Journal of Clinical Microbiology, May 1999, p. 1254-1259, Vol. 37, No. 5
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Prevalence of Mycobacterium avium in Slaughter Pigs in
The Netherlands and Comparison of IS1245
Restriction Fragment Length Polymorphism Patterns of Porcine
and Human Isolates
Ruud E.
Komijn,1,*
Petra E. W.
de Haas,2
Margriet M. E.
Schneider,3
Tony
Eger,4
Jan H. M.
Nieuwenhuijs,5
Remco J.
van
den Hoek,2
Douwe
Bakker,4
Fred G.
van Zijderveld,4 and
Dick
van
Soolingen2
National Inspection Service for Livestock and
Meat, 2270 JA Voorburg,1 Mycobacteria
Department, National Institute of Public Health and the
Environment, 3720 BA Bilthoven,2
Department of Internal Medicine, Subdivision of Infectious
Diseases and AIDS, University Hospital Utrecht, 3584 CX
Utrecht,3 Department of Bacteriology,
Institute for Animal Science and Health, 8200 AB
Lelystad,4 and Veterinary Health
Inspectorate, 2280 MK Rijswijk,5 The Netherlands
Received 11 May 1998/Returned for modification 9 July 1998/Accepted 25 January 1999
 |
ABSTRACT |
A significant increase in the incidence of caseous lesions in the
lymph nodes of slaughter pigs prompted a large-scale investigation in
five slaughterhouses in The Netherlands. In total, 158,763 pigs from
2,899 groups underwent gross examination. At least one pig with caseous
lesions in the submaxillary and/or mesenteric lymph nodes was observed
in each of 154 of the 2,899 groups examined (5%). In total, 856 pigs
(0.5%) were affected. As many as five pigs in each of 141 of the 154 positive groups (91.5%) had lymph node lesions. Greater numbers of
pigs with affected lymph nodes were found in 13 groups (8.5%). Four
pigs had lesions in the kidneys, liver, or spleen. Acid-fast bacteria
were detected by microscopic examination of 121 of 292 Ziehl-Neelsen-stained smears of caseous lesions (41%). In a follow-up
study, Mycobacterium avium complex (MAC) bacteria were
isolated from 219 of 402 affected lymph nodes (54.2%). Ninety-one of
the isolated strains were analyzed by restriction fragment length
polymorphism (RFLP) typing with insertion sequence IS1245
as a probe. All but 1 of these 91 strains contained IS1245 DNA, indicating that pigs in The Netherlands carried almost exclusively M. avium bacteria and no other bacteria of MAC. Only
one pig isolate exhibited the bird-type RFLP pattern. MAC isolates from
191 human patients in The Netherlands in 1996 were also typed by RFLP
analysis. Computer-assisted analysis showed that the RFLP patterns of
61% of the human isolates and 59% of the porcine isolates were at least 75% similar to the RFLP patterns of the other group of strains. This indicates that pigs may be an important vehicle for M. avium infections in humans or that pigs and humans share common
sources of infection.
| |
INTRODUCTION |
Severe Mycobacterium
avium complex (MAC) infections in humans, especially in human
immunodeficiency virus-positive and other immunodeficient individuals,
have been reported (8, 13). The origin of MAC infections in
humans is still a matter of speculation. Previous studies have shown
that the MAC bacteria are present in birds, soil, compost, water,
animals, pigs, and even cigarettes (2, 3, 5, 6, 8, 11, 19).
As suggested by the designation M. avium, infections
were once thought to be derived from birds. Later, serotyping showed
that only some of the MAC bacteria isolated from humans represent
serotypes 1, 2, and 3, which are the most common serotypes among bird
isolates (1, 7).
Recently, new molecular tools like restriction fragment length
polymorphism (RFLP) typing with the insertion sequence
IS1245 (IS1245 RFLP analysis) have become
available (2, 6, 12). Genotyping of M. avium
strains from various sources in Switzerland indicated that both pigs
and humans were infected with strains carrying a large number of
IS1245 elements (2). IS901 and
IS1245 RFLP typing showed that 47 M. avium
isolates from birds in The Netherlands invariably belonged to a
well-conserved separate taxon within MAC. Bird-type RFLP patterns were
observed only as an exception among isolates from other hosts (2,
12). These facts rule birds out as significant sources of
M. avium infections in humans in The Netherlands
(12).
