ABSTRACT
A total of 738 strains of Mycobacterium avium complex (MAC) were examined in biological experiments on poultry by use of PCR methods with primers for detection of the insertion sequence IS901. Serotype strains of MAC from all known 28 serotypes were examined. Further strains were isolated from human immunodeficiency virus (HIV)-negative and HIV-positive patients, 6 animal species, 17 bird species, and the environment. Of 165 strains virulent for poultry, characterized by generalized tuberculosis, 164 strains contained IS901, a result which is statistically highly significant (P, 0.01). The remaining 573 strains were nonvirulent; however, IS901 was present in 24 strains. From among 20 strains of serotypes 1, 2, and 3, IS901 was found in 15 strains, only 5 of which were virulent for poultry. The remaining 111 strains, of serotypes 4 to 28, were nonvirulent and did not incorporate IS901. None of the 152 strains isolated from humans was virulent for poultry, including 12 strains which were IS901 positive.
The importance of mycobacterial infections caused by strains of Mycobacterium avium complex (MAC) in animals and humans is continuously increasing (11, 18). In the human population, the condition is aggravated by the spread of human immunodeficiency virus (HIV) infection. In AIDS patients, the incidence of disseminated mycobacterial infection caused by MAC strains can reach up to 55% (34, 47). In poultry, swine, and cattle farms and in farmed red deer, avian tuberculosis imposes the highest financial losses (5, 9, 30, 39, 40).
In veterinary epidemiology, virulent strains inducing avian generalized tuberculosis are the most important. One of the oldest and most frequently used methods for virulence assessment of MAC strains in birds is the challenge assay with poultry; nevertheless, mice, guinea pigs, hamsters, and rabbits also have been used (2, 8, 10, 31, 42, 43, 51, 52). Serotyping has partly replaced time-consuming experimentation on laboratory animals. Originally, serotypes 1 and 2, virulent for birds, were classified as M. avium, and 13 nonvirulent serotypes were classified as M. intracellulare(39, 41). Later, a third virulent serotype of M. avium, serotype 3, was described (21). Piening et al. denominated those three MAC serotypes as the M. avium group, in accordance with previous studies. Strains of serotypes 12 to 20 were absolutely nonvirulent for poultry and were denominated as the M. intracellulare group. Strains of serotypes 4 to 6 and 8 to 11, causing changes only at the site of inoculation after intramuscular injection, were denominated as an intermediate MAC group (33).
As further serotypes were continuously investigated, in 1979 the number of serotypes belonging to M. intracellulare increased to 25 (serotypes 4 to 28) (49, 50). However, research results obtained in the early 1980s led to a new approach to the taxonomic classification of each MAC serotype. DNA-DNA hybridization was used to show the relationship of strains of serotypes 1 to 6 and 8 to 11 (3). These and other results led in 1990 to the suggestion for a new taxonomy for MAC strains, “wood pigeon” (mycobactin-dependent strains isolated from wood pigeons) and M. paratuberculosis strains. MAC strains of serotypes 1 to 6 and 8 to 11, and 21 were denominated as M. avium subsp.avium, wood pigeon strains were denominated as M. avium subsp. silvaticum, and M. paratuberculosis strains were denominated as M. aviumsubsp. paratuberculosis (44).
These results, especially for strains belonging to M. aviumsubsp. avium (serotypes 1 to 6, 8 to 11, and later 21), were supported by the sequencing of 16S rRNA and internal transcribed spacer 16S-23S ribosomal DNA from strains of all 28 serotypes (6, 12, 46). Restriction fragment length polymorphisms (RFLP) have been used to divide the MAC strains into the group M. aviumsubsp. avium, with serotypes 1 to 6 and 8 to 10, and the group M. intracellulare, with serotypes 7 and 11 to 21, according to McFadden et al. (24). Serotypes 1 to 6, 8 to 11, and 21 were further classified with 16S rRNA probes from Gen-Probe Inc., San Diego, Calif., as M. avium subsp.avium, and serotypes 7, 12 to 17, 19, 20, and 25 were classified as M. intracellulare (37, 38). The same results were later obtained in seven laboratories within a comparative study of the International Working Group on Mycobacterial Taxonomy (48).
