Paratuberculosis is a chronic, debilitating intestinal infection of ruminants caused by MAP. The disease is associated with notable economic losses in the beef and dairy industries because of increased culling, decreased value of culled cattle, diagnostic and treatment costs, and decreases in production.1-5 Serologic tests are an important component of paratuberculosis management programs when applied as herd-screening tools. Specificity estimates for commercially available paratuberculosis ELISAs are varied; reported estimates range from 95.3% to 99.8% in uninfected dairy cattle.6-11 Results from herd screening performed by use of a commercially available ELISA in beef cattle in Texas have indicated that the proportions of false-positive results were greater than that expected on the basis of reported assay specificities.12 A potential cause for these false-positive test results in beef cattle is their exposure to Mycobacterium spp that may have antigenic similarity to MAP and hence induce production of serum antibodies that cross-react with antigens in conventional serologic tests. These Mycobacterium spp are often referred to as environmental or atypical mycobacteria.13 In analyses of isolates obtained from bacterial cultures of feces from cattle and camelids, Cousins et al14 identified sufficient genetic homology among MAP and environmental mycobacteria to cause false-positive reactions on PCR tests for paratuberculosis.
Exposure to environmental mycobacteria has been associated with cross-reactions during intradermal tuberculin testing in both cattle and humans, and delayed-type hypersensitivity reactions have been used to characterize the geographic distribution of environmental mycobacteria exposure among people in the United States.15-18 The US Navy has measured responses to both PPD tuberculin and antigen from an environmental mycobacterial isolate (PPD-B tuberculin). The results of that screening program have been used to examine the incidence of tuberculosis, the extent of exposure to environmental mycobacteria, the potential for interference with tuberculosis test results by these environmental mycobacteria, and the geographic distribution of the environmental mycobacteria exposures. The screening program revealed that the prevalence of exposure to environmental mycobacteria is greatest in the southern and southeastern United States, including parts of Texas from which herds with high proportions of false-positive results for paratuberculosis (determined via ELISA) have been identified.
In a previous prevalence survey12 in Texas, several herds were identified for which the results via ELISA and microbial culture of feces were discordant, suggesting a high proportion of false-positive results. A subsequent study19 was performed in which environmental mycobacteria were isolated from bacterial cultures of feces collected in these herds with greater frequency than they were from bacterial cultures of feces collected in herds with low seroprevalence for paratuberculosis. The purpose of the study reported here was to further evaluate the effect of exposure to environmental mycobacteria on results of 2 commercial ELISAs for paratuberculosis in cattle.
Materials and Methods
Nineteen weaned Angus crossbred heifers (mean weight, 162.3 kg [357.1 lb]) were obtained from a single commercial cow-calf operation in central Texas. All calves were free from paratuberculosis, confirmed by results of microbial culture of feces and assessment of serum via ELISA-Aa and ELISA-B.b Calves were housed in concrete pens in groups of 9 or 10 with no direct contact with other cattle or exposure to soil. They were fed coastal hay ad libitum, received a 14% crude protein commercial creep supplement (approx 4.5 kg/head/d [10 lb/head/d]), and had free access to salt and mineral blocks. All animal research procedures were reviewed and approved by the Texas A&M University Laboratory Animal Care Committee.
Mycobacterial isolates were obtained from microbial cultures of feces collected from cattle in herds with high proportions of apparent false-positive results determined on the basis of high seroprevalence for paratuberculosis but low prevalence of MAP isolated via bacterial culture of feces and no history of MAP-as-sociated clinical disease. Isolates were obtained by use of radiometric culture methods in liquid medium,c as previously described.20 Briefly, the medium was supplemented with mycobactin J, egg yolk suspension, and antimicrobials. Fecal samples were decontaminated with 1.0% hexadecylpyridinium chloride and concentrated via filtration. The resulting filter membrane was placed into radiometric culture medium and evaluated weekly for growth by use of an ionization detector.d For acid-fast organisms, a PCR assay for the IS900 gene insertion element was used to identify MAP. Mycobacterial isolates that were negative for IS900 were termed environmental mycobacteria and identified via high-performance liquid chromatography of extracted mycolic acids.21 From among the mycobacteria species isolated, Mycobacterium terrae, Mycobacterium celatum, Mycobacterium scrofulaceum, Mycobacterium intracellulare, and Mycobacterium avium subsp avium were selected for inoculation on the basis of their prevalence in the herds that had discordant diagnostic findings and on previous clinical experience.