The current study was undertaken to determine the prevalence of MAC in
the lymph nodes of pigs. Furthermore, in order to examine the
significance of M. avium infections in slaughter pigs
with regard to public health aspects, the IS1245 RFLP
patterns of porcine isolates were compared with those of the
M. avium strains isolated from humans in The
Netherlands in 1996.
| |
MATERIALS AND METHODS |
Gross examination of pigs.
In an initial study, special
attention was given to the gross examination of the submaxillary and
mesenteric lymph nodes of pigs in five slaughterhouses during a 2-week
period at the end of 1996. The submaxillary lymph nodes were incised,
and the mesenteric lymph nodes were palpated. The following information
was collected: the farm identification numbers of the pigs slaughtered,
the number of pigs slaughtered per farm, the number of pigs with
caseous lesions in the submaxillary or mesenteric lymph nodes, and the number of pigs whose spleen, liver, or kidneys were also affected. Whenever possible, up to three specimens per group were studied by
microscopic examination of Ziehl-Neelsen-stained smears.
Sampling, culture, and identification of mycobacteria from
pigs.
In a follow-up study performed in early 1997, the presence
of mycobacteria in caseous lesions was determined by culture. For this
purpose, macroscopically positive submaxillary and mesenteric lymph
nodes were collected at six slaughterhouses and were frozen at
20°C. In the first part of the follow-up study, samples were taken
from each of three to four pigs in 44 groups in which several animals
were affected. In the second part of the follow-up study, 144 groups
with only one or two affected animals each were sampled. After arrival
at the laboratory, the samples were thawed, and direct smears were
produced. Ziehl-Neelsen-stained material was then examined
microscopically. In addition, cultures were grown from all lesions by
the following procedure: all lesions were ground, decontaminated by
oxalic acid-sodium hydroxide treatment, and inoculated onto
Löwenstein-Jensen medium, Stonenbrink egg medium, and Middlebrook
7H10 agar, followed by incubation for 4 weeks at 37°C.
Subcultures were made from colonies suspected of representing MAC
bacteria. The MAC bacteria of the subcultures were identified by the
following characteristics: growth after 2 to 4 weeks of incubation,
negative or doubtful acid phosphatase reaction, negative nitrate
reductase reaction, weakly positive catalase reaction (<45 mm) at room
temperature, variable catalase reaction at 68°C, negative
-d-galactosidase reaction, positive nicotinamidase and pyrazinamidase activities, and negative urease activity by the amidase
test of Bönicke. All but 1 of the 91 isolated MAC strains contained IS1245, which is characteristic of M. avium (2, 6, 12). To ensure this identification, 30 IS1245-containing MAC isolates were subjected to the
Accuprobe test specific for M. avium, and they were
found to be positive.
MAC bacteria from humans.
In 1996, 191 MAC isolates
originating from 35 peripheral laboratories were received at the
National Institute of Public Health and the Environment (RIVM) in The
Netherlands. This number covers at least 80% of all human MAC strains
isolated in The Netherlands in 1996.
Serotyping.
MAC isolates were tested by slide agglutination,
as described by Engel et al. (4), to determine their
serotypes. The panel of test sera represented serotypes 1 to 4 and 8.
DNA fingerprinting.
M. avium isolates were DNA
fingerprinted by RFLP typing; IS1245 was used as a probe, as
described previously (12, 16). Internal and external
molecular size markers and high-resolution gels (24 cm) were applied to
facilitate computer-assisted analysis.
Computer-assisted RFLP analysis.
