Elizabeth J. B. Manning

Molecular Epidemiology of Mycobacterium avium subsp. paratuberculosis Isolates Recovered from Wild Animal Species

ABSTRACT Mycobacterial isolates were obtained by radiometric culture from 33 different species of captive or free-ranging animals (n = 106) and environmental sources (n = 3) from six geographic zones within the United States. The identities of all 109 isolates were confirmed by using mycobactin J dependence and characterization of five well-defined molecular markers, including two integration loci of IS900 (loci L1 and L9), one Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis)-specific sequence (locus 251), and one M. avium subsp. avium-specific marker (IS1245), as well as hsp65 and IS1311 restriction endonuclease analyses. Seventy-six acid-fast isolates were identified as M. paratuberculosis, 15 were identified as belonging to the M. avium-M. intracellulare complex (but not M. paratuberculosis), and the remaining 18 were identified as mycobacteria outside the M. avium-M. intracellulare complex. Fingerprinting by multiplex PCR for IS900 integration loci clustered 67 of the 76 M. paratuberculosis strains into a single clade (designated clade A18) and had a Simpsons diversity index (D) of 0.53. In contrast, sequence-based characterization of a recently identified M. paratuberculosis short sequence repeat (SSR) region enabled the differentiation of the M. paratuberculosis isolates in clade A18 into seven distinct alleles (D = 0.75). The analysis revealed eight subtypes among the 33 species of animals, suggesting the interspecies transmission of specific strains. Taken together, the results of our analyses demonstrate that SSR analysis enables the genetic characterization of M. paratuberculosis isolates from different host species and provide evidence for the host specificity of some M. paratuberculosis strains as well as sharing of strains between wild and domesticated animal species.

Mycobacterium avium subsp. paratuberculosis Strains Isolated from Crohn's Disease Patients and Animal Species Exhibit Similar Polymorphic Locus Patterns

ABSTRACT Analysis of short sequence repeats of Mycobacterium avium subsp. paratuberculosis isolated from Crohns disease patients identified two alleles, both of which clustered with strains derived from animals with Johnes disease. Identification of a limited number of genotypes among human strains implies the existence of human disease-associated genotypes and strain sharing with animals.

Isolation of Mycobacterium avium subsp. paratuberculosis from free-ranging birds and mammals on livestock premises.

ABSTRACT Surveys for Mycobacterium avium subsp. paratuberculosis infection in free-ranging mammals and birds were conducted on nine dairy and beef cattle farms in Wisconsin and Georgia. Specimens were collected from 774 animals representing 25 mammalian and 22 avian species. Specimens of ileum, liver, intestinal lymph nodes, and feces were harvested from the larger mammals; a liver specimen and the gastrointestinal tract were harvested from birds and small mammals. Cultures were performed by using radiometric culture and acid-fast isolates were identified by 16S/IS900/IS1311 PCR and mycobactin dependency characteristics. M. avium subsp. paratuberculosis was cultured from tissues and feces from 39 samples from 30 animals representing nine mammalian and three avian species. The prevalence of infected wild animals by premises ranged from 2.7 to 8.3% in Wisconsin and from 0 to 6.0% in Georgia. Shedding was documented in seven (0.9%) animals: three raccoons, two armadillos, one opossum, and one feral cat. The use of two highly polymorphic short sequence repeat loci for analysis of 29 of the 39 strains identified 10 alleles. One allelic pattern broadly shared in domestic ruminants (“7,5”) appeared in approximately one-third of the wildlife M. avium subsp. paratuberculosis isolates studied. Given the few cases of shedding by free-ranging animals compared to the volume of contaminated manure produced by infected domestic ruminant livestock, contamination of the farm environment by infected wildlife was negligible. Wildlife may, however, have epidemiological significance for farms where M. avium subsp. paratuberculosis recently has been eliminated or on farms free of M. avium subsp. paratuberculosis but located in the geographic vicinity of farms with infected livestock.

Invasion and Persistence of Mycobacterium avium subsp. paratuberculosis during Early Stages of Johne's Disease in Calves

ABSTRACT Infection with Mycobacterium avium subsp. paratuberculosis causes Johnes disease in cattle and is a serious problem for the dairy industry worldwide. Development of models to mimic aspects of Johnes disease remains an elusive goal because of the chronic nature of the disease. In this report, we describe a surgical approach employed to characterize the very early stages of infection of calves with M. avium subsp. paratuberculosis. To our surprise, strains of M. avium subsp. paratuberculosis were able to traverse the intestinal tissues within 1 h of infection in order to colonize distant organs, such as the liver and lymph nodes. Both the ileum and the mesenteric lymph nodes were persistently infected for months following intestinal deposition of M. avium subsp. paratuberculosis despite a lack of fecal shedding of mycobacteria. During the first 9 months of infection, humoral immune responses were not detected. Nonetheless, using flow cytometric analysis, we detected a significant change in the cells participating in the inflammatory responses of infected calves compared to cells in a control animal. Additionally, the levels of cytokines detected in both the ileum and the lymph nodes indicated that there were TH1-type-associated cellular responses but not TH2-type-associated humoral responses. Finally, surgical inoculation of a wild-type strain and a mutant M. avium subsp. paratuberculosis strain (with an inactivated gcpE gene) demonstrated the ability of the model which we developed to differentiate between the wild-type strain and a mutant strain of M. avium subsp. paratuberculosis deficient in tissue colonization and invasion. Overall, novel insights into the early stages of Johnes disease were obtained, and a practical model of mycobacterial invasiveness was developed. A similar approach can be used for other enteric bacteria.