One milliliter of each mycobacterial isolate and 1 mL of an MAP isolate (obtained in the same manner as the other isolates) were each inoculated into 10 mL of mycobacterial growth broth.e Each culture was incubated until it became turbid, at which time it was evaluated for the presence of acid-fast bacteria. Ten milliliters of each pure culture was then inoculated into 100 mL of the growth broth. When the culture was turbid, it was again examined for the presence of acid-fast organisms. All resultant pure cultures were homogenized and the organisms killed by repeated passage through a 26-gauge needle and addition of formalin to a final concentration of 50% by volume. The cultures were shaken for 24 hours and each washed 3 times with PBS solution. One milliliter of each killed mycobacterial isolate was inoculated into a separate bottle of liquid culture medium to confirm that no viable organisms were present. Approximate organism concentrations for each isolate were determined visually; the number of organisms present in 3 separate 0.2-mm2 areas was counted by each of 2 observers, and the mean count (cells/mL) was calculated. Final concentration was estimated by multiplying the mean number of organisms per milliliter by the total volume of the killed product (Appendix). Cultures were centrifuged and decanted, and killed organisms were stored as pellets at room temperature (approx 21°C [70°F]) pending inoculation.
Calves were randomly assigned to receive 1 of 7 treatments: inoculation SC with 1 of the 5 environmental mycobacterial isolates (3 calves/isolate), MAP (positive control inoculum; 2 calves), or mineral oil (negative control inoculum; 2 calves). At the time of inoculation, an isolate was suspended in 1.5 mL of sterile water and mixed with 2.0 mL of mineral oil. The mixture was emulsified through a double Luer-lock needle to ensure mixing and facilitate injection. Each calf assigned to receive inoculation with MAP or 1 of the 5 environmental mycobacterial isolates was administered 1.2 mL of killed mycobacterial suspension in mineral oil. For all treatments, calves were sedated by use of xylazine hydrochloridef (0.3 mg/kg [0.14 mg/lb], IM) and then inoculated SC in the brisket region. Blood samples were collected via coccygeal or jugular venipuncture immediately prior to inoculation (week 0) and every 14 days thereafter for 10 weeks (at weeks 2, 4, 6, 8, and 10). Serum was separated from each blood sample and submitted for serologic evaluation by use of ELISA-A and ELISA-B. Laboratory personnel were unaware of calf identity (ie, treatment received) when processing samples. Results of the serologic tests were interpreted by use of the manufacturer's recommended cutoff values provided in the diagnostic kit instructions. Results from ELISA-A were classified as positive if the S:P ratio was ≥ 0.25. The S:P ratio was calculated as the ratio of the negative control OD subtracted from the sample OD to the negative control OD subtracted from the positive control OD. Results from ELISA-B were classified as positive if subtraction of the cutoff value (determined by adding 0.1 to the mean OD of the duplicate negative controls) from the sample OD and multiplication by 100 yielded a value that was > 0. The S: P ratios were calculated for ELISA-B for purposes of comparison of ELISAs and statistical analyses, not for results classification. Bacterial culture of feces was performed at the conclusion of the study by use of liquid culture mediac as previously described.19 At the conclusion of the study, all calves were evaluated for tuberculosis according to the Uniform Methods and Rules for Bovine Tuberculosis Eradication.22 All calves were injected with 0.1 mL of bovine PPD tuberculing in the caudal fold at the base of the tail; the area was palpated by 2 experienced veterinarians (AJR and JBO) 72 hours later to evaluate the reaction. Calves that had a suspect reaction on the basis of findings during palpation of the injection site underwent a comparative cervical test administered by regulatory officials from the Texas Animal Health Commission.