Analysis of the
IS1245 fingerprints was done with computer assistance, using
GelCompar software, version 4.1 (Applied Maths, Kortrijk, Belgium), as
described in a proposal for standardization of IS1245 RFLP
typing (16). The band positions of the
IS1245-containing restriction fragments were compared with
those of a set of internal molecular weight markers by superimposing
the autoradiograms of the IS1245 DNA fingerprints and the
autoradiograms of the internal markers. The patterns were compared by
the unweighted pair group method with the arithmetic averages
clustering method and with the Dice coefficient according to the
instructions of the manufacturer of GelCompar.
| |
RESULTS |
Examination of affected lymph nodes by microscopy and culture.
A total of 158,763 pigs in 2,899 groups were inspected during the
initial study at the end of 1996. Each of 154 groups (5%) included at
least one pig with caseous lesions in the submaxillary and/or
the mesenteric lymph nodes. Altogether, 856 pigs (0.5%) were affected.
For practical reasons, only 292 lesion smears were microscopically
examined. Acid-fast bacteria were seen in 121 of them. Five or fewer
pigs in each of 141 of the affected groups had caseous lesions. Greater
numbers of affected pigs were detected in the remaining 13 groups. The
average percentage of affected pigs in these 13 groups
amounted to 31, and the range was between 8 and 78%. Only
four pigs also had macroscopic deviations in the kidney, liver, or spleen.
In order to examine whether M. avium was the etiologic
agent of these caseous lesions, a follow-up study was planned for early 1997. In the first part of the follow-up study, 239 lymph nodes with
caseous lesions from pigs from 44 farms were examined (Table 1). These farms were not the same ones as
those in the initial study. MAC bacteria were isolated from 166 of the
lymph nodes (69%) from 39 of the groups examined (89%). Seventy-eight
percent of the affected mesenteric lymph nodes and 52% of the
submaxillary lymph nodes yielded growth of MAC bacteria. In the
second part of the follow-up study, lymph nodes from 163 pigs from 144 farms were examined (Table 1). MAC bacteria were isolated from 53 of the pigs (33%), which originated from 46 of all groups examined (32%). Forty-nine percent of the affected mesenteric lymph nodes and 23% of the submaxillary lymph nodes were found to be positive for
MAC by culture. From the mesenteric and submaxillary lymph nodes with a
positive culture for MAC bacteria, a total of 82 and 80% of the
samples, respectively, were also found to be positive by microscopic
examination (Table 1). However, also a total of 79 and 83% of the
mesenteric and submaxillary lymph nodes with negative cultures
for MAC bacteria, respectively, yielded acid-fast bacilli in the
microscopic examination (Table 1). Furthermore, rapidly growing
mycobacteria from 53 lymph nodes were cultured and were found to have
an orange pigment. These non-M. avium mycobacteria were
mainly (40 of the 53) isolated from the submaxillary lymph nodes.
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TABLE 1.
Correlation of culture results and microscopic
examination of affected lymph nodes in the first and second parts
of the follow-up studya
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Serotyping.
The serotypes of the MAC isolates were determined
by the slide agglutination method, and the results are given in Fig.
1. Most strains were of serotype 3 (18 strains) or 4 (20 strains), and 39 isolates did not react with the
panel of sera that we used. A minority of the isolates were of serotype
2, 8, or 4/8. No correlation was found between the serotypes and the
IS1245 RFLP patterns.
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FIG. 1.
Dendrogram of the 91 IS1245 RFLP patterns of
M. avium complex isolates from pigs. The columns
indicate (i) the farms where the pigs originated, (ii) the
slaughterhouses where the pigs were processed, (iii) the serotypes of
the isolates, and (iv) the IS1245 RFLP genotype family
(clade) to which the isolate belongs. The numbers at the top represent
percent relatedness.
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|
IS1245 RFLP typing of porcine isolates.
To get an
impression of the occurrence of IS1245 RFLP types in various
geographic regions, 10 to 20 isolates from pigs from each of the six
slaughterhouses enrolled in this study were genotyped. Figure 1 shows a
dendrogram of all 91 DNA fingerprint patterns. Only one of the
IS1245 RFLP patterns, consisting of three bands, represented
the bird-type DNA fingerprint (2, 6, 12). One other
MAC isolate was devoid of IS1245 DNA, indicating that this
strain represents a grouping other than M. avium in the
MAC. The number of copies for the other 89 strains ranged from 9 to 34, with an average of 21 per strain. In general, the degree of polymorphism among the DNA fingerprints of pig isolates was
large. However, most of the isolates could be grouped into
genotype families that shared a similarity of at least 75% among
the IS1245 RFLP patterns (Fig. 1).
