PROJ
TYPE:
ANIMAL HEALTH PROJ STATUS: NEW
START: 01 JUN 2005 TERM: 31
May 2008 FY: 2005
INVESTIGATOR: Habtemariam, T.; Yehualaeshet,
T.
PERFORMING INSTITUTION: Microbiology,
SUBJECT: Comparison and validation of IS900 and
putative sequence (Mptb52.16) as a diagnostic tool for Mycobacterium avium
subsp. paratuberculosis.
NON-TECHNICAL SUMMARY: Prolonged incubation
period of Mycobacterium avium subsp. paratuberculosis and
individual variability in subclinical and clinical disease expression makes the
diagnosis of the disease challenging. The goal of this study is to develop
a more rapid and sensitive DNA-based (conventional or real-time PCR-based
diagnostic test) to detect M. avium subsp. paratuberculosis in
milk. The specificity and susceptibility of real-time PCR based on IS900
and putative sequence as target will be compared.
OBJECTIVES: General:
Paratuberculosis (Johne's disease) is a chronic granulomatous enteric disease of
mainly ruminants caused by Mycobacterium avium subsp.
paratuberculosis. A prolonged incubation period and great
individual variability in subclinical and clinical disease expression are
characteristic of paratuberculosis. Excretion of the organism may occur
for prolonged periods (1 to 2.5 years) before the onset of clinical disease
(1,2). Thus, early diagnosis of infected animals is important to avoid
spreading of the disease, because control is dependent on detection and culling
of infected animals as early as possible. Until recently, only a few M.
paratuberculosis-specific genes and antigens/epitopes have been identified,
of which IS900 has been mainly documented followed by some putative sequences
(3). Several PCR assays based on IS900 have been developed for the
detection of M. avium subsp. paratuberculosis (4,5). PCR has
been used to improve the identification of microorganisms, especially where
traditional microbiological detection methods have serious limitations. The goal
of this study is to develop a more rapid and sensitive DNA-based (conventional
or real-time PCR-based diagnostic test) to detect M. avium subsp.
paratuberculosis in milk. We will compare the specificity and
susceptibility of real-time PCR based on IS900 and putative sequence as target.
Specific objectives: Objective 1: To optimize the conventional PCR and
real-time PCR-based diagnostic test of M. avium subsp.
paratuberculosis based on IS900- insertion segment and putative sequence,
Mptb52.16. Cost analysis, including material and labor, indicated an
approximately 50% higher cost per test for bacteriological culture than for the
conventional and real-time PCR tests. The real-time PCR (RT-PCR) method is
relatively simple and robust, and results can be achieved within 24 h. The
additional advantage of DNA-based assays is the detection of non-culturable
organisms where it is critical to detect all sources of infection.
Objective 2: Determine the diagnostic limit of DNA-based diagnostic
methods in spiked milk. Milk and fecal samples are the most common
specimens for paratuberculosis diagnosis. Milk is considered to be a
difficult specimen for the detection of organisms by PCR, due to the presence of
large amounts of fat and calcium ions. Detectable quantities of M.
avium subsp. paratuberculosis have previously been reported in the
milk of both clinically infected and subclinically infected cattle with Johne's
disease. Therefore, we selected to spike the milk as the first line of
sample to determine the specificity and limitation of RT-PCR as a diagnostic
tool. Objective 3: Compare and validate the real-time PCR using IS900-
insertion and putative sequence (Mptb52.16). The main goal of this
objective is to examine whether any of the fragments have better diagnostic
potential to diagnose M. avium subsp. paratuberculosis.
Purified DNA will be analyzed by conventional and real-time PCR targeting
IS900 and putative sequence to detect M. avium subsp. paratuberculosis
in milk.
APPROACH: The DNA-based RT- PCR will target IS900 segment and
putative sequence. The details of the sequence (AF503873, Mptb52.16), for
the primers and probe synthesis will be accessed from the GenBank.
Bacterial strains, which will be used in this investigation, are: Strain
*Source or reference Origin M. avium, 15769 ATCC chicken M.
paratuberculosis, 19698 ATCC (type strain) bovine M. paratuberculosis
43015 ATCC human M. scrofulaceum 19275 ATCC M. vaccae 15483 ATCC
cow's milk *Sources of bacterial strains are from ATCC, American Type Culture
Collection (Rockville, MD, USA); The strains of M. avium, scrofulaceum
and M. vaccae will be used as a negative control. Bacterial
suspension preparation and bacterial growth: After repeated treatment of cells
in the Branson ultrasonic cleaner to disintegrate of clumps an approximate
measure of cell number will be determined by using the optical density at 550 nm
or hematocytometer. Extraction of mycobacterial DNA from culture and
spiked milk: Due to the complex, difficult matrix involved, several
strategies will be considered for the DNA extraction. To overcome this
problem, organisms will be concentrated to a pellet by centrifugation and
resuspended in prewarmed in PBS. DNA will be isolated from growing
cultures and purified by using freeze thawing, enzymatic degradation, lysis,
phenol-chloroform treatment, and isopropanol precipitation. Samples of
milk (10 ml) will be spiked with 10 to 106 M. avium subsp.
paratuberculosis organisms, and the organisms will be concentrated by
centrifugation and resuspended in 2 ml of PBS. Real-time PCR assay.
RT-PCR will be conducted in a Smartcycler. To evaluate the
usefulness of the technique we will perform a parallel study of culture
detection and Ziehl-Neelsen stain of M. avium subsp.
paratuberculosis. Quantitation: The unknown targets will be
evaluated by using DNA samples containing various amounts of known template
(range, 101 to 106 copies). Personnel and Institutional capacity: The
experience of the first PI in molecular diagnosis of Mycobacterium bovis,
as a PhD research work, will be potential to solve the unformatted difficulties.
