1991;
Van Soolingen et al.. 1993).
The most widely used genetic marker
insertion sequence (IS) element IS6110, which is usually present in multiple copies in
M. tuberculosis (Thierry et al., 1990; McAdam et al., 1990; Hermans et al., 1990; Van
Soolingen et al., 1991). In contrast, M. bouis has been found to harbor often only one or
two copies of this element, thus limiting the power to discriminate
strains (Hermans et al., 1990, 1991; Van Soolingen et al., 1994).
(BT) has long been known as an animal disease; but its effects
and human health became apparent with the development of
of this chronic infection, other
test (Kantor, 1988; Kantor
about the impact of this zoonosis
of M. bovis infections
of the disease. The ability to
closely related Mycobacterium
methods (Runyon et al., 1974).
This procedure is time consuming,
(Crawford and Bates, 1984). The most
routes of tuberculosis
is the restriction
among patterns, which are
which are often
species of the M.
et al., 1990). The finding of various
complex bacteria has
for epidemiology of tuberculosis is the
M.I. Rontano et al. / Veterinary Microbiology 50 (1996) 59-71
Other repeated elements have been identified in M. tuberculosis complex: (a> the
polymorphic GC-rich repeat sequence (PGRS), such as, pTNB12 (Ross et al., 1992),
pTB233 (Doran et al., 1992) and pMBA2 (Bigi et al., 19951, (b) the direct repeat
sequence (DR) (Hermans et al., 19911, of which multiple copies are present flanking an
IS 6110 element and (c) the insertion sequence IS 1081 (Collins and Stephens, 1991).
Some of these have been used as genetic markers in epidemiology of bovine tuberculo-
sis (Skuce et al., 1994; Collins et al., 1993; Cousins et al., 1993; Van Soolingen et al.,
This study was undertaken to compare the usefulness of single and combined use of
IS6110, DR and PGRS for the differentiation of M. bouis isolates. The application of
this methodology for epidemiological surveillance was also evaluated.
2. Materials and methods
2.1. Mycobacterial strains
Eighty-five M. bouis isolates were selected. These isolates were from different
mammalian species: 3 from cats, 1 from a Camelidae (llama), 16 from humans, and 65
from bovines. There were 2 groups of epidemiological related isolates included, which
were identified as T52 and T53 from the herd 1 and 172, 174, 142 and 420 from the
All these strains were identified as M. bouis on the basis of growth in the presence of
pyruvate, colony morphology, biochemical and enzymatic tests (niacin, nitrate reduction,
catalase, urease, pyrazinamidase). Susceptibility to thiophene-2-carboxylic acid hydra-
zide (TCH), p-amine salicylic acid (PAS) were determined in glutamate-added egg solid
medium (without glycerol). In certain cases guinea pigs and rabbits were inoculated, in
order to confirm M. bouis identification. Most isolates were from different regions of
Argentina. An M. bouis isolate from a bovine in Costa Rica was also included (culture
identified as 4176). M. tuberculosis H37Rv and M. bovis AN5 were used as reference
2.2. DNA techniques
Extraction of DNA was performed as described by Van Soolingen et al., 1991.
Approximately 1 pg of genomic DNA was digested separately for 1 h at 37°C with 5 U
of AluI (Promega) or PvuII @omega). Restriction fragments were separated overnight
by electrophoresis on 0.8% agarose gel in 50 r&l Tris borate, 1 mM EDTA buffer
(TBE) at 45 V. Following electrophoresis DNA fragments were denatured by treating
the gel for 8 min with 0.25 M HCl, then 2 X 15 min with 0.5 M NaOH, 1.5 M NaCl and
neutralized 2 X 15 min with 1.5 M NaCl, 0.5 M Tris-HCl,
transferred to a nylon membrane by vacuum blotting using Miliblot-V transfer system
(Millipore, Bedford, MA, USA) and 10 X SSC (1 X SSC: 150 mM NaCl, 15 mM
trisodium citrate, pH 7.0) as transfer buffer. The DNA was fixed to membranes by
exposition to the UV light for 10 min.