A new approach to the differentiation of MAC strains was obtained with the description of repetitive insertion sequence IS900 inM. avium subsp. paratuberculosis strains and IS901 or IS902 in M. avium subsp.silvaticum strains (15, 19, 27). It was also found that MAC strains containing IS901 are more virulent for mice after intravenous infection (20). IS901was found in 97.8% of strains from birds, in 74.1% of strains from animals, and in 12.5% of strains from the environment. Examination of serotyped strains revealed IS901 only in strains of serotypes 1, 2, and 3 (28).
The presence of IS901 only in strains of serotypes 1, 2, and 3 and in strains virulent for mice suggests that the presence of IS901 may be connected to the virulence of MAC for birds. In this study, we therefore examined all available serotyped MAC strains and a range of strains isolated from birds, animals, the environment, and humans by using parallel challenge experimentation with poultry and an IS901 PCR detection method.
MATERIALS AND METHODS
Origin of MAC strains.A total of 738 MAC strains were examined (Tables 1, 2, and 3). Each field strain was isolated from one host or from one environmental sample. Serotyped strains were obtained from the culture collections of five laboratories. Field strains from animals, birds, and the environment were isolated in our laboratory or were obtained from eight other laboratories. Human strains were obtained from 29 other laboratories.
MAC strains
MAC strains isolated from animals and the environment
MAC strains isolated from humans
Serotype MAC strains.MAC strains (n = 131) (Table 1) of all 28 serotypes were examined. Each serotype strain from each laboratory was considered a separate strain due to long-term storage in various collections. There were 20 strains of serotypes 1, 2, and 3 and 111 strains of serotypes 4 to 28.
Field veterinary MAC strains.Fifty-six strains (Table 2) from 17 bird species and 38 locations were examined; 49 of these were isolated from parenchymatous organs with generalized tuberculosis, and 7 were isolated from unchanged parenchymatous organs or gut contents. In addition, 261 strains (Table 2) from six animal species and 89 locations were examined: 237 strains were isolated from swine, 12 were isolated from cattle, 10 were isolated from sheep, 1 was isolated from a goat, and one was isolated from a horse. Environmental strains (n = 138) (Table 2) were obtained from 51 infected swine and cattle herds and included samples of sawdust, drinking water, litter, stable environment, and feed.
Human MAC strains.A total of 152 strains (Table 3) isolated from HIV-negative and HIV-positive patients in 11 countries were examined. The strains were isolated from blood (19.6%), bone marrow (3.0%), feces (3.0%), lymph nodes (4.5%), sputum (67.6%), and gastric lavage (2.3%). The strains isolated from patients in the Czech Republic and Slovakia were divided, based on isolation period, into two groups: group A contained 29 strains isolated from 1968 to 1978 and stored at the Czechoslovak National Collection of Type Cultures in Prague, Czech Republic, and group B contained 20 strains isolated from 1996 to 1997 and provided in the first subculture by various laboratories.
Subculturing of MAC strains.Mycobacterial strains were subcultivated on Stonebrink, Löwenstein-Jensen, Herrold, and Middlebrook (7H11) solid media and in a liquid serum medium for mycobacterial cultivation (SEVAC, Prague, Czech Republic).
Virulence assessment of MAC strains.Strain virulence was assessed by biological experiments on 6- to 8-week-old pullets (two pullets for each strain) of the egg type breed ISA Brown after intramuscular injection with 1 to 5 mg of bacterial wet substance per kg of live weight (31). Gross lesions in parenchymatous organs (liver, spleen, kidney) were assessed 8 to 10 weeks after infection. The finding of organized, small tuberculous nodules which were poppy-seed to lentil size in both pullets was designated a positive finding.
PCR method (IS901).In this study, the PCR procedure of Kunze et al. was used (20). The primers (5′-GCAACGGTTGTTGCTTGAAA-3′ and 5′-TGATACGGCCGGAATCGCGT-3′) used for the detection of IS901 were derived from positions 76 and 1184 of fragment IS901, thus amplifying a PCR product of 1,108 bp.
Statistical evaluation.The χ2 test from the STAT Plus package (23) was used for statistical evaluation.
RESULTS
A total of 165 of 738 MAC strains tested were highly virulent for poultry in challenge experimentation (generalized tuberculosis); IS901 was detected in 164 strains (Table 1). No gross lesions were detected in 573 strains after infection, except for 5 strains isolated in swine that caused sporadic small nodules (up to four tubercles) of poppy-seed size to occur in the liver of infected poultry. Among 573 nonvirulent strains, IS901 was present in 24 strains. A highly significant (P, 0.01) relationship was found between the presence of IS901 and virulence for poultry after intramuscular infection (generalized tuberculosis).
