Serological cross-reactivity between Anaplasma marginale and an Ehrlichia species in naturally and experimentally infected cattle

Abstract

Seroconversion and cross-reactivity in cattle infected with Anaplasma marginale or a recently described Ehrlichia species (BOV2010 from British Columbia, Canada) were investigated. The study used 76 samples from 20 animals, a commercially available competitive enzyme-linked immunosorbent assay (cELISA) for bovine anaplasmosis, and an indirect fluorescent antibody test (IFAT). Blood smear examination and/or polymerase chain reaction assay were performed to confirm or rule out the presence of Anaplasma or Ehrlichia. Samples comprised 3 groups. Group 1 consisted of 24 samples from 9 cattle naturally infected with Ehrlichia sp. BOV2010. Group 2 had 13 samples from 3 A. marginale–infected cattle from Manitoba, Canada. Group 3 had 39 samples, consisting of 26 from 5 calves experimentally infected with Ehrlichia sp. BOV2010, 10 from 2 calves experimentally infected with A. marginale from cattle (Manitoba) or bison (Saskatchewan), and 3 from an uninfected calf. All samples from cattle naturally or experimentally infected with Ehrlichia sp. BOV2010 or A. marginale were seropositive for A. marginale by both cELISA and IFAT, except 3 calves euthanized at 28 and 33 days post-inoculation (DPI) that did not seroconvert. Antibodies were detected in 2 experimental animals inoculated with Ehrlichia sp. BOV2010, as early as 28 and 33 DPI by the cELISA and IFAT, respectively, and by 42 DPI for both tests. The current study demonstrates that the specificity of the recombinant major surface protein 5 (MSP5) antigen is not restricted to Anaplasma spp., which reduces the utility of the test for serological diagnosis of bovine anaplasmosis in regions where Ehrlichia sp. BOV2010–infected cattle might exist.

Keywords

competitive enzyme-linked immunosorbent assay indirect fluorescent antibody test

Introduction

Tick-borne pathogens are an increasing problem worldwide, including species of the blood-borne bacterial family Anaplasmataceae, which cause disease in animals and human beings.⁵ Species of Ehrlichia and Anaplasma are structurally, genetically, and immunologically similar.^1,5,15 Most Anaplasma spp. infect erythrocytes of domestic and wild ruminants. Three Anaplasma species infect other blood cells: Anaplasma phagocytophilum infects neutrophils, Anaplasma bovis infects monocytes, and Anaplasma platys infects platelets.¹⁵ Ehrlichia spp. infect leukocytes of a wide range of host species including human beings, domestic canines, ruminants, equids, and felids, except Ehrlichia ruminantium, which infects endothelial cells of domestic and wild ruminants. Ehrlichia ruminantium is distributed in sub-Saharan Africa and several islands in the Caribbean.¹ Anaplasma marginale causes bovine anaplasmosis in cattle. Although A. marginale is globally distributed, it is absent in Canada except for very few controlled incursions.¹¹ Both E. ruminantium and A. marginale are important globally to the cattle industry.

The diagnosis of Anaplasma and Ehrlichia infections may include identification of the organism in stained blood smears, polymerase chain reaction (PCR) analysis, and detection of antibodies using various serological tests. Examination of stained thin blood smears for evidence of rickettsemia is a common method used for diagnosing early or acute infections with Anaplasma.³¹ However, the evaluation of blood smears can be unreliable, particularly at low levels of rickettsemia. Molecular analyses using PCR and DNA hybridization assay have been used for the detection or confirmation of Anaplasma infection in seropositive cattle.^7,9 A sensitive and specific nested PCR targeting the major surface protein (msp5) gene has been developed to identify A. marginale in cattle.²⁶ Despite the availability of DNA-based assays for the detection of Anaplasma, serologic tests using major surface protein antigens are considered the most practical means of screening large numbers of cattle for evidence of infection.¹⁵ Several serological tests have been developed to detect infections of Anaplasma and Ehrlichia, including the competitive enzyme-linked immunosorbent assay (cELISA), indirect ELISA, card agglutination test, and indirect fluorescent antibody test (IFAT).^1,31 These tests may be hindered by lack of specificity within or between the several species of Anaplasma and Ehrlichia, which bear homologous families of immunodominant outer membrane proteins.¹