pH 7.5. DNA was
MS. Romano et al./ Veterinay Microbiology 50 (1996) 59-71
2. 1 -
1. 0- . -
3 4 5
8 9 10
11 12 13
14 15 16
A A E
A A A AAAAAI C
1 2 3 4 5 6 7 8
9 10 11
14 15 16
9 10 11
12 13 14
MI. Romano et al. / Veterinary Microbiology 50 (19%) 59-71
5 6 7 6 9 10 11 12 13 14 15 16
NBA BB CG
ccc A D E H
Fig. 1. RFLP analysis of selected M. bouis isolates with different representative
PuuII-digested DNA hybridized with: (A) IS6110, (B) DR. Fingerprints
with: (C) PGRS, (D) DR. Lane 1 H37Rv, lane 2 AN5, lane 3 107, lane 4 122, lane 5 130, lane 6 172, lane 7
52, lane 8 T53, lane 9 molecular mass standard, lane 10 9611, lane 11 9600, lane 12 1377, lane 13 124, lane
14 245, lane 15 9598, lane 16 4176. Numbers at the left indicate some sizes of standard DNA fragments.
Letters below the figures indicate the pattern assigned.
The IS6110 probe, a 245 bp fragment, was obtained by PCR with the INS1 and INS2
primers (Hermans et al., 1991). This sequence is located in a region to the right of the
PvuII site in IS6110. The pMBA2 probe is a plasmid
GC-rich repetitive sequence (PGRS) (Bigi et al., 1995). DR probe is a 36-mer
oligonucleotide (Hermans et al., 1991). Multiple copies of this sequence are the “hot
spot” of integration of IS6110. The IS6110
labeled with [ a- 32 P]dCTP using the Oligolabelling
manufacturer. The oligonucleotide probe DR was labeled with [ Y-~~P]ATP using T4
polynucleotide kinase (Ausubel et al., 1994).
The membranes containing 85 PuuII-digested
bridized with IS6110 and after stripping they were reprobed with DR. Forty-four
these isolates were also digested with AU.
hybridized with DR, then stripped and reprobed with pMBA2.
that contains a polymorphic
PCR product and pMBA2 plasmid were
kit (Pharmacia) as specified by the
M. boois genomic DNA were hy-
Membranes containing AluI digests were
2.4. Hybridization conditions
(sodium dodecyl sulfate), 2.5 X SSPE (1 X SSPE: 10 mM NaH2PG4,
mM EDTA, pH 7.4), 0.01% sodium pyrophosphate,
hybridization and washing conditions
were prehybridized 2 h at 65°C and hybridized
were prehybridized and hybridized in a solution containing 1% SDS
0.18 M NaCl, 1
acid. The O,l% polyanetolsulfonic
to the probe. The membranes
overnight at 65”C, with c~-~~P-labeled
M.I. Romano et al./ Veterinary Microbiology SO (1996) 59-71
5 67 8 9 10 11 12 13 14 15 16 17
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
AAACA c I
2 3 4 5 6
7 8 9 10 11 12 13 14 15 16 17
M.1 Roman0 et al. / Veterinary Microbiology 50 (19%) 59- 71
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
8 JAKDLMA 8 BOP AAAQR
Fig. 2. RFLP analysis of random M. bouis isolates. Fingerprints
(A) IS6110, (B) DR. Fingerprints
T253, lane 2 T256, lane 3 T257, lane 4 T258, lane 5 T259, lane 6 T260, lane 7 T261, lane 8 T263, lane 9
T264, lane IO T265, lane 11 T268, lane 12 T269, lane 13 T270, lane 14 T274, lane 15 T275, lane 16 T276.
lane 17 T278. Numbers at the left indicate some sizes of standard DNA fragments.
indicate the pattern assigned.