Differences in serologic response over time and among isolates were evaluated by use of a mixed-effects model with time modeled as a random effect. Pairwise comparisons were used to detect significant differences in serologic responses between treatment groups with Bonferroni adjustment for multiple comparisons. A first-degree autoregressive covariance structure was used to adjust for correlation of serologic responses among time points. By use of a Fisher exact test, proportions of calves with suspect reactor classification on the basis of caudal fold tuberculosis test results that had positive serologic results at any time during the study period and those that remained seronegative were compared. For all statistical tests, a value of P ≤ 0.05 was considered significant. Statistical analyses were performed by use of commercially available statistical software.h
Results
The ELISA results for the study calves varied among the different challenge isolates and between the 2 ELISA kits (Figures 1 and 2). Calves injected with mineral oil remained seronegative (as determined by use of both ELISAs) for the duration of the experiment. Of the 2 calves inoculated with killed MAP, both had positive results via ELISA-A at 2, 4, 6, 8, and 10 weeks after inoculation; 1 had positive results via ELISA-B at 6, 8, and 10 weeks after inoculation. Of the 3 calves inoculated with M scrofulaceum, 2 (calves 1 and 3) had positive results via ELISA-A at week 8, and all 3 had positive results at week 10. When sera from these 3 calves were evaluated by use of ELISA-B, 1 (calf 2) had a positive result on week 6, and 1 (calf 1) had positive results at weeks 6, 8, and 10. Among the 3 calves inoculated with M intracellulare, 1 had positive results via ELISA-A at week 2, and all 3 had positive results at weeks 4, 6, 8, and 10. None of the calves inoculated with M intracellulare had positive results via ELISA-B during the study period. One of 3 calves inoculated with M terrae had a positive result via ELISA-A at week 10. None of the serum samples from calves inoculated with M terrae yielded positive results via ELISA-B during the study period. Of the 3 calves inoculated with M avium, 1 had positive results via ELISA-A at weeks 2, 4, 6, 8, and 10, and 1 calf had positive results via ELISA-A at weeks 4 and 6. The third calf in this group did not have positive results via ELISA-A during the study period. None of the serum samples from these calves yielded positive results via ELISA-B during the study period. None of the serum samples from calves inoculated with M celatum yielded positive results via either ELISA-A or ELISA-B during the study period.
Mean ± SEM S:P ratios for crossbred beef calves during a 10-week period after inoculation with environmental mycobacteria (3 calves/isolate), MAP (positive control inoculum; 2 calves), or mineral oil (negative control inoculum; 2 calves) derived by use of a commercially available diagnostic test kit for paratuberculosis (ELISA-A).a Standard errors displayed are cumulative SEs calculated on the basis of all observations for each isolate. Results for M celatum are superimposed over those of the mineral oil control.
Citation: Journal of the American Veterinary Medical Association 230, 6; 10.2460/javma.230.6.896
Mean ± SEM S:P ratios for crossbred beef calves during a 10-week period after inoculation with environmental mycobacteria (3 calves/isolate), MAP (positive control inoculum; 2 calves), or mineral oil (negative control inoculum; 2 calves) derived by use of a commercially available diagnostic test kit for paratuberculosis (ELISA-B).b Standard errors displayed are cumulative SEs calculated on the basis of all observations for each isolate.
Citation: Journal of the American Veterinary Medical Association 230, 6; 10.2460/javma.230.6.896
For both ELISA-A and ELISA-B, S:P control ratios varied significantly (P < 0.001) among isolates over time adjusted for the correlation associated with repeated observations on the same individual (Tables 1 and 2). Mean S:P ratios for calves inoculated with MAP were significantly greater than those for the mineral oil– treated (negative control) calves (P < 0.001), M scrofulaceum–treated calves (P = 0.025), M celatum–treated calves (P < 0.001), M terrae–treated calves (P < 0.001), and M avium–treated calves (P < 0.001) by use of ELISA-A. Similarly, mean S:P ratios for calves inoculated with M intracellulare were significantly greater than those for the mineral oil–treated (negative control) calves (P < 0.001), M scrofulaceum–treated calves (P < 0.001), M celatum–treated calves (P < 0.001), M terrae–treated calves (P < 0.001), and M avium–treated calves (P < 0.001) by use of ELISA-A. Mean S:P ratios for calves inoculated with M scrofulaceum were significantly (P = 0.049) greater than those for M celatum–treated calves by use of ELISA-A. Mean S:P ratios for calves inoculated with MAP were significantly greater than those for the mineral oil–treated (negative control) calves (P = 0.008), M celatum–treated calves (P = 0.008), and M terrae–treated calves (P = 0.046) by use of ELISA-B. Similarly, mean S:P ratios for calves inoculated with M scrofulaceum were significantly greater than those for the mineral oil–treated (negative control) calves (P = 0.041), M celatum–treated calves (P = 0.008), and M terrae–treated calves (P = 0.046) by use of ELISA-B. For all calves, results of microbial cultures of feces performed at the end of the study were negative for mycobacteria.
