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Journal of Clinical Microbiology, March 1998, p. 777-782, Vol. 36, No. 3
Instituto Nacional de Tecnología
Agropecuaria, EEA Rafaela, Rafaela, Santa Fe,
Argentina,1 and
Animal Disease
Research Unit,
Received 21 August 1997/Returned for modification 15 October
1997/Accepted 17 December 1997
A competitive enzyme-linked immunosorbent assay using recombinant
major surface protein 5 (rMSP5-cELISA) of Anaplasma
marginale was validated in a naturally infected cattle herd in an
area of eastern Oregon where A. marginale is endemic. The
true positive and negative A. marginale infection status of
235 randomly selected cattle was determined by using a nested PCR
(nPCR) coupled with msp5 sequence analysis and
hybridization. Judgment of the reliability of the nPCR and
hybridization for detection of persistent infections was based on three
observations. First, the nPCR was able to detect as few as 30 infected
erythrocytes per ml. Second, the nPCR was able to consistently detect
low levels of rickettsemia in seven carrier cattle experimentally
infected with A. marginale. Third, msp5
sequence analysis showed >95% identity among 30 nPCR amplicons from cattle naturally infected with field strains of A. marginale. The nPCR and hybridization identified 151 infected and
84 uninfected cattle among the 235 animals tested. With a cutoff point
of 28%, the rMSP5-cELISA showed a sensitivity of 96% and a
specificity of 95%. These results indicate that the rMSP5-cELISA can
sensitively and specifically detect cattle with naturally acquired
persistent A. marginale infections and suggest that it is
an excellent assay for epidemiological studies, eradication programs,
and regulation of international cattle movement.
Anaplasma marginale is a
rickettsial hemoparasite transmitted to cattle biologically by ticks
and mechanically by flies and fomites (2, 18, 36, 45).
Following transmission, A. marginale invades and
multiplies within mature erythrocytes. During acute anaplasmosis,
rickettsemia levels exceed 109 infected erythrocytes per ml
and the resulting disease is characterized by anemia, weight loss,
abortion, and death (3, 13, 20). Recovery from acute
anaplasmosis results in persistent infection characterized by
repetitive cycles of rickettsemia ranging from approximately
102.5 to 107 infected erythrocytes per ml
(11, 14, 20). Persistently infected cattle serve as
long-term reservoirs for transmission within herds (11, 22,
45).
Anaplasmosis is an economically important disease affecting dairy and
beef cattle in most tropical and subtropical and many temperate
countries, including the United States (8, 33, 45).
Detection of persistently infected cattle is important to control the
movement of infected cattle into and from disease-free regions.
Microscopic examination by Giemsa stained blood smears, which is used
to confirm acute anaplasmosis, can only detect levels of
>106 infected erythrocytes per ml (1, 15).
Subinoculation of A. marginale-infected erythrocytes into
susceptible, splenectomized calves has been considered the "gold
standard" for detection of persistently infected cattle, but the
procedure is not practical for routine testing (25).
Serological tests, including complement fixation and card
agglutination, have been the most commonly used methods to detect A. marginale-infected cattle in the field (31, 34,
38) and are accepted as the basis for interstate and
international movement of animals (43). In addition, the
immunofluorescent-antibody test and enzyme-linked immunosorbent
assay (ELISA) have been utilized for epidemiological studies (9,
19, 28). All of these current tests for antibody detection use
crude antigens obtained from partially purified A. marginale
and lack the required sensitivity or specificity for a reliable
diagnosis (4, 9, 27, 28).
Major surface protein 5 (MSP5) is a 19-kDa surface protein highly
conserved among different strains of A. marginale and
A. ovis and in A. centrale (30,
37, 41). Both the native protein and a recombinant
MSP5 (rMSP5) fused to maltose binding protein (MBP)
(21) share an epitope recognized by monoclonal
antibody (MAb) AnaF16C1 (41). A competitive ELISA
(cELISA) based on serum antibody inhibition of MAb AnaF16C1 binding to
rMSP5 has been developed (21). The rMSP5-cELISA has a
demonstrated specificity of 100% (99% confidence interval of 98 to
100%) with sera from uninfected cattle in regions where A. marginale is not endemic (21). Additionally, under
experimental conditions, the cELISA will detect anti-A.