PROJECT CONTACT:
Email: teyehual@tuskegee.edu
URL: http://compepid.tuskegee.edu/
PROJ
TYPE:
USDA INHOUSE PROJ STATUS:
TERMINATED
START: 01 DEC 1998 TERM:
12 JUN 2002 FY: 2002
INVESTIGATOR: Stanker L
H; Ravva S V; Duffy B K
PERFORMING INSTITUTION:
OBJECTIVES: 1)
Develop methods to identify Salmonella, Campylobacter, and other
bacterial and parasitic pathogens, as they are found in animal waste, and define
their survival characteristics; 2) Develop methods to handle and treat animal
manure during production in order to preclude transmission of these pathogens to
land and/or crops for human food.
APPROACH: A combination
of microbiological and biochemical approaches will be employed to characterize
the biology of pathogens in animal waste. Pathogens in manure will be
studied by confocal and scanning electron microscopy to determine whether
biofilms are present. New methods for detection and identification of
pathogens will be developed. Pathogen reporter strains will be produced.
The survival of pathogens in manure will be studied by microbiology and
molecular biology approaches. New compounds and/or treatment methods for
minimizing pathogens will be developed and tested. This research should be
conducted in collaboration with the U.S. Salinity Laboratory in
5. Duffy, B., Sarreal,
C., Stevenson, R., Ravva, S., Stanker, L. Regrowth of pathogenic bacteria in
compost teas and risk of transmission to strawberry plants.
Proceedings of 2002 International Symposium: Composting and Compost
Utilization. 2002.
1. Ravva, S.V., Duffy, B.K., Stanker, L.H., Mandrell, R.E. Foodborne
pathogens in dairy environments. 30th United States-Japan
Cooperative Program in Natural Resources (UJNR) Protein Resources Panel Meeting.
October 15-19, 2001 in Tsukuba,
2. Stanker, L.H.,
PROJ
TYPE:
USDA INHOUSE PROJ STATUS: NEW
START: 13 JUN 2002 TERM:
31 MAY 2006 FY: 2005
INVESTIGATOR: Mcgarvey J
A; Ravva S V; Hernlem B J
PERFORMING INSTITUTION:
OBJECTIVES: 1. To
identify factors influencing survival and re-growth of human pathogenic bacteria
in a dairy system, including transfer of pathogens from manure and manure
by-products to crops. 2. To identify bacterial populations in the
manure environment. 3. To apply this information for designing pathogen
control strategies that will be both effective and
dairy-friendly.
APPROACH: Our overall
approach will be to examine pathogenic and non-pathogenic bacteria presence, and
their re-growth and transfer to agricultural crops during commercial dairy
operations, test these hypotheses in laboratory/greenhouse studies using
pathogens with bioluminescent and or antibiotic resistant markers and then
evaluate laboratory based pathogen control technologies in field trials at
commercial dairies. Rapid, sensitive pathogen detection methodologies that
are currently available will be adopted for evaluating the life cycle of
pathogen transmission on a typical
PROGRESS: 2003/10 TO
2004/09 1. What major problem or issue is being resolved and how are you
resolving it (summarize project aims and objectives)? How serious is the
problem? What does it matter? Americans suffer from 76 million cases
of food poisoning each year, causing 5,000 deaths, and costing $7 billion in
lost productivity. Most food poisoning is caused by bacteria such as those
present at high levels in manure. These include strains of E. coli,
Salmonella, and Campylobacter. Livestock also suffers from
transfer of bacterial pathogens, including Mycobacterium avium
paratuberculosis, responsible for Johne's Disease in cattle. Farm
animals and their manure are often present at high density near croplands.
Microbes from manure may be transferred to crops by aerosol, especially in
dust-, wind- and fog-prone areas. In addition, manure is often used as
fertilizer on crops for animal feed and fodder, as well as crops intended for
human consumption. This can allow direct contact between manure and
food/feed, as well as causing widespread contamination of soil. Over 80
million tons of animal manure are produced each year in the
1. Duffy, B., Sarreal,
C., Stevenson, R., Ravva, S., Stanker. L. Regrowth of pathogenic bacteria in
compost teas and risk of transmission to strawberry plants. F.C. Michel,
Jr., R.F. Rynk and H.A.J. Hoitink,editors. The JG Press, Inc.,
3. Mcgarvey, J.A.,
Bermudez, L.E. 2004. Differential gene expression in mononuclear phagocytes
infected with pathogenic and non-pathogenic mycobacteria. [No Journal name
given] 136(6):490-500.
PROJ
TYPE:
USDA COOPERATIVE AGREEMENT PROJ STATUS: NEW
START: 07 MAR 2005 TERM:
31 DEC 2006 FY: 2005
INVESTIGATOR: Mcgarvey J
A; Mitloehner F; Zhang R
PERFORMING INSTITUTION:
OBJECTIVES: The goal of
this project is to compare dairy waste treatment methodologies, especially
varying levels of aerobic and anaerobic treatment, as well as wastewater lagoon
circulation, aeration and covering for the reduction of pathogens and volatile
compound emission. These studies will provide greater understanding of how
pathogens persist or are destroyed under various conditions and simultaneously
elucidate the type of volatile organic compounds that are emitted during
treatment. The studies will involve lab scale aerobic and anaerobic
digesters, as well as on site dairy waste lagoon experiments conducted at an
operating dairy, and thus real world conditions. The results of these
studies will have impact on the way diary waste is treated for both human health
and environmental considerations.