Pairwise comparisons (with Bonferroni adjustment) of estimated marginal mean serum S:P ratios derived from all sample times from inoculation to conclusion of the study between isolates detected with ELISA-A in 19 crossbred beef calves inoculated with MAP or environmental mycobacteria. Comparisons are adjusted for correlation associated with repeated observations on the same calf.
| Inoculum | Mean S:P ratio | 95% confidence interval |
|---|---|---|
| Mineral oil* | 0.000A,B | NA |
| MAP | 0.500C | 0.244–0.756 |
| Mycobacterium avium | 0.188A,B | 0.111–0.266 |
| Mycobacterium scrofulaceum | 0.228B | 0.098–0.358 |
| Mycobacterium celatum* | 0.000A | NA |
| Mycobacterium terrae† | 0.064A,B | 0.000–0.152 |
| Mycobacterium intracellulare | 0.558C | 0.368–0.749 |
S:P values are constant; no CI reported.
Lower limit of 95% confidence interval is bound by zero.
NA = Value was constant at all sampling times.
A,B,CValues with different superscripts are significantly (P < 0.05) different.
Pairwise comparisons (with Bonferroni adjustment) of estimated marginal mean serum S:P ratios derived from all sample times from inoculation to conclusion of the study between isolates detected with ELISA-B in 19 crossbred beef calves inoculated with MAP or environmental mycobacteria. Comparisons are adjusted for correlation associated with repeated observations on the same calf.
| Inoculum | Mean S:P ratio | 95% confidence interval |
|---|---|---|
| Mineral oil* | 0.000A,B | NA |
| MAP | 0.500C | 0.244–0.756 |
| M avium | 0.188A,B | 0.111–0.266 |
| M scrofulaceum | 0.228B | 0.098–0.358 |
| M celatum* | 0.000A | NA |
| M terrae† | 0.064A,B | 0.000–0.152 |
| M intracellulare | 0.558C | 0.368–0.749 |
See Table 1 for key.
Caudal fold tests for tuberculosis resulted in identification of 10 calves as suspect reactors. This subgroup included 1 of 3 calves inoculated with M terrae, 2 of 3 calves inoculated with M avium, 2 of 3 calves inoculated with M scrofulaceum, all 3 calves inoculated with M intracellulare, and both calves inoculated with MAP. All of these calves yielded negative results via comparative cervical testing. None of the calves inoculated with mineral oil or M celatum had suspect reactions to the caudal fold test. The proportion of suspect reactors was greater (P = 0.001) among calves that had positive results via ELISA-A at least once, compared with those that had negative results via ELISA-A for all samples. The proportion of suspect reactors was not significantly different among those classified as seropositive via ELISA-B at least once during the study period and those that were seronegative via ELISA-B for all samples (P = 0.620).