marginale antibodies early in acute anaplasmosis and during
long-term persistence (21). However, the true sensitivity has not been defined with a statistically significant number of animals known to be positive, and neither sensitivity nor specificity has been evaluated for cattle from a region where A. marginale is endemic (21). Therefore, in this study, we
tested the hypothesis that the rMSP5-cELISA will sensitively and
specifically identify cattle persistently infected with A. marginale in a region where A. marginale is endemic. To
test this hypothesis required determination of the true infection
status of cattle within an area where A. marginale is
endemic. For this purpose, we optimized a nested PCR (nPCR), coupled
with sequence analysis and hybridization, to identify A. marginale msp-5 DNA in blood. Each of 235 cattle in a naturally
A. marginale-infected herd was identified as
A. marginale infected or uninfected by using the nPCR,
and sera collected at the same time point was tested for antibodies by
using the rMSP5-cELISA.
nPCR procedure.
The nPCR was optimized to identify
A. marginale msp5 DNA from blood. All reagents were
handled in a laminar-flow hood by using aerosol-resistant pipette tips
(ART, M Sensitivity of nPCR.
A calf (B9503) experimentally infected
with A. marginale Florida was used to determine the
minimal rickettsemia detected by the nPCR. Blood samples from calf
B9503 and from an uninfected control cow (Z35) were obtained in acid
citrate dextrose solution B and washed three times in
phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM
Na2HPO4, 1.8 mM KH2PO4,
pH 7.4). The buffy coat was removed, and erythrocytes were resuspended
in PBS at a final concentration of 3 × 109/ml
(Coulter Counter ZM). Standardized erythrocyte samples from calf B9503
were serially diluted in uninfected erythrocytes to maintain a constant
total erythrocyte count of 3 × 109/ml. The
rickettsemia of the diluted samples ranged between 3 × 107 (in undiluted samples) and 3 × 10 Consistent detection of A. marginale in
persistently infected cattle by nPCR.
Seven persistently infected
cattle (no. 803, 807, 808, 810, 811, 813, and B12) experimentally
inoculated with A. marginale Florida 15 months
previously were used to determine the ability of the nPCR to
consistently detect low rickettsemia levels within a cycle. Blood
samples from these long-term-infected cattle and from control cow Z35
were obtained in two sets on 3 consecutive days each at a 28-day
interval. Samples were prepared as described above and kept frozen at
Evaluation of rMSP5-cELISA in cattle from an A. marginale-infected herd.
The rMSP5-cELISA was evaluated in a
Hereford and Tarantais beef herd located in an area of eastern Oregon
where anaplasmosis is endemic. Acute anaplasmosis had been previously
observed in the herd, but the current prevalence was unknown.
Persistent A. marginale infection in the herd was
confirmed by an initial random sampling of 52 adult cattle in March
1996. At that time, 38% of the cattle were seropositive (data not
shown). Based on this preliminary data and assuming 90% sensitivity
and specificity for the cELISA, 235 randomly selected cattle were
sampled in August 1996 for determination of the true sensitivity and
specificity with narrow confidence limits (7, 12, 21). Ten
milliliters of blood in acid citrate dextrose solution B and 10 ml of
blood for serum collection were obtained by jugular venipuncture from
each of the 235 cattle. Whole-blood and serum samples were maintained
frozen at Sequence analysis.
To determine the degree of homology among
msp5 sequences of A. marginale in naturally
infected cattle and with the reference msp5 gene of the
Florida strain, a sample of nPCR products was randomly selected from
those that showed a 345-bp DNA size. Expecting that 90% of the 345 bp
nPCR product from natural infections would have at least 90% identity
to msp5 of A. marginale Florida, sequence information from 30 animals would enable determination of the extent of
sequence variation in the population with 95% confidence (7, 14,
41). The selected nPCR products were purified through a silica
gel column (Qia Quick; Qiagen) and sequenced. All sequences were
compared by using the Pileup system of the Genetics Computer Group
package from the University of Wisconsin, version 9.0 (6).
Hybridization assays.
A probe designed to hybridize to a
294-bp msp5 sequence internal to the 345-bp nPCR product was
constructed by using the following primers: internal forward,
5'-TACACGTGCCCTACCGACTTA-3' (msp5 positions 367 to 387); internal reverse, 5'-ATACCTGCCTTTCCCATTGAT-3'
(msp5 positions 660 to 640). The probe was labeled
with digoxigenin-11-ddUTP in the PCR and was used in Southern blot
assays in accordance with the manufacturer's (Genius; Boehringer
Mannheim, Indianapolis, Ind.) recommendations. For Southern blot
assays, nPCR products were electrophoresed in a 2% agarose gel and
transferred to a positively charged nylon membrane by alkaline (0.4 N
NaOH) capillary blotting. Membranes were incubated in a
prehybridization solution at 50°C for 4 h prior to addition of
the probe and then incubated in a hybridization solution with the
labeled msp5 probe overnight at 50°C. The membrane was
washed twice at room temperature in 2× SSC (1× SSC is 0.15 M NaCl
plus 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate. Two
high-stringency washes were performed at 70°C for 15 min in 0.5×
SSC-0.1% sodium dodecyl sulfate. Detection of bound probe was carried
out by using chemiluminescence.
rMSP5-cELISA.