APPROACH: Dairy waste
treatment methodologies are of concern because of human illnesses caused by
pathogens within manure and because of volatile organic gas emissions which
cause poor air quality in and around dairy operations. Pathogens such as
E. coli O157:H7. Salmonella spp., Campylobacter spp. etc.
have caused outbreaks associated with improper diary waste treatment. In
addition, improper treatment of diary waste can emit volatile chemicals into the
atmosphere, including volatile fatty acids (VFAs), volatile organic compounds
(VOCs), and ammonia. Furthermore, improper application of diary waste to
crop fields can result in the accumulation of sodium chloride, phosphate,
nitrate and nitrite. These chemicals not only impact crop production, but
also leach into ground water making it unsuitable for human or animal
consumption. To better understand how dairy waste can be treated to
eliminate pathogens, and decrease air emissions and unwanted groundwater
leaching, we will initiate bench scale aerobic and anaerobic digester
experiments. We will feed raw diary waste (manure and urine) into aerobic
and anaerobic reactors under different parameters such as temperature, amount of
dissolved oxygen, redox potential, retention time, etc. and analyze the starting
material and effluent for microbial content. We will also collect gasses
emitted from the waste treatment systems and analyze their chemical composition
using GC/MS. We will conduct experiments with manure that has been spiked
with known amounts of pathogenic bacteria including Salmonella, E.
coli O157:H7, Mycobacterium avium subsp. paratuberculosis,
etc. to determine the effect these methodologies have on pathogen growth and
survival. Lastly we will conduct experiments on a working 800-cow dairy
farm. This farm is uniquely suited for experiments on waste treatment
methodologies because it has two waste lagoons that receive waste from the same
source, but are run independently of each other, thus providing an experimental
treatment lagoon and a control lagoon. We will treat one lagoon under
conditions found to be ideal for the reduction of pathogen and volatile organic
compound emission, and chemical changes as they evolve. This work will not
only provide proof of principal in lab experiments but will show how these
concepts correlate to real world conditions on a working dairy. Documents SCA
with UC-Davis.
PROJ
TYPE:
ANIMAL HEALTH PROJ STATUS: NEW
START: 01 OCT 2001 TERM:
30 SEP 2006 FY: 2005
INVESTIGATOR: Cullor, J.
S.
PERFORMING INSTITUTION:
Population Health & Reproduction,
NON-TECHNICAL SUMMARY:
Detection of M. paratuberculosis in young cattle continues to be
difficult using current tests. In an effort to improve the sensitivity and
specificity of PCR for Johne's disease screen assays, we plan to investigate a
micro-colony PCR detection method.
OBJECTIVES: Our
objective is to assess and optimize the PCR confirmation of M.
paratuberculosis microcolonies. The primary hypothesis to be tested is
that PCR analysis can be performed on micocolonies of M. paratuberculosis
appearing between 7-14 days after spirally plated on Middlebrook agar plates.
The use of PCR on micro-colonies will circumvent the problems of
sensitivity and PCR inhibition when this technique is used directly on broth
cultures, manure or tissues.
APPROACH: Bovine manure
from clinically normal cows will first be spiked with up to 1x10(8) CFU/g of
M. paratuberculosis and decontaminated via the standard protocol used by
the California Animal Health Food Safety Laboratory (CAHFS,
No publications reported
this period.
PROJECT
CONTACT:
Name: Cullor, J. S. Phone:
559-688-1731. Fax: 559-686-4231
Email: mailto:jscullor@ucdavis.edu
URL: http://www.vmtrc.ucdavis.edu/dfsl/dfsl.html
PROJ
TYPE:
NRI COMPETITIVE GRANT PROJ STATUS: TERMINATED
CONTRACT/GRANT/AGREEMENT
NO: 98-35204-6535 PROPOSAL NO: 9802523
START: 01 DEC 1998 TERM:
30 NOV 2002 FY: 2003 GRANT YR: 1998
GRANT AMT:
$149,906
INVESTIGATOR: Gardner,
OBJECTIVES: 1. To
determine the effects of conditional dependence of test sensitivity when
combinations of tests are used for individual and herd diagnosis. 2. To
estimate sensitivity and specificity of two or more tests in combination when
there are more than two populations and no Gold standard.
APPROACH: To achieve
Objective #1, we will use existing data on tests for bovine paratuberculosis,
swine brucellosis, swine toxoplasmosis and beta-lactan residues in low milk.
As a model for other diseases, we will determine the optimal combination
of tests to determine an individual's and herd's paratuberculosis status,
incorporating decision and cost-benefit analyses, where ever appropriate.
For Objective #2, we will evaluate data sets that include results for
multiple tests for paratuberculosis and other animal diseases and use the
estimation-maximization algorithm and Gibbs sampler to perform
calculations.
PROGRESS: 1998/12 TO
2002/11. We have implemented a Bayesian method for estimation of
sensitivity and specificity when there are 2 correlated (dependent) tests and 2
populations with different prevalences and the true disease status is unknown
(i.e. there is no gold standard). All uncertainty in the accuracy of tests
and disease prevalence is modeled through the use of probability distributions.
Our previous work identified that a positive correlation between test
results lead to an overestimation of the accuracy of both tests using
traditional statistical methods that assume that the tests are conditionally
independent (uncorrelated). Correlation typically occurs when the 2 tests
measure similar biologic processes e.g. serum antibody responses. The
model has been applied to the evaluation of 2 serologic tests for toxoplasmosis
in pigs and we have shown that the model yields unbiased estimates of test
sensitivity and specificity. We have developed a web-based interface using
an HTML form for implementation of methods to estimate test sensitivity and
specificity in the absence of a gold-standard. The programs can
incorporate data obtained from several populations, results of multiple tests
and can account for data from a reference population in which the true status
(infected or not infected) or each individual is known exactly. Two
estimation methods are used and both assume test independence conditional on the
infection status of individuals and constant test accuracy in each population.
The program is available at www.epi.ucdavis.edu/diagnostictests/
IMPACT: 1998/12 TO
2002/11 The development of a web-based program will allow for more
widespread implementation of these methods by scientists involved in the
assessment of accuracy of animal diagnostic tests.
PUBLICATIONS: 1998/12 TO
2002/11
1.