DNA hybridized with:
from AM-digested with: (C) PGRS, (D) DR. Lane 1
Letters below the figures
probes. They were washed twice for 5 min in 1 X SSC, 0.1% SDS at room temperature,
followed by 2 X 15min washes in 0.1 X SSC, 0.1% SDS at 65°C. With the oligonu-
cleotide probe the membranes were prehybridized
overnight at 52°C. These blots were washed 2 X 5 min in 6 X SSC, 0.1% SDS at room
temperature and 2 X 5 min in 6 X SSC, 0.1% SDS at 52°C. Molecular size markers used
were supercoiled ladder&@ PhiX17CHueIII
and h-HindIII. H37Rv-PuuII was also used as molecular markers. In Figs. 1 and 2 only
some molecular sizes were indicated.
2 h at 52°C and then hybridized
(Gibco BRL, Gaithersburg, MD, USA)
Eleven different patterns were identified
isolates hybridized with the 245-bp
fragment at the right of the PvuII site of the IS6110. Seventy (82%) of these isolates
were of pattern A. They contained only one IS61 10 element,
hybridization fragment (pattern A; see Fig. 1A and Fig. 2A). The remaining
were contained in 10 different patterns which presented
fragment in a different position. In some cases there were slight differences
patterns, for example pattern C presented one fragment of 1.8 kb (Fig. 1A lane 16) and
pattern B presented one fragment of 1.7 kb (Fig. 1B lane 17). The origin of these 15
isolates were as follows: cats (31, camelidae (11, human (2) and bovines (9). Pattern A
isolates were all from bovines or from humans. Both groups of epidemiologically
isolates presented IS6110 pattern A (Table 1 and Fig. 1A; isolates 172 lane 6 and T52
lane 8). M. tuberculosis H37Rv/PuuII DNA contained
ments which were used as molecular size markers. The sizes of M. tuberculosis H37Rv
fragments after digestion with PuuII were 5.0, 3.8, 2.9, 2.1, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3
DNA from 85 M. bouis
This probe hybridizes with a
detected in a 1.9 kb
either two fragments
11 IS6110 hybridizing frag-
66 M.I. Roman0 et al./ Veterinary Microbiology SO C 1996159-71
RFLP types assigned by combining the patterns
IS6110 DR/ PuuII
PGRS DR/ Ah1
Cluster I a
Cluster II ’
Cluster III ’
Cluster IV d
Cluster V ’
Cluster Vl ’
North East and Center
’ 4 isolates from North East and I from Center.
h 4 isolates from Center (herd 2).
’ 3 isolates from Center.
’ 2 isolates from North East.
e 2 isolates from Center (herd I).
’ 3 isolates from Center.
and 0.9 kb (Fig. 1A lane 1). With IS6110
because they showed a unique patterns (Table 2).
The Puull-digested DNA from 85 M. bollis
(DR/PuuII). This probe detected
most M. bouis isolates. The longest fragment presented the highest polymorphism.
band situated in the middle was much less intense than the others (Fig. 1B and Fig. 2B).
H37Rv presented a distinctive pattern (Fig. IB lane 1). The three major
patterns were called A. B and C. Pattern A was presented in 37 isolates (43%), including
probe 6 isolates (7%) were differentiated
isolates was also probed with DR
12 patterns. These patterns reveled 3 fragments in
M.I. Roman0 et al. / Veterinary Microbiology 50 (1996) 59-71
Comparison of different genetic markers for molecular epidemiology of bovine TB
Probe and restriction
(% of isolates)
% of isolates
IS61 IO/ PouII
isolates and pattern C in 18 isolates (13% and 21%, respectively).
was similar to pattern C, but its longest fragment was slightly heavier than the longest
band observed in pattern C (data not shown). The isolates from herd 2 presented pattern
A and the isolates from herd 1 presented pattern B (Table 1 and Fig. 1B; isolates 172
lane 6 and T53 lane 8 >. DR/PuuII probe differentiated
analyzed because they showed unique patterns (Table 2).
Hybridization with DR/AZuI identified 22 different patterns in 44 isolates analyzed
(Table 1 and Fig. 1D and Fig. 2D). M. tuberculosis H37Rv strain presented a distinctive
pattern (Fig. 1D lane 1). Pattern A was observed in 9 isolates (20%), pattern B in 10 and
pattern C in 4 isolates (22% and 9%, respectively),
as 3 isolates (Tables 1 and 2). Each group of epidemiologically
herd 1 and herd 2 had a distinctive pattern. The isolates from herd 2 were grouped in
pattern B, while the isolates from herd 1 were grouped in pattern G (Table 1 and Fig.