Serum samples from the 235 cattle and
from experimentally infected and uninfected calves used as controls
were evaluated by the rMSP5-cELISA. The test was performed as
previously described (21). Individual wells of flat-bottom
plates (Immulon II; Dynatech Laboratories) were coated with 1 µg of
amylose resin-purified rMSP5-MBP fusion protein in 100 µl of
carbonate-bicarbonate coating buffer, pH 9.6. After overnight storage
at 4°C and warming to room temperature, plates were blocked with 0.5 M PBS containing 10 g of fraction V bovine serum albumin, 15 g of glycine, and 40 g of sucrose per liter. Following four washes
with 200 µl of PBS per well, test sera adsorbed for 30 min with 5 µg of dried MBP per 100 µl of serum were added in duplicate and
incubated for 30 min. After four washes with PBS, 0.08 µg of
horseradish peroxidase-conjugated MAb ANA F16C1 in 0.5 M PBS-1%
fraction V bovine serum albumin was added to each well and incubated
for 15 min. After an additional four washes with PBS, 50 µl of
0.5-µg/ml o-phenylenediamine dihydrochloride in substrate
buffer (0.2 M Na2HPO4, 0.1 M citric acid) was
added to each well and incubated for 10 min. Reactions were stopped
with 25 µl of 2 N NH2SO4, and optical density
at 492 nm (OD492) was determined with a Multiskan Plus II
reader. Results were expressed as percent inhibition based on the
following formula: 100 Sensitivity of nPCR.
The sensitivity of the nPCR was
determined by using 10-fold dilutions of A. marginale-infected erythrocytes into a 3 × 109/ml normal-erythrocyte suspension. Infected-erythrocyte
levels ranged from a starting concentration of 3 × 107/ml in undiluted samples to a final concentration of
3 × 10 Consistent detection of A. marginale in
persistently infected cattle with nPCR.
After acute A. marginale infections, cyclical multiplication of the rickettsiae
in persistently infected cattle is characterized by fluctuations
between 102.5 and 107 infected erythrocytes per
ml with low levels of rickettsemia for about 5 to 8 days of every 5 to
6-week cycle (11, 14, 20). Therefore, the ability of the
nPCR to detect A. marginale in persistently infected
cattle with daily consistency and over the time period during
which rickettsemia levels fluctuate periodically was evaluated.
For this experiment, two sets of blood samples, each obtained on 3 consecutive days, were obtained at a 28-day interval from seven cattle
experimentally infected 15 months previously with A. marginale Florida. The primary PCR detected zero to three infected
cattle on each of the 6 days on which they were analyzed, while the
nPCR was able to detect all seven persistently infected cattle on each
of the 6 days on which they were analyzed (Fig. 2).
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Detection of Cattle Naturally Infected with Anaplasma
marginale in a Region of Endemicity by Nested PCR and a
Competitive Enzyme-Linked Immunosorbent Assay Using Recombinant
Major Surface Protein 5
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
P; Molecular Bio-Products, Inc.). Blood samples were thawed,
and 300 µl was used for DNA isolation in accordance with the
manufacturer's (Purogene, Gentra Systems, Inc.) recommendations. DNA
of each sample was resuspended in 100 µl of hydration solution to
give approximately 100 µg of DNA per ml. Primers were designed by
using the published sequence of msp5 from A. marginale Florida (GenBank accession no. M93392) and were as
follows (5'-to-3' sequence and gene location): external forward,
5'-GCATAGCCTCCCCCTCTTTC-3' (msp5 positions 254 to
273); external reverse, 5'-TCCTCGCCTTGCCCCTCAGA-3'
(msp5 positions 710 to 692); internal forward,
5'-TACACGTGCCCTACCGACTTA-3' (msp5 positions 367 to 387). Bovine lactogen primers (bPL) used in the nPCR for msp5-negative samples to ensure the presence of amplifiable
DNA in the sample were as follows (5'-to-3' sequence and gene
location): external forward, 5'-GATACCATGGCAATATACAAC-3'
(bPL positions 91 to 111); external reverse,
5'-GAGCCACTCTGAGATGATG-3' (bPL positions 458 to
440); internal forward, 5'-GTTAGCCTGGGGTTAGCAA-3'
(bPL positions 420 to 438). Two PCR rounds in a final
volume of 25 µl were carried out with a commercial kit (PCR master
kit; Boehringer Mannheim) in a Perkin Elmer thermal cycler. The PCR
master solution contained 25 U of Taq DNA polymerase in 20 mM Tris-HCl, 3 mM MgCl2, 0.01% (vol/vol) Brij 35, and 0.4 mM each deoxynucleoside triphosphate (pH 8.3) in a volume of 0.5 ml.
