Objective—To compare sensitivity of several methods
of bacteriologic culture of pooled bovine fecal
samples for detection of Mycobacterium paratuberculosis
and evaluate homogeneity in number of
M paratuberculosisin pooled fecal samples.
Sample Population—Feces from 10 dairy cows that
shed M paratuberculosis at various concentrations
and 1 dairy cow known to be free of infection with
Procedure—5 fecal pooling methods, 2 culture methods,
and 2 pool sizes were evaluated. Each pooled
sample contained 1 infected sample and 4 or 9 uninfected
Results—Sensitivity of detection of M paratuberculosis was
greater with smaller pool size (5 vs 10 samples/
pool). Detection sensitivity was also associated
with concentration of bacteria in the infected sample.
Results indicated that, compared with concurrent
bacterial culture of individual infected samples, 37 to
44% of pooled samples with low bacterial concentrations
yielded positive culture results and 94% of
pooled samples with high bacterial concentrations
yielded positive results.
Conclusions and Clinical Relevance—Bacteriologic
culture of pooled fecal samples may provide a valid
and cost-effective method of detecting M paratuberculosis
infection in cattle herds. (Am J Vet Res
Objective—To evaluate the in vitro susceptibility of various field isolates of Mycobacterium avium subsp paratuberculosis (MAP) to gallium nitrate.
Sample—10 isolates of MAP, including 4 isolated from cattle, 2 isolated from bison, 1 isolated from an alpaca, and 3 isolated from humans.
Procedures—The in vitro susceptibility to gallium nitrate was tested by use of broth culture with detection of MAP growth by means of a nonradiometric automated detection method. For each MAP isolate, a series of 7 dilutions of gallium nitrate (concentrations ranging from 200 to 1,000μM) were tested. Gallium nitrate was considered to have caused 90% and 99% inhibition of the MAP growth when the time to detection for culture of the MAP stock solution and a specific concentration of gallium nitrate was delayed and was similar to that obtained for culture of the MAP stock solution (without the addition of gallium nitrate) diluted 1:10 and 1:100, respectively.
Results—Gallium nitrate inhibited MAP growth in all 10 isolates. The susceptibility to gallium nitrate was variable among isolates, and all isolates of MAP were inhibited in a dose-dependent manner. Overall, the concentration that resulted in 90% inhibition ranged from < 200μM for the most susceptible isolates to 743μM for the least susceptible isolates.
Conclusions and Clinical Relevance—Gallium nitrate had activity against all 10 isolates of MAP tested in vitro and could potentially be used as a prophylactic agent to aid in the control of MAP infections during the neonatal period.
Objective—To evaluate sensitivity and specificity of a
new ELISA for antibodies against Mycobacterium
avium subsp paratuberculosis.
Design—Cross-sectional observational survey.
Sample Population—Serum samples from 590 cattle
that were infected with M avium subsp paratuberculosis
and 723 cattle that were not infected.
Procedure—Serum samples were tested by use of
an ELISA for antibodies against M avium subsp
Results—Sensitivity of the test varied from 15.4 to
88.1%, depending on the clinical stage and bacterial
shedding status of the cattle.
Conclusions and Clinical Relevance—Results
obtained with use of the new ELISA agreed favorably
with those of a previous ELISA. Practitioners must be
aware of variability in the sensitivity of the test, which
depends on the clinical and shedding status of the
cattle, because this may affect interpretation of test
results. (J Am Vet Med Assoc 2001;218:1163–1166)
Objective—To evaluate sensitivity of microbial culture
of pooled fecal samples for detection of
Mycobacterium avium subsp paratuberculosis (MAP)
in large dairy herds and assess the use of the method
for estimation of MAP prevalence.
Animals—1,740 lactating cows from 29 dairy herds
Procedure—Serum from each cow was tested by
use of a commercial ELISA kit. Individual fecal samples
were cultured and used to create pooled fecal
samples (10 randomly selected fecal samples/pool; 6
pooled samples/herd). Sensitivity of MAP detection
was compared between Herrold's egg yolk (HEY) agar
and a new liquid culture method. Bayesian methods
were used to estimate true prevalence of MAP-infected
cows and herd sensitivity.