Serotype MAC strains.Only 15 of 131 serotype MAC strains were found to contain IS901 (Table 1). These were all serotype 1, 2, or 3. Another five isolates of serotype 1, 2, or 3, however, were IS901 negative. Only 5 of 15 IS901-positive strains of serotypes 1, 2, and 3 induced generalized tuberculosis in poultry. The remaining 111 strains of other serotypes (4 to 28) were nonvirulent for poultry.
MAC strains from birds.Of 56 MAC strains isolated from birds, 49 were IS901-positive strains which were all fully virulent for poultry in challenge experimentation (Table 2). The other seven, IS901-negative strains, isolated from unchanged tissues of parenchymatous organs or intestinal contents of the examined birds, were nonvirulent.
MAC strains from animals.A total of 261 MAC strains were isolated from six animal species (Table 2). A total of 107 were IS901-positive strains, 105 of which (98.1%) were virulent for poultry. No IS901 was detected in the DNA of 154 animal strains, and of these strains, only 1 was virulent for poultry. Five strains induced sporadic changes in the liver of infected poultry in the form of small nodules. Of 236 animal strains originating from swine, 90 were IS901-positive strains, 88 of which (97.8%) were virulent for poultry. Of 146 IS901-negative strains, only 5 induced sporadic nodules in the liver, the remaining strains being nonvirulent. A relationship between IS901 and virulence for poultry was found in all other strains isolated from wild pigs, cattle, sheep, goats, and a horse, except for one of the strains (IS901 negative) isolated from a goat, which did induce generalized tuberculosis in poultry.
Environmental MAC strains.Of 138 environmental strains, 5 contained IS901 and were virulent for poultry (Table 2); 3 of these strains were isolated from drinking water, 1 was from stable scrapings, and 1 was from the sawdust of swine bedding. All five IS901-positive strains from the environment were isolated from three herds of swine in which infection by these strains was diagnosed. None of the other 133 strains contained IS901, nor were they virulent for poultry.
MAC strains from humans.All 152 MAC strains from humans were nonvirulent for poultry, including 12 IS901-positive strains (Table 3). Of 98 strains isolated from HIV-negative patients, IS901 was detected in 8 strains, which were all isolated from sputum. From 54 HIV-positive patients, four IS901-positive strains were detected (three strains from sputum and one strain from blood). MAC strains from the Czech Republic and Slovakia were divided into two groups based on the date of their isolation. Group A comprised 29 strains isolated from 1968 to 1978; IS901 was detected in 6 strains (5 strains originated from Slovakia). Group B contained 20 strains isolated from 1996 to 1997; no IS901 was detected.
Statistical evaluation of the results.Monitoring of strain virulence for poultry together with the presence of IS901 in the strains revealed that the presence of IS901 in strains virulent for poultry was of statistically higher significance than that in nonvirulent strains (P, 0.01). In addition, the frequency of strains with IS901 in individual hosts was analyzed. Statistically highly significant differences (P, 0.01) were found for the presence of IS901 in birds and animals, animals and humans, birds and environment, animals and environment, and birds and humans. No statistical differences were found for the presence of IS901 in strains isolated from the environment and from humans. Study of the relationship between IS901-positive isolation and HIV infections did not reveal any significant difference for the occurrences of IS901-positive strains in HIV-positive and HIV-negative patients.
DISCUSSION
The virulence of MAC strains for poultry in this study was tested by intramuscular administration (31). The use of intraperitoneal infection in parallel with intramuscular infection is advisable for efficient assessment of virulence (C. Thoen [U.S. Department of Agriculture, Ames, Iowa], personal communication). Intravenous infection is a further alternative; however, inoculation with strains of serotypes 8 and 9 resulted in mycobacteremia and the death of the experimental poultry without prior formation of tubercles in parenchymatous organs (infection type Yersin). Other studies with the oral or intramuscular route of infection showed the strains of these serotypes to be nonvirulent for poultry (31). In addition, oral infection is impractical, as it prolongs the experiment for several months (16).