The first round used 12.5 µl of the master solution, 1 µl of 20 µM msp5 external primers, 5.5 µl of water, and 5 µl of
purified DNA. The second round of amplification used 12.5 µl of
the PCR master solution, 1 µl of 20 µM msp5 external reverse and msp5 internal forward primers, 9.5 µl of
sterile water, and 1 µl of the PCR product from the first round.
Cycling conditions were preheating at 95°C for 3 min and 35 cycles of
95°C for 30 s, 65°C for 58 s, and 72°C for 30 s
with final extension at 72°C for 10 min for each round. PCR and nPCR
products were visualized in a 2% agarose gel following electrophoresis
in 0.4 M (N-morpholino)propanesulfonic acid buffer and
staining with 0.015% ethidium bromide. A 458-bp band was expected
after the PCR, and a 345-bp band was expected after the nPCR.
Conditions for the bovine lactogen nPCR were identical to those
described for the msp5 PCR and nPCR. A 347-bp product was
expected after the lactogen nPCR.
2
(in 109-diluted samples) infected erythrocytes per ml.
Samples were maintained frozen at 
20°C until processed for nPCR.
20°C until processed for nPCR.
20°C until processed by nPCR and rMSP5-cELISA,
respectively.
[(mean OD492 of test
serum × 100)/mean OD492 of negative control].
RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
2/ml in samples diluted 109.
The lowest A. marginale dilution with a detectable
primary PCR product of 458 bp was 103, while an nPCR
product with the expected 345-bp size was detectable at a
106 dilution (Fig. 1). This
sensitivity is equivalent to 30 parasites per ml of blood for the nPCR
(approximately 106% rickettsemia).
View larger version (26K):
[in a new window]
FIG. 1.
Minimum A. marginale rickettsemia
detectable with PCR and nPCR. Tenfold dilutions, starting at 3 × 107 infected erythrocytes per ml, were prepared in a
suspension of 3 × 109 normal erythrocytes per ml. W,
water; M, molecular size markers; 
, uninfected control.
View larger version (69K):
[in a new window]
FIG. 2.
Consistent detection of A. marginale
infection in seven persistently infected cattle by nPCR. DNA was
obtained from the blood of cattle experimentally inoculated with
A. marginale Florida 15 months previously. Animal
identities are indicated on the left. , uninfected control; w, water.
Lanes 1, 2, and 3 represent samples obtained on 3 consecutive days.
Lanes 4, 5, and 6 are samples obtained 28 days later, again on 3 consecutive days.
msp5 sequence analysis. To ensure that hybridization is a valid method to verify the specificity of nPCR-amplified products, msp5 sequence variation was determined in a statistically significant number of animals in the study herd. Analysis of the sequences of 30 randomly selected nPCR products with the expected 345-bp size showed over 95% identity of the sequences compared with the Florida strain msp5 sequence (data not shown). Of the 30 samples, 2 were identical to the Florida strain msp5 sequence, while the remaining 28 had consistent single-base-pair changes. Based on these results, hybridization was considered a valid method of confirming the specificity of nPCR amplicons.