1D; isolates 172 lane 6 and T53 lane 8). With DR/AluI
isolates (Table 2).
When these membranes were hybridized
probe identified fragments with a size range of about 0.2 to 10 kb (Fig. 1C and Fig. 2C).
Only fragments larger than 2 kb were analyzed. PGRS identified
The predominant pattern was observed in 12 isolates (27% pattern A). Patterns B and C
had 4 isolates each (9%) (Tables 1 and 2). The epidemiologically
herd 2 were included in PGRS pattern V and the isolates from herd 1 presented pattern
A (Table 1 and Fig. 1C; isolates 172 lane 6 and T53 lane 8). With PGRS 32% of the
isolates was differentiated. The M. bouis reference strain AN5, as well as, a M. bovis
obtained a from Costa Rican bovine, had distinctive
1D and C, strains AN5 lane 2 and 4176 lane 16). M. bouis BCG also presented
distinct pattern with these probes (data not shown).
strain M. bouis AN5 (Fig. 1B lane 2). Pattern B was presented in 11
M. bovis BCG pattern
5 isolates (6%) from 85
the remaining patterns had as much
related isolates from
was differentiated 41% of the
with a PGRS-type probe, pMBA2, this
21 different patterns.
related isolates from
DR/AZuI and PGRS patterns (Fig.
M. I. Romano et ul. / Veterinary Microbiology 50 ( 19961 5% 71
DNA it was possible to increase differentiation
isolates analyzed. Fifty-six percent of these isolates had a distinctive
remaining isolates were grouped in clusters (Tables 1 and 2). Six clusters from different
regions of Argentina were found: (a) cluster I: 4 bovines from the Northeast and one
from the Central region, (b) cluster II: four epidemiologically
2 (isolates 172, 174. 142 and 4201, also from the Central region, (cl cluster III: three
bovines from the Northeast region, (dl cluster IV: two bovines from the Central region,
(e) cluster V: two epidemiologically related isolates from herd 1 (isolates T51 and T53)
from the Central region and (f) cluster VI: three bovines from the Central region (Table
All the isolates in clusters had one copy of the IS6110 detected in the 1.9 kb PuuII
fragment (pattern A), although the clusters
Furthermore, one isolate (strain 52) was identified as different of the isolates grouped in
cluster VI using the DR/PouII probe (Table 1).
If only DR/AluI and PGRS patterns are considered,
be grouped into 30 RFLP types, where 54% of the isolates presented a distinctive
pattern (Table 21.
the patterns generated by DR and PGRS in Ah1 and PuclII digested
up to obtain 31 RFLP types from 44
RFLP type. The
related isolates from herd
having a different DR/PuuII patterns.
these 44 M. boLlis isolates can
IS6110 probe was used on 85 M. hods isolates from Argentina, and 11 patterns were
found. However, 82% of isolates had a single copy of this element detected in the same
PuuII band. This situation further limits the use of this marker in epidemiological
surveillance of bovine tuberculosis.
The DR probe was used against PuuII and AluI digested genomes. Twelve DR/PvuII
patterns were obtained from 85 isolates. The major pattern included 43% of the isolates.
Therefore, a better differentiation was obtained
According to Hermans et al. (1991) the three fragments
probe in M. bouis BCG are: (1) a 1.9 kb fragment, containing
the DR region at the right of this element, (2) a 4 kb fragment, containing
IS6110 and the DR region at the left of this element, (3) a 2.9 kb fragment, which is
located right from the 1.9 kb PcuII DR-containing
M. bouis isolates presented 3 similar fragments, but included variations.
longest band presented the highest polymorphism.
much less intense than the others, possibly because this fragment has less DR repeat
units. When DR/AluI was used the differentiation
polymorphism was detected compared to DR/PuuII.
phism was detected.