4. Pouillot R, Gerbier
G,
PROJ
TYPE:
NRI COMPETITIVE GRANT PROJ STATUS: TERMINATED
CONTRACT/GRANT/AGREEMENT
NO: 2001-35204-10874 PROPOSAL NO: 2001-02494
START: 15 OCT 2001 TERM:
31 OCT 2003 FY: 2005 GRANT YR: 2002
GRANT AMT:
$205,000
INVESTIGATOR: Gardner,
NON-TECHNICAL SUMMARY:
Trade in animals and animal products is in part dependent on the validity of
assurances that exporting herds and countries provide about the infection status
of their animals. Our objective is to develop Bayesian models that can be
used to make improved inferences about freedom from infection for a single herd
or multiple herds in a country/region. We will use beta distributions to
model uncertainty and variability in test sensitivity and specificity, and
prevalence. For the single-herd model, we will use bovine paratuberculosis
as our example because of current interest in the Voluntary Johne's Disease Herd
Status Program (VJDHSP) and the difficulties inherent in differentiating low
prevalence from non-infected herds. For the multiple-herd model, we will
use survey data on porcine reproductive and respiratory syndrome, infectious
bovine rhinotracheitis and bluetongue to validate our model. There are
often extensive surveillance data that can complement negative survey findings,
but there are no standardized methods for incorporating these data into
assessment of freedom from infection. Our final objective is to develop
Bayesian methods for incorporation of non-survey data and adjustment for
time-dependent changes in the quality of survey and surveillance data. We
will use data on bovine spongiform encephalopathy, brucellosis and tuberculosis
for these assessments. The methods developed will be adaptable to most
infectious livestock diseases, including those of interest to commodity groups
such as the VJDHSP and to those that are federally regulated such as
brucellosis.
OBJECTIVES: To develop
Bayesian methods for: (1) Certification of the status of a single herd with
respect to prevalence and freedom from infection (incorporating the prior
probability of infection and uncertainly and variability in test sensitivity and
specificity). (2) Certification of status of a group of herds with respect
to within-herd prevalence, proportion of infected herds and freedom from
infection (incorporating the prior probability of infection, uncertainty and
variability in test sensitivity and specificity, and the possible clustering of
positive test results at the herd level). (3) Incorporation of non-survey
data and adjustment for time-dependent changes in the quality of survey and
surveillance data.
APPROACH: We will use
beta distributions to model uncertainty and variability in test sensitivity and
specificity, and prevalence. Gibbs sampling will be used to combine prior
distributions and herd-level test results into a posterior prevalence
distribution. For the single-herd model, we will use bovine
paratuberculosis as our example because of current interest in the Voluntary
Johne's Disease Herd Status Program (VJDHSP) and the difficulties inherent in
differentiating low prevalence from non-infected herds. For the
multiple-herd model, we will use survey data on porcine reproductive and
respiratory syndrome, infectious bovine rhinotracheitis and bluetongue to
validate our model. PROGRESS: 2001/10 TO 2003/10. Trade in animals
and animal products is in part dependent on the validity of assurances that
exporting herds and countries provide about the infection status of their
animals. We developed Bayesian hierarchical models for determining
infection status and infection prevalence given testing of all or a sample of
animals from a single herd with an imperfect test. Expert prior
information about herd infection status, diagnostic test accuracy are
incorporated into the model. Posterior versus prior probabilities are
presented as a curve, summarizing the probability of infection over a range of
possible prior probability values. The model has been applied to serologic
data for paratuberculosis in dairy herds. For the multiple herd setting,
we created a model that allows for 3 levels of inference: probability that a
country (region) is free of infection, the proportion of infected herds in an
infected country, and the within-herd prevalence. The model uses test
results from animals sampled in a two-stage cluster sample of herds. The
model was validated with simulated data and applied to surveys of
1. Suess EA,
3. Hanson TE, Johnson
WO,
6. Johnson WO, Su CL,
9. Branscum AJ,
Name: Gardner,
Email: iagardner@ucdavis.edu
PROJ
TYPE:
ANIMAL HEALTH PROJ STATUS: NEW
START: 15 APR 2005 TERM:
14 APR 2009 FY: 2005
INVESTIGATOR: Gardner,
OBJECTIVES: 1. To
quantify and evaluate the role of the environmental bio-burden of
Mycobacterium avium subsp. paratuberculosis (Map) in the
transmission of Johne's disease on dairy farms. 2. To evaluate optimal
testing strategies for detection of Map in dairy herds.
APPROACH: Objective 1,
numbers of Map bacteria in multiple environmental locations in dairy herds will
be quantified using Herrold's egg yolk medium and the Trek ESP II system.
Cultures which yield values that are very high shedders will be quantified
further by serial dilution to estimate the most probable numbers of bacteria in
the samples. Objective 2, a stochastic simulation model will be modified
to evaluate various testing (including use of environmental samples) and
sampling methods for detection of Map in dairy herds.
PROGRESS: 2005/01 TO
2005/12. The specific objectives of the study are to 1) quantify
concentrations of Map, as measured by culture on Herrolds egg yolk medium (HEYM)
and in liquid culture (Trek ESP II), in environmental samples including recycled
lagoon water used for flushing cow alleyways, 2) collect environmental Map data
biannually with the goal of correlating changes in current values with changes
in cohort specific Map risk when females born in the herd at the time of
sampling are in the second or later lactation, and 3) quantify the Map colony
counts on HEYM tubes where results are recorded as too numerous to count (TNTC),
equivalent to approximately more than 70 colonies per tube. We started our
investigation in a dairy with about 2700 lactating cows (aggregated in 12
strings), 300 dry cows and about 150 pregnant heifers. Currently, fecal
culture prevalence and ELISA seroprevalence of cows (n=976) at dry-off are 9.4%
and 4.6%, respectively. Testing of fecal slurry samples in February 2005
collected from alleyways using a standardized protocol showed that 100% (12/12)
were positive on HEYM, including 25% (3/12) alleyways had at least one HEYM tube
that had Map colonies that were TNTC. Repeat testing of the herd in
October 2005 using the Trek ESP II system yielded similar findings. In
November 2005, we investigated logistical aspects of detection of TNTC cows in
this herd. All environmental samples were positive by quantitative
real-time (qrt) PCR and one sample was positive using the qualitative PCR
available through the Minnesota Diagnostic Laboratory. We are currently
doing follow-up testing of 3 strings identified as having different likelihoods
(high, moderate and low, respectively) of having cows that are shed Map.