Successful control of Johne's disease in nine dairy herds: Results of a six-year field trial

The objective was to evaluate if a standardized Johnes disease control program significantly reduced the prevalence of cattle infected with Mycobacterium avium ssp. paratuberculosis in dairy herds with a moderate to high initial infection prevalence of >or=10% ELISA-positive adult cattle. Nine Wisconsin dairy herds of diverse sizes and management styles completed the 6-yr study. The control program involved changes to heifer rearing practices in combination with a routine testing program. For heifers, the program specifically required 1) segregated maternity pens for ELISA-positive and ELISA-negative cattle; 2) removal of calves from the maternity pen in <2h; 3) use of colostrum only from individual ELISA-negative cows (no colostrum pooling); 4) hygienic collection of colostrum; 5) feeding of pasteurized milk as milk replacer or on-farm pasteurized milk until weaning; and 6) minimizing contact with manure from the adult cattle until weaning. The testing program was designed to detect the most infectious cattle by using a commercial ELISA once on every adult during each lactation. Producers were required to cull cows with strong-positive ELISA results before the next calving and to label cows with low- to medium-level ELISA results and manage them to limit infection transmission. Outcomes were measured by comparing the apparent prevalence based on ELISA or fecal culture in the whole herd and in first-lactation cohorts at 2 time points: before implementation of the control program and at the end of the trial. The combined results from the 9 herds showed a significant reduction in ELISA-positive cows, from 11.6% at the start of the trial to 5.6% at conclusion of the trial. The apparent prevalence decline among first-lactation cows was greater and was evident by ELISA (10.4 vs. 3.0%) and by fecal culture (17.0 vs. 9.5%). Although variations among farms were observed, the collective results demonstrated that bovine paratuberculosis can be controlled in dairy herds through effective heifer husbandry practices in combination with diagnostic testing to identify, for culling or management, cows most likely infectious.

Rapid and Reliable Method for Quantification of Mycobacterium paratuberculosis by Use of the BACTEC MGIT 960 System

ABSTRACT A simple method for the enumeration of viable Mycobacterium paratuberculosis cells was developed and evaluated using the MGIT 960 culture system. For each of 12 M. paratuberculosis strains isolated from either cattle or humans, single-cell suspensions of M. paratuberculosis cells were adjusted to an optical density at 600 nm of 1.00 (107.6 to 108.2 cells/ml), and serial dilutions were prepared. Standard curves were established by relating the MGIT time-to-detection data to the log10 CFU for these suspensions using standard plate counting and BACTEC 460 results as reference methods. Universal and strain-specific standard quantification curves were generated. A one-phase exponential decay equation best fit the universal standard curve and strain-specific curves (R2 of 0.96 and >0.99, respectively). Two subgroups within the universal curves were distinguished: one for laboratory-adapted strains and the other for recently isolated low-passage bovine strains. The predictive errors for log10 estimations using the universal standard curve, each subgroups standard curve, and strain-specific curves were ±0.87, ±0.45, and ±0.31 log10 units, respectively. CFU estimations by all three standard curves were highly reproducible, regardless of the M. paratuberculosis strain or inoculum volume. In comparison with the previously described BACTEC 460 M. paratuberculosis counting method, quantification with MGIT 960 was less expensive, more rapid, more accurate, and more sensitive (<10 CFU). This MGIT counting method has broad applications for studies requiring the quantification of viable M. paratuberculosis cells, such as drug susceptibility testing or environmental survival studies.

Efficient Differentiation of Mycobacterium avium Complex Species and Subspecies by Use of Five-Target Multiplex PCR