Different authors have explored the usefulness
bouis typing. Skuce et al. (1994) have used the repetitive elements IS6110, IS1081 and
a PGRS (pTNB 12) to study the epidemiology
The discriminatory power of IS1081 was very low. With IS6110,
were obtained. The major pattern clustered 52% of isolates, corresponding
here than with the IS6110
detected by the DR/PuuII
the IS61 10 element and
the left arm of
fragment. The patterns of the
The band situated at the middle was
With PGRS, similar high polimor-
further because more
of repetitive DNA sequences for M.
of bovine tuberculosis in Northern Ireland.
10 different patterns
to a single
M.I. Romano et al. / Veterinary Microbiology 50 (1996) 59-71
copy of IS6110.
isolates analyzed belonged to the major pattern and that they had one copy of I.56110
detected in 1.9 kb fragment (pattern A), showing
IS6110. It is important to notice that the IS6110 probe used by Skuce et al. (1994) is
the entire IS61 10 element, while we used a fragment
IS6IIO. The entire IS6110 probe hybridizes
isolates: one 5’ (a fragment of approximately
the PvuII site of the IS6110. Both fragments also contain a part of the DR region. The
longest DR/PuuII hybridizing fragment which contains
observed to have more polymorphism (Fig. 1B and Fig. 2B). Taking this into account,
the entire IS6110 probe can detect more polymorphism
right PuuII arm of IS6110. On the contrary, Skuce et al. (1994) using PGRS only found
12 different patterns among 109 isolates, while 21 different patterns among 44 isolates
were found by us. Even though different PGRS probes (pTNB12 and pMBA2) were
used, they seem to generate the same fingerprint
taken into account that PGRS fingerprints are difficult to analyze, because bands with
different intensities and very slight polymorphism
results could also be attributed to the fact that Skuce et al. (1994) worked with samples
from more restricted geographic areas than we.
Collins et al. (1993) used IS6110 and IS1081 to identify RFLP types among 160 M.
bovis isolates, mainly from New Zealand. Nearly all these isolates had the same RFLP
type with IS 1081. The authors used an IS 6110 probe that comprises both PvuII arms of
IS6110 and these analyses demonstrated that 50% of isolates corresponded
IS6110 pattern. The major polymorphism
isolates, can be explained in the same way as above.
Cousins et al. (1992) analyzed 36 human, bovine, porcine and wild life M. bovis
isolates from Australia. Again, IS6110 and PGRS were used. In that study the IS6110
had low power to distinguish isolates of M. bovis. From 36 isolates probed with PGRS,
20 types were found and the major pattern clustered 44% of the isolates. This is in
agreement with the data obtained from Argentine
Van Soolingen et al. (1994) used IS6110 (same probe used by us>, DR/AluI
PGRS to analyze 153 isolates from human, bovine, and wild life M. bovis isolates. They
analyzed Argentine, Dutch and Saudi Arabian
isolates, 69% contained a single IS6110 copy detected on a 1.9 kb Puu II fragment.
They also analyzed Dutch and Saudi Arabian isolates, mostly from animal sources other
than cattle, and only 40% of them had one copy of the IS6110 element detected on a 1.9
kb PvuII fragment. They suggested that different animal reservoirs of M. bovis may
M. bovis types. In Argentina most of M. bovis isolates are from cattle
and human. The human infection is due to transmission
These authors analyzed the M. bovis isolates, containing
same chromosomal position with PGRS and DR/AluI.
differentiation indicating that they could be used to trace the infection routes.