Group-level serum ELISA results correlated well with qrtPCR data.
Samples from 17 ELISA positive cows within these strings were submitted
for HEYM culture and qrtPCR testing in December 2005.
IMPACT: 2005/01 TO
2005/12. Cows shedding large numbers of Map pose a tremendous risk for
transmitting the organism to other cattle on the farm, and they may also
contribute to passive (pass through) fecal shedding of Map by uninfected cows,
and thus false-positive fecal culture results. That some fecal culture
results (low and moderate shedders) might be false positives is a major paradigm
shift for management of bovine paratuberculosis and also has implications for
evaluation of serologic tests for Map because most investigators use fecal
culture as the gold standard.
PUBLICATIONS: 2005/01 TO
2005/12.
Berghaus RD, Farver TB, Anderson
RJ, Jaravata CC,
Name: Gardner,
Email: iagardner@ucdavis.edu
PROJ
TYPE:
NRI COMPETITIVE GRANT PROJ STATUS: EXTENDED
CONTRACT/GRANT/AGREEMENT
NO: 2003-35204-13374 PROPOSAL NO: 2003-02398
START: 01 AUG 2003 TERM:
31 JUL 2006 FY: 2005 GRANT YR: 2003
GRANT AMT:
$157,000
INVESTIGATOR: Gardner,
NON-TECHNICAL SUMMARY:
Correct classification of herd status for animal pathogens is an integral
component of disease control programs, risk-factor studies, health certification
programs, and risk analyses related to animal movements. Our long-term
research goal is to develop new methods to assess test accuracy for animal
diseases, to improve classification of herd disease status and apply these
methods to herd testing for bovine paratuberculosis. First, we will
develop methods that use quantitative test results in the overall assessment of
herd-level sensitivity and specificity. To achieve this objective we will
use ELISA values for paratuberculosis infected and non-infected cattle and
develop a generalized linear model that includes animal and herd-level risk
factors. Second, we will develop Bayesian methods for estimating the
herd-level sensitivity and specificity of testing systems for animal diseases
when there are two conditionally independent or dependent tests. We expect
that Bayesian methods will be superior to traditional frequentist methods for
assessing the accuracy of herd-level tests when there is no "gold standard" and
where tests are conditionally dependent. We will use paired ELISA and
fecal culture results from three paratuberculosis studies. We will assess
cut-off dependent and independent approaches for herd classification.
Methods developed in the proposed research will be applicable to many
infectious animal diseases where quantitative test results are available and
where herd-level interpretation of test results in the basis of classification
of disease risk.
OBJECTIVES: Develop and
apply methods that account for the quantitative nature of test results in the
overall assessment of herd-level sensitivity and specificity. Develop and
apply Bayesian methods for estimating the herd-level sensitivity and herd-level
specificity of testing systems for animal diseases when there is one test or two
conditionally independent or dependent tests. APPROACH: For objective 1, we will
develop a general linear mixed model that discriminates the quantitative test
scores from infected and non-infected animals. The model will allow for
the possibility of animal-level risk factor (covariate) information such as age,
lactation number, an indicator of purchased versus raised etc., and for
herd-level information such as average age, type of ownership etc. In
addition, we will extend this univariate model to a multivariate model that
allows for several diagnostic tests. The model will be applied to ELISA
and culture data for bovine paratuberculosis. Once this initial study is
completed using "gold standard" information, we will extend the problem to the
situation where the true status of individual animals is unknown. This
will be achieved with use of a "mixture" model that is based on weighted average
of the densities of test results corresponding to infected and non-infected
animals. For objective 2, we will develop and compare cutoff-based and
cutoff-independent methods of assessing herd-level sensitivity and specificity.
The latter method allocates a herd as positive if the posterior
probability that the herd is infected, given all observed data, is larger than a
specified value e.g. 95%. The methods will be applied to one test in a
single population, two tests in one population, and two tests in two or more
populations. We will also allow for the possibility of sequential and
simultaneous use of multiple tests. Our Bayesian approach will involve
Gibbs sampling, a Markov chain Monte Carlo simulation technique, that allows for
incorporation of existing information about test sensitivity and specificity,
and disease prevalence.
PROGRESS: 2005/01 TO
2005/12. Our long-term goal is to develop new methods to assess test
accuracy for animal diseases, to improve classification of herd disease status
and apply these methods to herd testing for bovine paratuberculosis. As
part of our first objective, we developed and applied Bayesian methods for
estimating the herd-level sensitivity and herd-level specificity of testing
systems for animal diseases when there is one test or two conditionally
independent or dependent tests. We considered 4 different sampling
schemes: a single test case and three sequential test cases. The
corresponding herd-level characteristics were calculated and compared with
different sample sizes, sampling schemes, animal-level sensitivity, specificity,
and cut-off values. Models were developed to incorporate animal or
herd-level risk factors and models with small herd size are also considered.
We compared posterior estimates of animal and herd-level characteristics
for these four sampling schemes with simulated data. Two examples, one for
bovine paratuberculosis and a second for Salmonella in pig herds, were
used to demonstrate application of the methods in field studies (Su et al.,
2006). As part of our second objective, we developed a Bayesian approach
to sample size calculations for cross-sectional studies designed to estimate
sensitivity and specificity of one or more diagnostic tests. Sample size
calculations can be made for common study designs such as one test in one
population, two conditionally independent or dependent tests in at least 2
populations, and three tests at least 2 populations. We determined a
sample size combination that yields high predictive probability, with respect to
the future study data, of accurate and precise estimates of sensitivity and
specificity. We also consider hypothesis testing for demonstrating the
superiority or equivalence of one diagnostic test relative to another. The
predictive probability can also be computed when the sample size combination is
fixed in advance, thereby providing a power-like measure for the future study.
The method is straightforward to implement using the S-Plus/R library
emBedBUGS together with WinBUGS (Branscum et al., 2006).