ABSTRACT Infections caused by the Mycobacterium avium complex (MAC) are on the rise in both human and veterinary medicine. A means of effectively discriminating among closely related yet pathogenetically diverse members of the MAC would enable better diagnosis and treatment as well as further our understanding of the epidemiology of these pathogens. In this study, a five-target multiplex PCR designed to discriminate MAC organisms isolated from liquid culture media was developed. This MAC multiplex was designed to amplify a 16S rRNA gene target common to all Mycobacterium species, a chromosomal target called DT1 that is unique to M. avium subsp. avium serotypes 2 and 3, to M. avium subsp. silvaticum, and to M. intracellulare, and three insertion sequences, IS900, IS901, and IS1311. The pattern of amplification results allowed determination of whether isolates were mycobacteria, whether they were members of the MAC, and whether they belonged to one of three major MAC subspecies, M. avium subsp. paratuberculosis, M. avium subsp. avium, and M. avium subsp. hominissuis. Analytical sensitivity was 10 fg of M. avium subsp. paratuberculosis genomic DNA, 5 to 10 fg of M. avium subsp. avium genomic DNA, and 2 to 5 fg of DNA from other mycobacterial species. Identification accuracy of the MAC multiplex was evaluated by testing 53 bacterial reference strains consisting of 28 different mycobacterial species and 12 nonmycobacterial species. Identification accuracy in a clinical setting was evaluated for 223 clinical MAC isolates independently identified by other methods. Isolate identification agreement between the MAC multiplex and these comparison assays was 100%. The novel MAC multiplex is a rapid, reliable, and simple assay for discrimination of MAC species and subspecies in liquid culture media.

Paratuberculosis in key deer (Odocoileus virginianus clavium).

Paratuberculosis was diagnosed in an endangered Key deer (Odocoileus virginianus clavium) in November 1996. Between 10 April 1997 and 28 September 2000, the Key deer population was monitored for infection with Mycobacterium avium subsp. paratuberculosis by necropsy of available carcasses (n = 170), fecal cultures, and serology. One additional clinically affected Key deer was discovered in July 1998, and M. avium subsp. paratuberculosis was cultured from the feces of one live, asymptomatic deer. The results of this study provided sufficient evidence to consider the Key deer herd infected with M. avium subsp. paratuberculosis at very low prevalence.

Culture Phenotypes of Genomically and Geographically Diverse Mycobacterium avium subsp. paratuberculosis Isolates from Different Hosts

ABSTRACT Mycobacterium avium subsp. paratuberculosis causes paratuberculosis (Johnes disease) in ruminants in most countries. Historical data suggest substantial differences in culturability of M. avium subsp. paratuberculosis isolates from small ruminants and cattle; however, a systematic comparison of culture media and isolates from different countries and hosts has not been undertaken. Here, 35 field isolates from the United States, Spain, Northern Ireland, and Australia were propagated in Bactec 12B medium and Middlebrook 7H10 agar, genomically characterized, and subcultured to Lowenstein-Jensen (LJ), Herrolds egg yolk (HEY), modified Middlebrook 7H10, Middlebrook 7H11, and Watson-Reid (WR) agars, all with and without mycobactin J and some with sodium pyruvate. Fourteen genotypes of M. avium subsp. paratuberculosis were represented as determined by BstEII IS900 and IS1311 restriction fragment length polymorphism analysis. There was no correlation between genotype and overall culturability, although most S strains tended to grow poorly on HEY agar. Pyruvate was inhibitory to some isolates. All strains grew on modified Middlebrook 7H10 agar but more slowly and less prolifically on LJ agar. Mycobactin J was required for growth on all media except 7H11 agar, but growth was improved by the addition of mycobactin J to 7H11 agar. WR agar supported the growth of few isolates. The differences in growth of M. avium subsp. paratuberculosis that have historically been reported in diverse settings have been strongly influenced by the type of culture medium used. When an optimal culture medium, such as modified Middlebrook 7H10 agar, is used, very little difference between the growth phenotypes of diverse strains of M. avium subsp. paratuberculosis was observed. This optimal medium is recommended to remove bias in the isolation and cultivation of M. avium subsp. paratuberculosis.

TESTING FOR MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS INFECTION IN ASYMPTOMATIC FREE-RANGING TULE ELK FROM AN INFECTED HERD

Forty-five adult tule elk (Cervus elaphus nannodes) in good physical condition were translocated from a population located at Point Reyes National Seashore, Marin County (California, USA), to a holding pen 6 mo prior to release in an unfenced region of the park. Because infection with Mycobacterium avium subsp. paratuberculosis (Mptb) had been reported in the source population, the translocated elk underwent extensive ante-mortem testing using three Johnes disease assays: enzyme linked immunosorbent assay (ELISA); agar gel immunodiffusion assay (AGID), and fecal culture. Isolation of Mptb was made from fecal samples in six of 45 elk (13%). All AGID results were negative while ELISA results for 18 elk (40%) were considered elevated. Elevated ELISA results or Mptb isolation from fecal samples were obtained for 22 of 45 elk (49%); these elk were euthanized and necropsied. Mycobacterium avium subsp. paratuberculosis was isolated from tissue in 10 of 22 euthanized elk (45%); of these 10 cases of con-firmed infection, eight had elevated ELISA results (80%) and four were fecal culture positive (40%). One of 10 cases had histopathologic lesions consistent with Mptb infection. Mycobacterium avium subsp. paratuberculosis was also isolated from tissue from one of eight fetuses sampled. The number of tule elk found to be infected was unexpected, both because of the continued overall health of the source herd and the normal clinical status of all study animals.