The M. avium isolates subjected to RFLP typing
originated from 91 pigs from 75 farms. A single pig from each of 63 farms was examined, and two or three pigs from each of 12 farms were
inspected. In the case of 11 of the 12 multiple isolates from the same
farm, two or more different DNA fingerprints were found (Fig. 1). This indicates the presence of multiple M. avium strains in
pigs from 11 of 12 farms from which more than one porcine M. avium isolate was obtained. In contrast, identical DNA
fingerprints were found among isolates from different farms. In total,
nine clusters, with a cluster size of two to six isolates, comprised 30 strains originating from 28 farms in a widespread geographic area.
Comparison of RFLP patterns of human and porcine M. avium isolates.
In 1996, 191 MAC isolates from the same
number of human patients were subjected to IS1245-based RFLP
typing in the framework of an epidemiological population-based study on
MAC infections in The Netherlands (13). Forty-eight of
the 191 isolates (25%) lacked IS1245 DNA, indicating that
these strains do not represent M. avium but represent
other groupings within MAC. Computer-assisted analysis helped
compare the 90 porcine M. avium isolates with the 143 IS1245-containing human M. avium isolates
from 1996. Nine genotype families were defined on the basis of at least
75% similarity between the IS1245 RFLP patterns of human
and porcine isolates. The occurrence of isolates from both sources in
these nine clades is given in Table 2. In
total, 59% of the pig isolates and 61% of the human strains were in
common genotype families. The largest family (clade 7502) comprised 21 isolates from pigs and 83 isolates from humans (Fig.
2). Two genotype families comprised only
four human isolates (clade 7507; data not shown) and only 22 pig
isolates (clade 7508; Fig. 1).
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TABLE 2.
Occurrence of MAC isolates from humans and pigs in
IS1245 RFLP genotype families with at least 75% similarity
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FIG. 2.
Dendrogram of IS1245 DNA fingerprints of pig
and human isolates in clade 7502. The numbers at the top represent
percent relatedness.
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| |
DISCUSSION |
The average prevalence of caseous lesions in slaughtered pigs was
0.5%, which is unexpectedly high, taking into account the fact that
positive pigs were selected only by eye on the basis of deviations in
lymph nodes. In an earlier study in Switzerland, Offermann
(10) isolated M. avium from the mesenteric
lymph nodes from 48 of 345 (13.9%) healthy slaughter pigs without any
lesions in these lymph nodes. Therefore, the true prevalence of
M. avium in slaughter pigs in The Netherlands might be
much higher.
Molecular typing and computer-assisted analysis facilitate the
comparison of human and porcine isolates on a large scale. Although no
identical DNA fingerprints of porcine and human origin were found, 60%
of the isolates from both sources had a similarity of at least 75%
among the IS1245 RFLP patterns. This means that, for
IS1245 RFLP patterns consisting of 20 bands, at least 15 band positions are shared. Taking into account the high degree of
IS1245-based polymorphism among M. avium strains in general, this justifies the conclusion that
humans and pigs are infected with the same types of M. avium strains. It is currently not clear whether humans and pigs
share common sources of infection or that pork products prepared
without appropriate heating may infect susceptible humans. Long-term
epidemiological studies are needed to examine this hypothesis. Such
studies might find direct links between the consumption of contaminated
pork products and infections in humans. However, such studies are
complicated by the fact that pigs from various parts of The Netherlands
are slaughtered at about 26 large and 30 small slaughterhouses
scattered over the whole country. In addition, approximately 70% of
the pork and pork products are exported.
Isolation of M. avium by culture is considered
the "gold standard" test for the diagnosis of porcine mycobacterial
infections. A sensitivity for microscopic examination of
Ziehl-Neelsen-stained smears of 15% for MAC culture-positive lymph
nodes has been reported by Margolis et al. (9). In the
follow-up part of the current study, a much greater sensitivity was
found by microscopic examination: in total, 80% for the
submaxillary lymph nodes and 82% for the mesenteric lymph nodes.