Currently, the cELISA is the primary test used for diagnosing infection with A. marginale in cattle. The IFAT also can be used as a diagnostic test for anaplasmosis.³¹Results of the IFAT have been demonstrated to have 90.8% agreement with DNA hybridization results for A. marginale infection in carrier cattle.¹⁰ Another study reported 96% agreement between an indirect ELISA and IFAT in detecting antibodies against A. marginale in Brazilian cattle.¹⁸ However, the ELISA is generally preferred to the IFAT because the ELISA can be semiautomated making it possible to evaluate larger numbers of serum samples, and the reading of results is less subjective.¹⁸ The cELISA, based on the use of a monoclonal antibody (mAb) that recognizes major surface protein 5 (MSP5) in A. marginale, Anaplasma centrale, and Anaplasma ovis, was validated using a nested PCR procedure, with estimates of sensitivity and specificity of 96–100% and 95–100%, respectively.^14,26 The cELISA test kit^a is commercially available for the diagnosis of bovine anaplasmosis in cattle. In a previous study, cattle experimentally infected with A. phagocytophilum tested positive using this cELISA test kit.⁶ In another study, cross-reactivity was not demonstrated between human or dog antibodies to A. phagocytophilum and the recombinant MSP5 antigen of A. marginale when tested by cELISA.²⁵ However, cross-reactivity was evident when recombinant MSP5 antigens of A. phagocytophilum or A. marginale were incubated with heterologous sera using an indirect ELISA format.²⁵

A newly described Ehrlichia genotype (BOV2010) was recently detected in naturally infected cattle from British Columbia, Canada.⁸ Based on the results of cELISA,^a the cattle were originally suspected of being infected with A. marginale.¹¹ The present study was conducted to investigate the specificity of serological diagnosis of A. marginale and Ehrlichia sp. BOV2010 in cattle and the potential for confounding interpretation of cELISA and IFAT results used to detect bovine anaplasmosis.

Materials and methods

Samples

Samples of fresh or frozen serum, as well as ethylenediamine tetra-acetic acid whole blood and/or DNA samples extracted from blood were assigned to 1 of 3 groups, based on origin of the samples (Table 1). All animal use was conducted under the approval of the University of Saskatchewan Committee on Animal Care and Supply in accordance with guidelines of the Canadian Council on Animal Care.²¹

Table 1.

Source and grouping of serum samples tested by competitive enzyme-linked immunosorbent assay (cELISA) and indirect fluorescent antibody test (IFAT) for Anaplasma marginale.*

	Source animals for samples			No. of positive samples
Group	No.	Animals	No. of samples tested	cELISA	IFAT
1	9	Field cattle (British Columbia) naturally infected with Ehrlichia sp. BOV2010	24	24	24
2	3	Field cattle (Manitoba) naturally infected with A. marginale	13	13	13
3	5	Cattle (A, C, E–G) experimentally infected with blood from 7 of the naturally Ehrlichia sp. BOV2010–infected cattle included in Group 1 (above)	26	11	9
	2	Cattle (D, H) experimentally infected with Manitoba or Saskatchewan stabilate of A. marginale	10	2	2
	1	Uninfected control	3	0	0

For group 1, infection was confirmed by polymerase chain reaction (PCR) in a previous study (7 animals)⁸ or current study (2 animals). For group 2, infection was confirmed by PCR in an earlier study.¹¹ For group 3, molecular analysis results for 7 animals (A, C–G, I) were reported in a previous study.⁸ Molecular analysis of samples from animal H was conducted during the current study.