A. marginale status of cattle from the herd in which A. marginale is endemic. To determine the true A. marginale infection status of cattle from the selected herd, blood samples from all 235 cattle were analyzed initially by nPCR, followed by hybridization if any ethidium bromide-stained bands were detectable. An animal was defined as a true positive when nPCR resulted in a product of 345 bp that hybridized to the A. marginale msp5 probe. An animal was defined as a true negative when there was no amplicon or when the nPCR amplicon did not hybridize to the msp5 probe. When no amplicon was present with msp5 primers, the presence of amplifiable DNA was confirmed in all of the animals by using primers for the bovine lactogen gene. A 345-bp band in the nPCR hybridized to the msp5 probe in dot and Southern blots in 151 (64%) of 235 samples. A representative Southern blot is shown in Fig. 3. The 345-bp nPCR product specifically hybridized to the msp5 probe (Fig. 3, lanes 1 and 2). Occasionally, the nPCR product had multiple bands (Fig. 3, lane 5) or a single band with a smaller apparent molecular size than 345 bp (Fig. 3, lanes 3 and 4), none of which hybridized to the probe. In total, 84 animals (36%) did not have an nPCR product or had a product which did not hybridize to the probe. All samples which did not result in a band in the msp5 nPCR had amplifiable DNA, as determined by the presence of a 347-bp nPCR product from the bovine lactogen gene (data not shown).
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rMSP5-cELISA. Serum samples from all 235 cattle were analyzed by rMSP5-cELISA. Percentages of inhibition by the serum samples were widely distributed between 0 and 99% (Fig. 4). For infected (nPCR-positive) cattle, these values ranged from 16 to 99%, while those for uninfected (nPCR-negative) cattle ranged from 0 to 42%, with the exception of one serum with 75% inhibition. Overlap of the percent inhibition of infected and uninfected cattle ranged from 16 to 42% (Fig. 4). Clearly, the cutoff point selected to discriminate between true-positive and true-negative sera is arbitrary and will affect the calculation of sensitivity and specificity. The cutoff point selected for this study as the threshold which best discriminates infected from uninfected cattle was 28% inhibition. With this cutoff point, the agreement between the nPCR and rMSP5-cELISA results was 96% with a kappa value of 0.91, indicating excellent agreement (12, 39). Six of 151 true-positive cattle were falsely negative by the cELISA, with 16, 19, 20, 20, 23, and 25% inhibition. Of 84 true-negative cattle, 4 were false positives by the rMSP5-cELISA, with 32, 32, 42, and 75% inhibition (Table 1). Thus, with a cutoff of 28% inhibition, the sensitivity of the rMSP5-cELISA was 96% (95% confidence interval of 91 to 98%) (12) and its specificity was 95% (95% confidence interval of 88 to 98%) (12). The prevalence of A. marginale in the study herd, based on nPCR-hybridization, was 64%. Based on this, the rMSP5-cELISA had predictive values for positive and negative results of 96 and 93%, respectively.
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DISCUSSION |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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In this study an rMSP5-cELISA was validated by using an nPCR coupled with analysis of the amplicon by DNA sequencing and hybridization to determine the true A. marginale status of naturally infected cattle. The reliability of the nPCR and hybridization as a gold standard is based on three observations. First, the nPCR was able to detect 30 infected erythrocytes per ml of blood, which is a 10- to 100-fold increase in sensitivity over the previously described quantitative PCR assay, RNA probe assay, PCR-ELISA, and hybridization assay (10, 14-16). Second, nPCR was able to consistently detect low levels of rickettsemia in seven persistently infected cattle both on a daily basis and at an interval of 28 days during which the rickettsemia levels were predicted to cycle. Third, msp5 sequence analysis showed high conservation among 30 nPCR amplicon sequences from naturally infected cattle in the herd in which A. marginale is endemic. The sequences were over 95% identical to the reference, A. marginale Florida, validating the use of hybridization to confirm the specificity of nPCR amplicons. A similar level of msp5 sequence conservation has recently been demonstrated at two separate peaks of rickettsemia within one animal (14). Thus, the nPCR with hybridization is a highly sensitive and specific method of detecting A. marginale-infected animals.
After primary A. marginale infection, cattle which survive acute anaplasmosis remain persistently infected for life, independently of re-exposure to the rickettsia (10, 32), serving as reservoirs for transmission by ticks in the field (11, 22, 45). Accurate immunologic identification of persistently infected animals in areas where A. marginale is endemic is difficult. First, persistent infection is characterized by very low-level cyclic multiplication of A. marginale with rickettsemia fluctuating between 102.5 and 107 infected erythrocytes/ml (14, 20). Antibody levels in cattle persistently infected at this low level are difficult to detect with current serological tests (17, 29, 42). Second, cattle in regions where A. marginale is endemic can be exposed to multiple rickettsial and ehrlichial agents that may induce antibodies cross-reactive with A. marginale proteins (40), causing false-positive serology results (5, 24, 35). This problem has been encountered with the 32-kDa diagnostic antigen of Cowdria ruminantium (26). In contrast to sera from animals in areas where C. ruminantium is not endemic, false-positive reactions to the C. ruminantium 32-kDa antigen were detected in regions where C. ruminantium is endemic, presumably due to cross-reactivity with Ehrlichia spp. (26).