However, 81% of all samples with a negative MAC culture result
also yielded acid-fast bacilli by microscopic examination. Furthermore,
large differences between the predictive value of positive
microscopic examinations of submaxillary lymph nodes (26%) and that of
positive microscopic examinations of mesenteric lymph nodes (71%) were
observed. This low predictive value regarding positive
microscopic examinations of the submaxillary lymph nodes is presumably
due to a high prevalence of other, non-MAC bacteriological infections
caused by injuries as a result of fighting and/or cutting of dents. In
our study we found more than 50 positive cultures that yielded
orange-pigmented acid-fast mycobacterial rapid growers.
The occurrence of IS1245 is restricted to M. avium (6, 12). Only 1 of 91 porcine isolates lacked
IS1245 DNA in this study, revealing that the porcine MAC
isolates almost invariably represented true M. avium.
Among the human isolates, 25% of the strains did not hybridize to the
IS1245 probe. This indicates that a proportion of the human
MAC isolates much larger than that of the porcine isolates represented
other groupings within MAC. This presumably reflects the fact
that humans have sources of infection not shared with pigs.
The identification of the IS1245-negative MAC strains is
described elsewhere (13).
In the current study, MAC isolates from pigs at one farm were usually
infected with various genotypes of M. avium, and
identical fingerprints were found among isolates from pigs from
different farms. This suggests that there is no ongoing transmission
among pigs but, rather, that pigs are infected from environmental
sources, and these may be shared by farms at different geographic
locations. In a study by Engel et al. (5) of three farms in
The Netherlands in 1977, M. avium serotype 2 was
isolated from 12 of 13 pigs on one farm and occasionally from pigs on
two other farms. Since serotypes 1, 2, and 3 were commonly found among
bird isolates, this finding at that time strongly suggested a role of
birds in the transmission of "avian" tuberculosis. However, in our
previous study (12), we found multicopy
IS1245 RFLP patterns among M. avium serotype
2 and 3 strains apart from the frequently found bird-type RFLP pattern.
The multicopy patterns clearly do not represent the bird type RFLP
pattern. This means that serotyping is not a reliable method of
recognizing M. avium strains that originate in birds.
The serotyping results in this study also reflect this. Although
21 of the 91 porcine isolates represented serotype 2 or 3, only one
of these 21 strains had the bird-type IS1245 RFLP
pattern. This finding, combined with the fact that 47 M. avium strains from birds in The Netherlands
invariably exhibited the bird-type IS1245 RFLP pattern
(12), excludes birds as significant sources of MAC
infections in pigs.
Engel et al. (5) used a pig infection model to demonstrate
that tuberculous lymphadenitis can be induced by feeding pigs compost.
However, it is assumed that compost can no longer be suspected as a
main factor in the etiology of M. avium
infections in pigs, because compost is disinfected nowadays by
heating and is thought not to contain viable M. avium bacteria.
In this study, slaughter pigs were examined by selecting lymph
nodes with caseous lesions. Macroscopically negative lymph nodes
and the dissemination of MAC infections to other organs must be
examined to estimate the true prevalence of MAC bacteria in pigs.
Furthermore, detailed studies are needed to further investigate possible sources of infection at farms with a high incidence of MAC-positive pigs.
| |
ACKNOWLEDGMENTS |
We acknowledge the contributions of the inspection teams of the
slaughterhouses in Boxtel, Doetichem, Druten, Roosendaal, Rotterdam,
and Zevenaar, The Netherlands, for gross examination and collection of
the lymph nodes of slaughter pigs in the bacteriological survey.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: National
Inspection Service for Livestock and Meat, P.O. Box 3000, 2270 JA
Voorburg, The Netherlands. Phone: 31-70-3578806. Fax: 31-70-3578806. E-mail: r.e.komijn{at}rvv.agro.nl.
| |
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Journal of Clinical Microbiology, May 1999, p. 1254-1259, Vol. 37, No. 5
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