Statistically high significance (P, 0.01) was found between virulence and the presence of IS901 in all freshly isolated strains from birds, animals, and the environment. However, no such association was seen in reference serotype and human MAC strains stored for a long time. Strains of serotypes 1, 2, and 3 incorporated IS901-negative nonvirulent strains, IS901-positive nonvirulent strains, and IS901-positive virulent strains (Table 1). Some of these strains were isolated several decades ago, and the loss of virulence has occurred gradually due to mechanisms so far unknown. Anz and Meissner assigned the loss of virulence for poultry of some MAC strains of serotypes 1, 2, and 3 to their adaptation to laboratory conditions (1). Loss of virulence in mycobacterial strains during laboratory passage is well known. The first BCG vaccine was obtained in this way (14).
The virulence of MAC strains for poultry has been associated with various factors, including their growth type (rough and smooth colony types) and the presence of plasmids (13, 22, 26). Our results indicate that although the virulence of MAC strains is multifactorial, IS901 plays a significant role. All of these virulence factors (including IS901) could be lost during a long-term laboratory passage and storage.
All MAC strains isolated from humans were avirulent for poultry. However, 12 of these were IS901 positive. Humans as well as animals are not typical hosts for IS901-positive strains virulent for poultry. This fact is in agreement with previous serotyping studies suggesting that the passage of MAC in humans leads to a loss of virulence for poultry (2). A reduction of virulence was also observed during storage of these strains and subculturing at 37°C, a temperature lower than a bird's body temperature (42°C). This difference may be another factor in virulence attenuation (40). Consistent virulence in MAC strains isolated from animals may be due to the short duration of the infection associated with a relatively short life expectancy of infected animals. In contrast, MAC infection in humans may be subclinical and therefore involve a long-term process (up to several years), thereby exposing the strains for a longer time to “unfavorable conditions.” When field strains of MAC are examined, it is necessary to take into account a relatively broad spectrum of hosts. Our results are consistent with previous studies (7, 20, 36) which have indicated that IS901-positive strains are most frequently isolated from birds (Tables 1 and 2), possibly because birds are the natural hosts of avian tuberculosis.
Our study revealed that IS901 was present in only 3.6% of the 138 environmental strains examined (Table 2). Pigs are in contact with IS901-negative MAC strains through drinking water, sawdust, and other substances (17). Our findings agree with those of other authors (4, 7, 31, 35), that is, IS901-negative MAC strains not virulent for poultry prevail in the environment. Nevertheless, certain environmental conditions can serve as a reservoir for IS901-positive MAC strains.
The frequency of IS901-positive isolation in human and environmental strains was relatively low, suggesting similar infection sources. Immunocompetent humans, although in contact with MAC strains in the environment, are not usually infected under normal conditions (25). It is interesting that 6 of 12 strains originated from the former Czechoslovakia from 1968 to 1978 (Table 3), when avian tuberculosis was frequent in birds, pigs, and other farm animals. Results published during this period showed a large proportion (up to 40%) of MAC strains of serotypes 1 and 2 isolated from humans and subsequently shown to be virulent for poultry (2).
Gene probes (Accu-Probe) have been used for the rapid identification ofM. avium subsp. avium and M. intracellulare. However, the results do not correspond to the virulence of the strains for poultry, as M. avium subsp.avium includes the IS901-positive serotypes 1 to 3 but also serotypes 4 to 6, 8 to 11, and 21, which do not contain IS901 and are not virulent for poultry (37, 38, 45, 48). In the Czech Republic and Slovenia, serotypes 4, 8, and 9 (nonvirulent for poultry) are the most commonly isolated from swine and the environment (29, 30). In veterinary laboratories, strain virulence for poultry can be readily and rapidly assessed by the PCR method for the detection of IS901, thereby circumventing the need for biological experiments on poultry (20). Biological experiments could be used for virulence assessment of strains which are stored in collections for long periods. Therefore, a rapid diagnostic method using PCR would be useful for reducing the spread of virulent MAC strains among other animals in infected herds and for minimizing financial losses.
ACKNOWLEDGMENTS
We are grateful to those who are mentioned in Tables 1, 2, and 3for providing some of the strains for this study and to Z. Rozsypalova and M. Fisakova for technical assistance. We thank Tim Bull (St. George's Hospital, Medical School, University of London, London, United Kingdom) for critical reading of the manuscript.
This work was supported by the National Agency for Agricultural Research, Ministry of Agriculture, Czech Republic (grant EP 7172/97).
FOOTNOTES
- Received 7 July 1999.
- Returned for modification 14 September 1999.
- Accepted 10 November 1999.
- Copyright © 2000 American Society for Microbiology
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