The previously reported specificity of 100% for the rMSP5-cELISA was obtained by using 261 serum samples from cattle known to be uninfected in A. marginale-free regions (21). In this study, with 28% inhibition as the cutoff point, the specificity of the rMSP5-cELISA in the study herd in an area where A. marginale is endemic was 95%. As with C. ruminantium, the cause of the 5% false positives in this study could be due to cross-reactivity with other, related organisms in the region. False-positive reactions might also be due to recently resolved A. marginale infections, with persistence of serum antibodies. However, there are no reports of spontaneous clearance with sterile immunity under natural conditions (23, 44), and antibiotic use was not reported in the herd during the study period. Additionally, in this study, there was no evidence of A. marginale clearance in any of 15 infected cattle found to be positive by the nPCR and rMSP5-cELISA in both March and August of the same year. Finally, a false-positive reaction might be a result of a specific reaction between anti-MBP antibodies present in bovine serum and the MBP-rMSP5 antigen, blocking the binding of MAb AnaF16C1 to rMSP5 by steric hindrance. This has previously been demonstrated, requiring an MBP adsorption step prior to addition of sera to the test antigen (21). While the adsorption of sera was performed as previously described to eliminate nonspecific reactions (21), we cannot rule out the possibility that residual MBP antibodies provided some inhibition which would result in false-positive reactions.
The rMSP5-cELISA was able to detect cattle naturally infected with A. marginale with a sensitivity of 96%. Other serological tests for anaplasmosis, including card agglutination and complement fixation, have reported sensitivities of 84 and 79%, respectively (17). However, these values were not based on stringently defined true-positive animals, as was done in this study. Most of them used microscopic detection of A. marginale or comparison with other serology results as a gold standard (4, 9, 17, 29). A total of six false-negative reactions (4%) were detected by the rMSP5-cELISA. These might occur as a consequence of recent primary infection. Under experimental conditions, the rMSP5-cELISA was not able to consistently detect anti-A. marginale antibodies until 16 to 27 days postinoculation, depending on the dose and route of inoculation (21). While the ability of nPCR to detect early infection was not evaluated, early detection of A. marginale infection is expected since an RNA probe with less sensitivity than the nPCR identified the rickettsia 2 days after inoculation (10). Thus, cattle with a recently acquired infection would be identified positively by the nPCR and as falsely negative by the rMSP5-cELISA prior to days 16 to 27. Low responders or nonresponders also should be considered as a possible cause of false-negative reactions. This could explain the results obtained with one infected animal (no. 1012), which was falsely identified as negative by the rMSP5-cELISA at both sampling times.
The rMSP5-cELISA has high sensitivity and specificity when stringent definitions are used to determine true-positive and -negative cattle in a herd in which A. marginale is endemic. The results suggest that it is an excellent assay for eradication programs and regulation of interstate and international movement of cattle. Additionally, the ability of the rMSP5-cELISA to accurately detect individually infected animals will facilitate epidemiologic investigations, particularly in areas where the rickettsia is expanding through movement of infected animals into disease-free regions.
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ACKNOWLEDGMENTS |
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This work was supported by USDA/NRICGP grant 95-38411-2473, USDA Cooperative Agreement 58-5348-3-367, USDA-CWU grant 5348-32000-008-00D, USDA-FAS-ICD-RSED grant BR-17, the Washington Animal Disease Diagnostic Laboratory, and the Instituto Nacional de Tecnología Agropecuaria, Argentina.
We acknowledge Beverly Hunter, Carla Robertson, Kay Morris, Willard Harwood, and Emma Karel for technical assistance, Victor Vanzini for assistance with statistical analysis; and Patricia Rasmussen and Odillon Vidotto for assistance with animal sampling. Special thanks to Gerald and Bonnie Colton for kind permission to sample the study herd.
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FOOTNOTES |
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* Corresponding author. Mailing address: Washington Animal Disease Diagnostic Laboratory, Washington State University, Pullman, WA 99164-7034. Phone: (509) 335-3045. Fax: (509) 335-7424. E-mail: tfm{at}vetmed.wsu.edu.
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REFERENCES |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Journal of Clinical Microbiology, March 1998, p. 777-782, Vol. 36, No. 3
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