IMPACT: 2005/01 TO
2005/12. Correct classification of herd status for animal pathogens is an
integral component of disease control programs, risk-factor studies, health
certification programs, and risk analyses related to animal movements.
This classification is primarily dependent on the interpretation of
diagnostic tests results at the herd-level. Our approach is based on
Bayesian methods which allow incorporation of prior information and knowledge
about test performance into the current study. Our research findings allow
users greater flexibility in study planning and data analysis than are sometimes
available with traditional statistical approaches. Wherever possible, we
have written code in the shareware program WinBUGS so that it can be used
widely. Code for these problems is available to users on our website, www.epi.ucdavis.edu/diagnostictests/
PUBLICATIONS: 2005/01 TO
2005/12
1. Branscum AJ, Johnson
WO,
Name: Gardner,
Email: iagardner@ucdavis.edu
PROJ
TYPE:
ANIMAL HEALTH PROJ STATUS: TERMINATED
START: 01 OCT 2000 TERM:
30 SEP 2004 FY: 2003
INVESTIGATOR: Gardner,
NON-TECHNICAL SUMMARY:
If countries and regions are able to "certify" freedom from important animal
pathogens, trade opportunities may increase and product export costs may
decrease. To develop a Bayesian statistical approach (using Gibbs
sampling) to quantification of disease freedom. The output from the model
will be probability distributions that can be used to make inferences about the
proportion of diseased herds, within-herd prevalence, and the probability that a
country is free of disease. The research will be involve collaboration
with others in the
OBJECTIVES: 1. Develop a
Bayesian approach to certify disease freedom of a country/region that
incorporates uncertainty in probability estimates. 2. Compare frequentist
and Bayesian approaches to certify disease freedom using common data sets and to
compare sample size requirements for surveys with both
approaches.
APPROACH: 1. The
Bayesian approach will be implemented with the Gibbs sampler, an interactive
Markov-chain Monte Carlo method. The mathematical calculations will
incorporate the prior probability that a country is free of disease, the
uncertainty in sensitivity and specificity estimates and the possible clustering
of positive test results at a herd level. The output will be a
probabilistic estimate of disease freedom. 2. Frequentist and Bayesian
estimates will be compared with common published data sets on porcine
reproductive and respiratory syndrome and Newcastle Disease. The effect of
selected prior distributions for the Bayesian approach will be evaluated.
Sample sites used in frequentist calculations for surveys will be compared
with estimates that we will derive using Bayesian approaches.
PROGRESS: 2000/10 TO
2004/09 Quantitative methods to certify freedom from animal pathogens (no
change from what was submitted last year (see below): Quantitative approaches
are needed to allow scientifically-valid inferences about freedom of animals
from important pathogens that affect animal trade locally, regionally and
internationally. Freedom in the context of these inferences includes a
herd prevalence of a pathogen less than a threshold value (e.g. <0.2% of
infected herds). In a related project, we have developed Bayesian models to make
probabilistic inferences for multiple herds and for single herds. The
single herd model has both a binomial sampling version (relevant to a small
sample of the herd e.g. 10% of animals) and a hypergeometric version (relevant
to testing of the entire herd. In the model, expert prior information
about the infection status of the herd, diagnostic test accuracy (sensitivity
and specificity) and within-herd prevalence are used, when such data are
available. Post-test probabilities versus pre-test probabilities of
infection are presented in the novel form of a curve, summarizing the
probability of infection over a range of possible prior probability values.
The primary objective of this study is to collect field data to validate
the suitability of the Bayesian models under field conditions. The model
is currently being evaluated using serologic and fecal culture data for
Mycobacterium paratuberculosis infection in 29 herds in the Central
Valley of California. ELISA testing of 60 adult cows in each of the
selected herds has been completed. Results were interpreted as per
standard procedures at the California Animal Health and Food Safety Laboratory:
<0.2 = negative, 0.2 to 0.35 = suspicious, and >0.35 = positive. For
the 29 herds, the median number of positive or suspicious animals out of the 60
cows tested was 3 (range, 0 to 15). Five herds had no positive cows in 60
tested. We have completed follow-up ELISA testing of all adult cows in 2
of the herds with 0/60 positive on the initial screening and found 15/332 (4.5%)
and 16/1144 (1.4%) ELISA-positive/suspicious samples. Fecal samples are
currently being cultured from the 31 reactors to try and unequivocally establish
whether these 2 herds are infected.
IMPACT: 2000/10 TO
2004/09. Our initial findings indicated that screening of 60 lactation-2
or older cows with ELISA in large California dairy herds (median size, 700 cows)
provides a reasonable balance between cost of testing and failing to detect low
prevalences of M. paratuberculosis infection.
PUBLICATIONS: 2000/10 TO
2004/09.
1.
Name: Gardner,
Email: iagardner@ucdavis.edu
PROJ
TYPE:
ANIMAL HEALTH PROJ STATUS: NEW
START: 01 APR 2005 TERM:
31 MAY 2007 FY: 2005
INVESTIGATOR: Cullor, J.
S.
PERFORMING INSTITUTION:
Population Health and Reproduction.
NON-TECHNICAL SUMMARY:
In order to insure animal health, food safety and security, and supply needed
scientific information to develop better management practices and support and
enforce regulatory decisions, there is a need for a broad based detection system
to rapidly detect potential vectors of animal and foodborne diseases on the
dairy farm environment. This project seeks to develop a rapid and high
sample throughput detection system that can be applied to a wide variety of
on-farm sample types including milk, cattle feed and water for the control of
infectious agents.
OBJECTIVES: The overall
goal of this project is to further develop, optimize and characterize the
sensitivity, specificity and ease of performance of an automated DNA extraction
method coupled with PCR technology on a variety of on-farm sample types, for the
identification of infectious agents, or potential vectors of infectious
diseases. These sample types include 1.) mastitic milk, for the direct and
rapid detection of various bacterial pathogens including Mycoplama bovis
and streptococcus species 2.) cattle feed, for the detection of prohibited
ingredients including ruminant DNA to prevent the spread of Mad Cow disease and
3.) agricultural water for the presence of potential waterborne pathogens
including E. coli 0157 and Salmonella which can be shed by
asymptomatic cattle.