Discussion
The results of the present study have indicated that cattle may develop an immune response after experimental inoculation with environmental mycobacteria, which may generate false-positive results when the cattle are tested by use of an ELISA for paratuberculosis. This is consistent with the findings of Jørgensen,23 who evaluated the effects of oral exposure to various environmental mycobacteria, including M avium and M intracellulare, on serologic tests for paratuberculosis used approximately 25 years ago. Jørgensen also reported that this exposure resulted in suspect reactions to ID caudal fold injection of bovine PPD, which is similar to the results of our study and other investigations15-17 that assessed the effects of exposure to environmental mycobacteria on tuberculin sensitivity in cattle. The false-positive ELISA reactions attributable to environmental mycobacteria exposure appear to markedly alter the reliability of conventional interpretation of such test results in affected beef cattle herds. Additionally, the frequency of false-positive results differed between the 2 ELISAs. Overall, at least 1 false-positive reaction was detected in 9 of 15 calves during the study by use of ELISA-A, compared with 2 of 15 calves by use of ELISA-B. However, 1 of the calves inoculated with MAP remained seronegative throughout the study, according to results of ELISA-B. These results suggest that current specificity estimates within herds with potential exposure to environmental mycobacteria may be too high.
The source of exposures to these mycobacteria is unclear, but beef cattle are most likely exposed to saprophytic environmental mycobacteria while grazing. Alternatively, cattle may be exposed via aerosolization of mycobacteria associated with water sources.24 This mode of exposure has been reported to be the most common among humans with nontuberculous mycobacterial disease.25 It should be noted that the route of exposure in the experiments of this report was unnatural and that the organisms were formalin-killed; either factor might account for differences in immunologic responses, compared with those after natural exposure, although the responses in the present study were consistent with findings in the field. The level of exposure is likely affected by environmental factors and can be predicted geographically. Falkinham et al26 determined that the frequency of isolation of mycobacteria including M avium, M intracellulare, and M scrofulaceum from water sources was greater in the southeastern United States than in northeastern states. Furthermore, it is likely that there are differences in susceptibility and serologic responses among breeds and individuals after exposure to environmental mycobacteria. Exposure to mycobacteria could also alter the host response to subsequent exposure to pathogenic mycobacteria including MAP.24
Our findings are important because they suggest that false-positive ELISA results may overestimate the prevalence of paratuberculosis in beef cattle in some regions. To date, paratuberculosis prevalence estimates in beef cattle have largely been based on results of testing via ELISA and range from 0.4% to 8.6%.5,12,27,28 Many of these estimates are based on results from samples evaluated by use of ELISA-A.5,12,27 A substantial number of these estimates are from cattle in the southern United States, which appears to be associated with increased probability of exposure to environmental mycobacteria.26 Interestingly, in a national survey to determine the prevalence of serum antibodies against MAP in cattle, Texas had the lowest proportion of seropositive herds among those sampled.27
Although several significant differences in serologic reactions to different mycobacterial isolates were detected in the present study, it is important to note that these results were not derived from a randomly selected population and the serum S:P ratios were not normally distributed. Analysis of residuals did not identify severe departures from normality, but the limited sample size and relatively short duration of observation in our study likely limit the stability of the model used to test these hypotheses. Therefore, it would be inappropriate to use the results of the present study to predict how serologic responses to environmental mycobacteria exposure might differ at additional time points or with alternative means of exposure. The data indicated changes in S:P ratios over a fixed period following experimental exposure and highlight cumulative differences in S:P ratios and relative differences in rate of S:P ratio increase following exposure to different mycobacteria.
The results of the present study indicated that exposure of cattle to environmental mycobacteria, specifically M avium, M intracellulare, and M scrofulaceum, may cause them to have false-positive results via 2 ELISAs that are widely used for diagnosis of paratuberculosis. Furthermore, there may be differences in the frequency of such false-positive reactions between these 2 ELISAs. In our study, the association of the isolates with false-positive reactions did not result from random mycobacterial isolate selection, but instead purposeful selection of isolates collected from herds with high seropositivity in the absence of supporting clinical history or culture results. Results of the present study and previous work suggest that the potential for natural exposure of cattle to Mycobacterium spp is high and appears to be associated with geographic differences in risk of exposure. On the basis of our findings, we suggest that veterinarians consider their geographic location and previous experiences when deciding which screening test for paratuberculosis to use. Producers should be forewarned that false-positive results may occur and that final management decisions should be based on findings of confirmatory tests if initial ELISA results are not consistent with herd clinical history.