In the present study the hybridization with DR/AluI
different patterns, respectively, among 44 isolates. We believe that the use of these two
probes is adequate for M. bovis typing for the following reasons: (1) by combining
In the present study, we found that 82% of Argentinean
a lower discriminatory power of
from the right PuuII arm of
with two PvuII fragments
4 kb) and one 3’ (a fragment of 2.9 kb) of
in M. bovis
the left IS6110 arm was
than the probe that binds to the
(data not shown). It should also be
are observed. The differences in
to the major
to Argentine in these isolates compared
isolates. From 42 Argentine
from cattle (Latini et al., 1990).
a single IS61 10 copy at the
Both probes gave a good
and PGRS showed 22 and 21
M. I. Romano et ~11. / Veterinatyv Microbiology 50 f 1996) 59-71
patterns it was possible to differentiate
from different geographic origin, such as, M. bouis AN5 and the isolate from a Costa
Rican bovine had distinctive DR/AluI and PGRS patterns.
related isolates were correctly identified (Table I), (3) the use of IS61 10 did not provide
new differentiation over that of PGRS and DR/AfuI.
the additional differentiation of only one isolate of 44 taken (isolate 52, Table
However, the digestion of all isolates with two enzymes complicated
In summary. we conclude that it is better to use only A~KI digested DNA and to
hybridize first with DR (because the patterns are simple to analyze) and then with PGRS
probe. It could be considered a sufficiently
These results, along with the recording and tracing of cattle movements,
tribute to a better knowledge of the origin of M. bouis infection in herds from different
areas. This information could help to define strategies for bovine tuberculosis
lance or eradication in different regions or countries.
54% of the isolates (Table 21, (2) the isolates
The use the DR/PuKII provided
the procedure and
sensitive procedure to differentiate isolates.
This work was partially
Stockholm, Sweden. We are grateful to Osvaldo Rossetti in whose unit this work was
carried out, to Haydee Gil and Manuel
Bemardelli and Omar Latini for providing of M. boois isolates and to Eleonora Campos
for her critical reading.
supported by the International Foundation for Science,
Rodriguez for technical help, to Amelia
Ausubel. F.M., Brent, R., Kingston,
Current protocols in molecular biology. Vol. 1. Wiley, New York, Inc. 6.4.6.
Bigi, F.. Romano, M.I., Alito. A. and Cataldi. A., 1995. Cloning of a novel polymorphic
DNA from Mycobacrerium botit Res. Microbial.,
Collins, D.M., Erasmuson, S.K., Stephens, D.M.. Gary, F.Y. and de Lisle, G.W.. 1993. DNA fingerprinting
Mycobacterium bow’s strains by restriction fragment
ISIOXI and IS6/10. J. Clin. Microbial.. 31: 1143-l 147.
Collins, D.M. and Stephens, D.M.. 1991. Identification
bock. FEMS Microbial. Lett., 83: 1 I- 16.
Collins. D.M. and de Lisle, G.W.. 1987. BCG identification
Microbial., 133: 1431- 1434.
Collins, D.M. and de Lisle. G.W., 1985. DNA restriction endonuclease
other members of the tuberculosis complex. J. Clin. Microbial.,
Collins, D.M. and de Lisle, G.W.. 1984. DNA restriction endonuclease
sis and Mycobacterium bouis BCG. J. Gen. Microbial.,
Cousins. D.V., Williams. S.N., Ross, B.C. and Ellis, T.M.. 1993. Use of a repetitive element isolated from M.
in hybridization studies with M. boris: a new tool for epidemiological
tuberculosis. Vet. Microbial.. 37: l-7.
Cousins. D.V.. Francis, B.R., Cow, B.L., Collins. D.M.. McGlashan,
1990. Tuberculosis in captive seals: bacteriological
tuberculosis complex. Res. Vet. Sci.. 4X: 196-200.
R.E., Moore, D.D., Seidman, J.G.. Smith, J.A. and Struhl. K., 1994.
CC rich repetitive
analysis and hybridization with insertion element
of an insertion sequence, ISIONI, in Myobacterium
by DNA restriction fragment patterns. J. Gen.
analysis of Mycobacterium
analysis of Mycobacterium
studies of bovine
C.H., Gregory. A. and MacKenzie. R.M.,
studies on an isolate belonging to the Mycobacterium
M.I. Roman0 et al. / Veterinary Microbiology 50 (1996) 59-71
Doran, T.J., Hodgson,
DNA sequence from Mycobacterium bouis. FEMS L&t., 96: 179-186.