Results—Estimated sensitivity for pooled fecal
samples among all herds was 0.69 (25 culture-positive
pools/36 pools that were MAP positive).
Sensitivity increased as the number of culture-positive
samples in a pool increased. The HEY agar
method detected more infected cows than the liquid
culture method but had lower sensitivity for
pooled fecal samples. Prevalence of MAP-infected
cows was estimated to be 4% (95% probability
interval, 2% to 6%) on the basis of culture of
pooled fecal samples. Herd-level sensitivity estimate
ranged from 90% to 100% and was dependent
on prevalence in the population and the sensitivity
for culture of pooled fecal samples.
Conclusions and Clinical Relevance—Use of pooled
fecal samples from 10 cows was a cost-effective tool
for herd screening and may provide a good estimate
of the percentage of MAP-infected cows in dairy
herds with a low prevalence of MAP. (Am J Vet Res
Objective—To evaluate the effect of vaccination of calves with a killed Mycobacterium avium subsp paratuberculosis (MAP) vaccine on colonization of tissues following oral MAP exposure.
Animals—12 healthy Holstein calves.
Procedures—At 14 days after birth, calves received the MAP vaccine (1.0 mL, SC) or saline (0.9% NaCl) solution (1.0 mL, SC [control treatment]). Each calf received 1.2 × 109 CFUs of live MAP orally 21 and 22 days after vaccination. Prior to vaccination and at subsequent intervals, a blood sample was collected for ELISA detection of antibodies against MAP and for whole blood, antigen-specific, interferon (IFN)-γ–release assay. Nine weeks after MAP challenge, calves were euthanized and various tissue samples were collected for mycobacterial culture. Interferon-γ production in prescapular lymph node cells was measured following in vitro stimulation with MAP antigens.
Results—Calves were seronegative for anti-MAP antibodies at all times. Compared with the findings in control calves, antigen-specific IFN-γ production in circulating lymphocytes and prescapular lymph node cells from vaccinated calves was significantly higher. Culture of tissues from vaccinated calves yielded significantly fewer CFUs of MAP (2,417 CFUs/g), compared with tissues from control calves (15,709 CFUs/g). Furthermore, significantly fewer tissue samples from vaccinated calves yielded MAP in culture (21.8 tissues/calf), compared with findings in control calves (27.6 tissues/calf).
Conclusions and Clinical Relevance—Inoculation of calves with a killed MAP vaccine was associated with reduced colonization of intestinal tissues following experimental exposure to MAP. Use of the vaccine could potentially reduce transmission of MAP to calves in infected herds.
Objective—To evaluate sensitivities at the herd level
of test strategies used in the Voluntary Johne's
Disease Herd Status Program (VJDHSP) and alternative
test strategies for detecting dairy cattle herds
infected with Mycobacterium paratuberculosis.
Design—Nonrandom cross-sectional study.
Sample Population—64 dairy herds from
Pennsylvania, Minnesota, Colorado, Ohio, and
Wisconsin. Fifty-six herds had at least 1 cow shedding
M paratuberculosis in feces; the other 8 herds
were free from paratuberculosis.
Procedure—For all adult cows in each herd, serum
samples were tested for antibodies to M paratuberculosis with
an ELISA, and fecal samples were submitted
for bacterial culture for M paratuberculosis. Sensitivities
at the herd level (probability of detecting infected herd)
of various testing strategies were then evaluated.
Results—Sensitivity at the herd level of the testing
strategy used in level 1 of the VJDHSP (use of the
ELISA to test samples from 30 cows followed by confirmatory
bacterial culture of feces from cows with
positive ELISA result) ranged from 33 to 84% for
infected herds, depending on percentage of cows in
the herd with positive bacterial culture results. If follow-
up bacterial culture was not used to confirm positive
ELISA results, sensitivity ranged from 70 to
93%, but probability of identifying uninfected herds
as infected was 89%.
Conclusions and Clinical Relevance—Results suggest
that the testing strategy used in the VJDHSP will
fail to identify as infected most dairy herds with a low
prevalence of paratuberculosis. A higher percentage
of infected herds was detected if follow-up bacterial
culture was not used, but this test strategy was associated
with a high probability of misclassifying uninfected
herds. (J Am Vet Med Assoc 2002;220: 1053–1057)