ABBREVIATIONS
| MAP | Mycobacterium avium subsp paratuberculosis |
| PPD | Purified protein derivative |
| S:P ratio | Test sample to positive control sample ratio |
| OD | Optical density |
HerdChek, IDEXX Laboratories Inc, Westbrook, Me.
ParaCheck, CSL/Biocor, Omaha, Neb.
BACTEC 12B medium, BD Diagnostic Systems, Franklin Lakes, NJ.
BACTEC 460, Johnston Laboratories, Towson, Md.
Middlebrook 7H9 broth, Becton-Dickinson, Franklin Lakes, NJ.
Vedco, St Joseph, Mo.
Tuberculin PPD Bovis, Intradermic, USDA, Animal and Plant Health Inspection Service, Riverdale, Md.
SPSS, version 11.5 for Windows, SPSS Inc, Chicago, Ill.
References
- 1
Chi J, VanLeeuwen JA, Weersink A, et al. Direct production losses and treatment costs from bovine viral diarrhoea virus, bovine leukosis virus, Mycobacterium avium subspecies paratuberculosis, and Neospora caninum. Prev Vet Med 2002;55:137–153.
- 2↑
Groenendaal H, Galligan DT. Economic consequences of control programs for paratuberculosis in midsize dairy farms in the United States. J Am Vet Med Assoc 2003;223:1757–1763.
- 3
Ott SL, Wells SJ, Wagner BA. Herd-level economic losses associated with Johne's disease on US dairy operations. Prev Vet Med 1999;40:179–192.
- 4
Pence M, Baldwin C, Black CC III. The seroprevalence of Johne's disease in Georgia beef and dairy cull cattle. J Vet Diagn Invest 2003;15:475–477.
- 5
Lombard JE, Garry FB, McCluskey BJ, et al. Risk of removal and effects on milk production associated with paratuberculosis status in dairy cows. J Am Vet Med Assoc 2005;227:1975–1981.
- 6
Reichel MP, Kittelberger R, Penrose ME, et al. Comparison of serological tests and faecal culture for the detection of Mycobacterium avium subsp paratuberculosis infection in cattle and analysis of the antigens involved. Vet Microbiol 1999;66:135–150.
- 7
Sockett DC, Conrad TA, Thomas CB, et al. Evaluation of four serological tests for bovine paratuberculosis. J Clin Microbiol 1992;30:1134–1139.
- 8
Collins MT, Sockett DC, Ridge S, et al. Evaluation of a commercial enzyme-linked immunosorbent assay for Johne's disease. J Clin Microbiol 1991;29:272–276.
- 9
Kalis CHJ, Barkema HW, Hesselink JW, et al. Evaluation of two absorbed enzyme-linked immunosorbent assays and a complement fixation test as replacements for fecal culture in the detection of cows shedding Mycobacterium avium subspecies paratuberculosis. J Vet Diagn Invest 2002;14:219–224.
- 10
Collins MT, Wells SJ, Petrini KR, et al. Evaluation of five antibody detection tests for diagnosis of bovine paratuberculosis. Clin Diagn Lab Immunol 2005;12:685–692.
- 11
McKenna SL, Keefe GP, Barkema HW, et al. Evaluation of three ELISAs for Mycobacterium avium subsp paratuberculosis using tissue and fecal culture as comparison standards. Vet Microbiol 2005;110:105–111.
- 12↑
Roussel AJ, Libal MC, Whitlock RL, et al. Prevalence of and risk factors for paratuberculosis in purebred beef cattle. J Am Vet Med Assoc 2005;226:773–778.
- 13↑
Hornick DB, Schlesinger LS. Mycobacterioses other than tuberculosis. In:Hausler WJ, Sussman M, ed.Topley and Wilson's microbiology and microbial infections. Vol 3. New York: Oxford University Press, 1998;419–442.
- 14↑
Cousins DV, Whittington R, Marsh I, et al. Mycobacteria distinct from Mycobacterium avium subsp. paratuberculosis isolated from the faeces of ruminants possess IS900-like sequences detectable IS900 polymerase chain reaction: implications for diagnosis. Mol Cell Probes 1999;13:431–442.