APPROACH: 1.) To
evaluate a automated DNA extraction protocol to be used on milk, cattle feed and
agriculture water for its ability to abrogate the effects of inhibitory agents
and rapidly process numerous samples. 2.) To further develop PCR primers
and florescent probes to detect and identify various waterborne infectious
agents including E. coli 0157 and Salmonella. 3.) To
optimize and characterize each DNA extraction /PCR assay for sample throughput,
lower limit of detection, sensitivity, specificity and ease of performance using
laboratory spiked samples.
PROGRESS: 2005/01 TO
2005/12. An automated DNA extraction system based on magnetic bead
technology has been purchased, set-up, and its use, including training, offered
to other research units within the
PUBLICATIONS: 2005/01 TO
2005/12.
No publications reported
this period
PROJECT
CONTACT:
Name: Cullor, J. S. Phone: 559-688-1731. Fax:
559-686-4231
Email: jscullor@ucdavis.edu
PROJ
TYPE:
ANIMAL HEALTH PROJ STATUS: NEW
START: 01 APR 2005 TERM:
31 MAY 2010 FY: 2005
INVESTIGATOR: Gardner,
NON-TECHNICAL SUMMARY:
There are many deficiencies in existing knowledge about the transmission and
detection of Map in cattle herds, including how to most effectively use
currently-available tests for diagnosis of Map infection. This project
will assess persistence of the Map on dairy farms and methods for quantification
of bioburden.
OBJECTIVES: 1. To
quantify and evaluate the role of the environmental bio-burden of
Mycobacterium avium subsp. paratuberculosis (Map) in the
transmission of Johne's disease on dairy farms. 2. To evaluate optimal
testing strategies for detection of Map in dairy herds.
APPROACH: For objective
1, numbers of Map bacteria in multiple environmental locations in dairy herds
will be quantified using Herrold's egg yolk medium and the Trek ESP II system.
Cultures which yield values that are very high shedders will be quantified
further by serial dilution to estimate the most probable numbers of bacteria in
the samples. For objective 2, a stochastic simulation model will be
modified to evaluate various testing (including use of environmental samples)
and sampling methods for detection of Map in dairy herds.
PROGRESS: 2005/01 TO
2005/12 The objectives of the study were to evaluate the effectiveness of
various testing methods for detection of Map-infected herds at the initial step
of herd classification, and 2) to determine the most cost-effective testing
strategies for detection of Map-infected herds for various herd demographic
factors. A stochastic simulation model was used to compare the
cost-effectiveness of 7 currently-available testing methods for detection of
Map-infection in
No publications reported
this period
PROJECT
CONTACT:
Name: Gardner,
Email: iagardner@ucdavis.edu
PROJ
TYPE:
STATE PROJ STATUS: TERMINATED
START: 27 SEP 2002 TERM: 26
SEP 2003
INVESTIGATOR: Gardner,
PERFORMING
INSTITUTION: Medicine and Epidemiology.
SUBJECT: Field evaluation of
fecal pool cultures for detection of Mycobacterium paratuberculosis in large
dairy herds.
NON-TECHNICAL SUMMARY: Johne's Disease is a chronic
infectious and wasting bacterial disease of ruminants caused by Mycobacterium
avium subspecies paratuberculosis (MAP) and occurs worldwide.
This project will apply new epidemiologic approaches to test evaluation
and prevalence estimation for Mycobacterium paratuberculosis in dairy
herds using field data.
APPROACH: 1. Determine the sensitivity of pooled fecal
culture by 2 methods (TREK and traditional HEY culture) relative to individual
fecal culture. 2. Estimate the prevalence of infected herds and
within-herd prevalence in
PROGRESS: 2002/09 TO 2003/09 We evaluated the sensitivity of
pooled fecal culture for detection of Mycobacterium avium subsp.
paratuberculosis (Map) in large dairy herds and assessed the utility of
the method for estimation of Map prevalence. We used a cross-sectional
study design that involved random sampling of 60 cows in each of 29
IMPACT: 2002/09 TO
2003/09. Use of fecal pools from 10 cows was a cost-effective tool for
herd screening and might provide a good estimate of the percentage of
Map-infected cows in low prevalence herds.
PUBLICATIONS: 2002/09 TO
2003/09
Saraya Tavornpanich, Ian A. Gardner, Randall J. Anderson, Sang Shin,
Robert H. Whitlock, Terry Fyock, John M. Adaska, Richard L. Walker, Sharon K.
Hietala. 2004. Evaluation of pooled fecal culture for detection of
Mycobacterium avium
PROJECT
CONTACT:
Name: Gardner,
Email: iagardner@ucdavis.edu
PROJ
TYPE:
STATE PROJ STATUS: TERMINATED
START: 01 JAN 2003 TERM: 31
DEC 2004
INVESTIGATOR: Adaska, J. M.
PERFORMING INSTITUTION:
CA Animal Health and Food Safety Laboratory System (CAHFS).
PROGRESS: 2003/01 TO
2004/12 The main objective of the project was to determine whether or not
the causal organism of Johne’s disease, Mycobacterium avium ssp.
paratuberculosis (MAP), was present and viable in pre- and
post-pasteurized samples of hospital milk being fed to calves on calf ranches.
We initially enrolled five calf ranches but one dropped out after the
initial sampling period leaving data from four for analysis. Three of the
calf ranches each collected milk from approximately forty dairies while the
fourth collected milk from 15 dairies. All four ranches reported that they
heated milk to approximately 165degreesF and had holding time of 2-3 minutes.