Hermans, P.W.M., van Soolingen, D., Bik, E.M., de Haas, P.E.W., Dale, J.W. and van Embden, J.D.A., 1991.
Insertion element IS987 from Mycobacterium bock BCG is located in a hot-spot integration
insertion elements in Mycobacterium tuberculosis complex strains. Infect. Immun., 59: 2695-2705.
Hermans, P.W.M., van Soolingen, D., Dale, J.W., Schuitema,
Embden, J.D.A., 1990. Insertion element IS986 from Mycobacterium
diagnosis and epidemiology of tuberculosis. J. Clin. Microbial.,
Kantor, I.N. and Alvarez, E., 1991. Current status of bovine tuberculosis
Special Publication No. 10, Pan American Zoonoses Center, Martinez, Argentina.
Kantor, I.N., 1988. The status of bovine tuberculosis in Latin America and the Caribbean.
No. 8, Pan American Zoonosis Center, Martinez, Argentina.
Latini, M.S., Latini, O.A., Lopez, M.L. and Cecconi, J.O., 1990. Tuberculosis
Arg. Torax, 51: 13-16.
McAdam, R.A., Hermans, P.W.M., van Soolingen, D., Zainuddin,
Dale, J.W., 1990. Characterization of a Mycobacterium
IS3 family. Mol. Microbial., 4: 1607-1613.
Ross, B.C., Raios, K., Jackson, K. and Dwyer, B., 1992. Molecular cloning of highly repeated DNA element
from Mycobacferium tuberculosis and its use as an epidemiological
Runyon, E.H., Wayne, LG. and Kubica, G.P., 1974. Family II Mycobacteriaceae.
Gibbons (Editors), Bergey’s manual of determinative
Baltimore, p. 681.
Skuce, R.A., Brittain, D., Hughes, M.S., Beck, L.A. and Neill, S.D., 1994. Genomic
Mycobacterium bouis from cattle by restriction fragment length polymorphism
Thierry. D., Brisson-Noel, A., Vincent-L&y-Frebault,
Characterization of a Mycobacferium tuberculosis
diagnosis. J. Clin. Microbial., 28: 2668-2673.
Van Soolingen, D., de Haas, P.E.W., Haagsma, J., Eger, T., Hermans, P.W.M., Ritacco, V., Alito, A. and van
Embden, J.D.A., 1994. Use of various genetic markers in differentiation
Strains from animals and humans and for studying
Microbial., 32: 2425-2433.
Van Soolingen, D., de Haas, P.E.W., Hermans, P.W.M., Groenen, P.M.A. and van Embden, J.D.A.,
Comparison of various repetitive elements as genetic markers for strain differentiation
Mycobacterium tuberculosis. J. Clin. Microbial., 29: 2578-2586.
Van Soolingen, D., Hermans, P.W.M., de Haas, P.E.W.,
Occurrence and stability of insertion sequences in Mycobacrerium tuberculosis complex strains: evaluation
of an insertion sequence-dependent DNA polymorphism
Clin. Microbial., 29: 2578-2586.
J. and Bates, J.H., 1984. Phage typing of mycobacteria.
A.L.M., Davies, J.K. and Radford, A.J., 1992. Characterization
In: G.P. Kubica and L.G. Wayne (Editors),
Part A, Marcel Dekker, New York, pp. 123-132.
of a novel repetitive
A.R.J., McAdam, R.A., Catty, D. and van
tuberculosis: A useful tool for
in Latin America and the Caribbean.
bovina en seres humanos. Rev.
Z.F., Catty, D., van Embden, J.D.A. and
tuberculosis insertion sequence belonging to the
tool. J. Clin. Microbial.,
In R.E. Buchanan and N.E.
8” ed. The Williams and Wilkins Co.,
analysis. J. Clin. Microbial.
S., Guesdon, J. and Gicquel, B., 1990.
sequence IS6110, and its application in
of Mycobacterium bouis strains
of bovine tuberculosis. epidemiology J. Clin.
and epidemiology of
Soll, D.R. and van Embden, J.D.A., 1991.
as a tool in the epidemiology of tuberculosis. J.