- 15
Corner LA, Pearson CW. Response of cattle to inoculation with atypical mycobacteria isolated from soil. Aust Vet J 1979;55:6–9.
- 16
Corner LA. The duration of the response of cattle to inoculation with atypical mycobacteria. Aust Vet J 1981;57:216–219.
- 17
Pearson CW, Corner LA, Lepper AW. Tuberculin sensitivity of cattle inoculated with atypical mycobacteria isolated from cattle, feral pigs and trough water. Aust Vet J 1977;53:67–71.
- 18
Edwards LB, Acquaviva FA, Livesay VT, et al. An atlas of sensitivity to tuberculin, PPD-B, and histoplasmin in the United States. Am Rev Respir Dis 1969;99:1–132.
- 19↑
Roussel AJ, Fosgate GT, Manning EJB, et al. Association of fecal shedding of mycobacteria with high ELISA-determined seroprevalence for paratuberculosis in beef herds. J Am Vet Med Assoc 2007;230:890–895.
- 20↑
Collins MT, Kenefick KB, Sockett DC, et al. Enhanced radiometric detection of Mycobacterium paratuberculosis by using filter-concentrated bovine fecal specimens. J Clin Microbiol 1990;28:2514–2519.
- 21↑
Butler WR, Guthertz LS. Mycolic acid analysis by high-performance liquid chromatography for identification of Mycobacterium species. Clin Microbiol Rev 2001;14:704–726.
- 22↑
Animal and Plant Health Inspection Service Web site. Bovine tuberculosis eradication: uniform methods and rules. Available at: www.aphis.usda.gov/vs/nahps/tb/tb-umr.pdf. Accessed Aug 1, 2006.
- 23↑
Jørgensen JB. Pathogenicity and immunogenicity of atypical mycobacteria for calves: a short summary. Rev Infect Dis 1981;3:979–980.
- 24↑
Shield MJ. The importance of immunologically effective contact with environmental mycobacteria. In:Ratledge C, Stanford J, ed.The biology of the mycobacteria: immunological and environmental aspects. Vol 2. New York: Academic Press, 1983;343–415.
- 25↑
Fry KL, Meissner PS, Falkinham JO. Epidemiology of infection by nontuberculous mycobacteria. VI. Identification and use of epidemiologic markers for studies of Mycobacterium avium, M intracellulare, and M scrofulaceum. Am Rev Respir Dis 1986;134:39–43.
- 26↑
Falkinham JO, Parker BC, Gruft H. Epidemiology of infection by nontuberculous mycobacteria. I. Geographic distribution in the eastern United States. Am Rev Respir Dis 1980;121:931–937.
- 27↑
Dargatz DA, Byrum BA, Hennager SG, et al. Prevalence of antibodies against Mycobacterium avium subsp paratuberculosis among beef cow-calf herds. J Am Vet Med Assoc 2001;219:497–501.
- 28
Braun RK, Buergelt CD, Littell RC, et al. Use of an enzymelinked immunosorbent assay to estimate prevalence of paratuberculosis in cattle of Florida. J Am Vet Med Assoc 1990;196:1251–1254.
Appendix
Results of manual counts of mycobacteria isolates to establish concentration of organisms prior to final centrifugation and inoculum preparation.
| Isolate | Cells/mL* | Volume (mL) | Estimated total No. of cells |
|---|---|---|---|
| MAP† | ND | 42 | ND |
| Mycobacterium intracellulare | 4.90 × 108 | 45 | 2.21 × 1010 |
| Mycobacterium celatum‡ | 3.53 × 106 | 40 | 1.41 × 108 |
| Mycobacterium avium | 1.77 × 108 | 43 | 7.61 × 109 |
| Mycobacterium terrae† | ND | 40 | ND |
| Mycobacterium scrofulaceum | 1.79 × 108 | 45 | 8.06 × 109 |
Cells were counted in 3 separate 0.2-mm2 areas by each of 2 observers and averaged to determine mean cells per milliliter.
Cells were too clumped for an accurate count.
Some large clumps and chains of cells were not counted.
ND = No data.