We were able to identify MAP by PCR in about 5% of pre-pasteurization
samples and from the same percentage of post-pasteurization samples. There
was no agreement between which samples were positive pre-pasteurization and
which were positive post-pasteurization. It needs to be kept in mind that
PCR identifies the presence of DNA and can be positive even in samples where no
viable or infectious organisms are present. By culture we have been able
to identify viable organisms in 2.5% of the total samples with an equal number
found in pre-pasteurization samples and post-pasteurization samples. The
results of this study indicate that hospital milk collected from several dairies
and fed to calves on calf ranches is a potential source of infection of calves
with the causal organism of Johne’s disease. We were able to isolate MAP
from post-pasteurization samples indicating that the pasteurization methods
currently in use on the calf ranches in this study (165 degrees F for 2-3
minutes) may not be adequate to kill MAP in hospital milk. Because the
pasteurization equipment was not evaluated or monitored during the course of the
study however it is possible that technical problems with the equipment
resulting in contamination of post-pasteurization samples or inadequate
temperature or time of pasteurization may have resulted in inadequate killing of
MAP. The significance and benefit to the dairy industry from this work is
that a major question, whether dilution, by combining hospital milk from several
dairies, would result in MAP being undetectable and presumably an insignificant
source of infection in milk fed to calf ranch calves. Further, the study
answered the question of whether or not on-farm pasteurization eliminates 100%
of the viable organisms in those same milk samples. 3. Conclusions:
Mycobacterium avium ssp paratuberculosis was detected by PCR from
approximately 5% of hospital milk fed to calves on four
PUBLICATIONS: 2003/01 TO
2004/12
No publications reported
this period
PROJECT
CONTACT:
Name: Adaska, J. M. Phone: 559-688-7543. Fax:
559-686-4231
Email: jmadaska@ucdavis.edu
PROJ
TYPE:
STATE PROJ STATUS: TERMINATED
START: 01 JAN 2002 TERM:
31 DEC 2002 FY: 2003
INVESTIGATOR: Cullor, J.
S.; Smith, W.; Kirk, J.
PERFORMING INSTITUTION:
Population Health & Reproduction.
NON-TECHNICAL SUMMARY:
The detection of M. paratuberculosis (Johne's disease) in young cattle
continues to be difficult using current tests. There is a lack of good
screening assays that are fast, inexpensive and sensitive enough to regularly
and frequently screen cattle. This project seeks to improve Johne's
disease screening assays by modifying the conventional media culturing method in
ways to shorten the time required for results, while maintaining low costs, ease
of performance and the sensitivity/specificity which is characteristic of
conventional culturing methods.
OBJECTIVES: Conventional
media culturing for M. paratuberculosis involves decontaminating the
sample with various agents and antibiotics followed by subculturing onto solid
agar slants which are incubated for many weeks to months before visible colonies
appear. This project examines the hypothesis that the culturing step can
be modified by the substitution of spiral plating on Middlebrook agar and the
addition of micro-colonial morphology screening to obtain primary culture
results more rapidly.
APPROACH: The first
objective is to assess the ability of the spiral plating micro-colony morphology
screening method (SP/MCM) to return results more rapidly than the conventional
culture method using 'spiked' manure samples. Once this is confirmed the
second objective is to assess the SP/MCM method on actual field samples from
herds with high incidence of Johne's disease.
PROGRESS: 2002/01 TO
2002/12. Previous results funded by other grants, suggested that a
modification in the way that manure samples were plated and screened could
provide a more rapid detection of Mycobacterium paratuberculosis, however
these results were obtained from manure samples containing very high numbers of
the organism. In order to compare the modified culture technique to
conventional culture methods, additional studies were necessary to optimize the
sensitivity (lower limit of detection) of the assay. Because M.
paratuberculosis has an extremely prolonged growth rate, clinical samples
are first "decontaminated" with agents that will suppress the growth of other
faster growing bacteria. However these agents can also suppress the growth
of mycobacteria to varying degrees. Several methods to decontaminate the
samples prior to plating are being investigated and optimized so that the very
low numbers of mycobacteria that may be present in the naturally infected animal
can be detected. Results to date suggest that a commonly used
decontamination agent, HPC (hexadecylpyridinium chloride) continues to suppress
the growth of mycobacteria after being plated and during incubation, thus
hindering the detection of very low numbers in the sample.
IMPACT: 2002/01 TO
2002/12. Johne's is a contagious bacterial disease of ruminants that has
significant animal health, economic, and potential human health consequences.
This grant provides additional funding to investigate and optimize the
sensitivity of a more rapid culture method for the detection of the M.
paratuberculosis from bovine manure.
PUBLICATIONS: 2002/01 TO
2002/12
No publications reported
this period
PROJECT
CONTACT:
Name: Cullor, J. S. Phone: 559-688-1732. Fax:
559-688-4231
Email: jscullor@ucdavis.edu
PROJ
TYPE:
STATE PROJ STATUS: TERMINATED
START: 01 JAN 2002 TERM:
31 DEC 2002 FY: 2003
INVESTIGATOR: Gardner,
OBJECTIVES: This project
will examine whether Bayesian methods can be successfully applied to analysis of
Johne's Disease herd-test results thereby better accounting for uncertainty in
test performance, prevalence estimates and the herd-specific pretest (prior)
probability of JD.
APPROACH: This will be
accomplished by developing and expanding an existing Bayesian model for
certification of Johne's disease. This will include addressing multiple
stages of disease, hypergenometric sampling and use of multiple
tests.
PROGRESS: 2002/01 TO
2002/12. We have developed a Bayesian computer model to allow improved
inferences about the infection status of dairy herds with Mycobacterium
paratuberaculosis, the causative agent of Johne's disease, based on herd
testing. The model links laboratory test results from a sample of cows or
the entire cow herd with test accuracy data and prior information from the herd
to provide updated inferences about the probability that the herd is infected
and within-herd prevalence. In the model, uncertainty in all model inputs
is modeled as a probability. The model currently is being validated with
serum enzyme-linked immunosorbent assay (ELISA) and fecal culture data from 29