Group 1

Twenty-four serum samples (1–5 per animal) were collected at different times from 9 adult field cattle from British Columbia naturally infected with Ehrlichia sp. BOV2010. Whole blood and/or extracted DNA obtained from each animal were analyzed by PCR to confirm infection with Ehrlichia sp. BOV2010. Diagnosis of infection in 7 animals from this group was described in a previous study,⁸ whereas infection of the other 2 animals was confirmed in the present study following the PCR protocol described previously.⁸

Group 2

Thirteen serum samples (1–6 per animal) were collected at different periods from 3 field cattle from Manitoba naturally infected with A. marginale (2009) and stored at −20°C. Infection with A. marginale in these animals had been detected by cELISA and confirmed by PCR analysis of whole blood.¹¹

Group 3

A series of paired whole blood and serum samples were collected from 8 splenectomized calves experimentally infected with A. marginale or Ehrlichia sp. BOV2010. Five calves were inoculated with single or pooled aliquots of citrated whole blood collected from 7 of the naturally infected British Columbia cattle included in group 1 as described previously.⁸ Two positive control calves were inoculated with stabilates of A. marginale from cattle (Manitoba) or bison (Saskatchewan). One calf remained uninfected as a negative control. Samples were collected prior to inoculation and at 14, 28, 33, 42, 56, 70 and 84 days post-inoculation (DPI) or until euthanasia, whichever occurred first.

Serological tests

Serum samples from all 3 groups were tested for antibodies to A. marginale using the commercial cELISA Anaplasma antibody test kit.^a Kit controls and test samples were tested in triplicate following the manufacturer’s instructions. The results of the test were considered valid when the negative control had a mean OD_650nm range of 0.4–2.1 and the percent inhibition of the positive control was ≥30%. The percent inhibition was calculated as percent inhibition = 100 − [(ABS S/ABS N) ×100], where ABS is absorbance at 650 nm, S = sample, and N = negative control. Samples with percent inhibitions <30% and ≥30% were considered negative and positive, respectively.

The IFAT antigen blood smear slides were prepared from whole blood obtained by serially infecting 2 splenectomized calves with A. marginale as described previously²² with some modifications. Blood from the positive control splenectomized calf (D) inoculated with a Manitoba isolate of A. marginale as described for the group 3 samples above was monitored daily by examination of blood films stained with Romanowsky stain variant.^b At 42 DPI, once parasitemia reached approximately 38%, washed red blood cells from this animal were suspended in sterile phosphate buffered saline (PBS; 1:2 v/v), and a total volume of 87 ml of this suspension was inoculated intrajugularly into a second recipient splenectomized calf (I). Calf I had remained as a negative uninfected control for 42 days but was subsequently used as the recipient to produce the Anaplasma antigen slides for IFAT. When parasitemia in calf I reached 51% at 13 DPI, blood was collected for preparation of antigen blood smears, and the animal was euthanized. This procedure was followed to reduce the incubation period required to maximize parasitemia while minimizing concomitant antibody response and nonspecific background staining in the IFAT. Antigen blood smears were prepared as described previously.²² Briefly, blood was centrifuged at 1,250 × g for 10 min to remove plasma and buffy coat. Erythrocytes were washed 3 times in PBS (pH 7.4); after washing, cells were suspended in PBS containing 1% bovine serum albumin (1:3 v/v). Smears were prepared, wrapped in aluminum foil, and stored at −70°C until used. In addition to the smears prepared with the Manitoba isolate, antigen blood smears prepared similarly with an A. marginale strain from Brazil were also used. To perform the IFAT, slides were thawed at room temperature and fixed in absolute methanol for 3 min. Sera were titrated at serial 2-fold dilutions from 1:80 to 1:640. Twenty-five microliters of diluted sera from positive or negative controls or test samples were added to squares delineated on the blood smear slides by a hydrophobic pen.^c Slides were incubated at 37°C for 30 min. After incubation, slides were washed 3 times in PBS and then incubated for 30 min at 37°C with a fluorescein isothiocyanate (FITC)-labeled rabbit anti-bovine immunoglobulin G (IgG)-heavy and -light chains conjugate (FITC-anti-IgG-H+L)^d diluted 1:600 in PBS containing 0.01% Evans blue stain. Slides were washed 3 times for 10 min each in PBS, drained, and then mounted with fluorescence Slow Fade reagent.^e Slides were examined for FITC fluorescence by fluorescence microscopy using 40× or 100× objectives and incident light wavelength of 450–490 nm.^f Slides were graded as negative (–) or positive (+) using control sera from uninfected and A. marginale–infected calves, respectively, as described below. A negative reaction gave a greenish or red counter staining, while a specific positive reaction was detected as green fluorescent bodies within the infected erythrocytes with fluorescence equal to or higher than the weak positive control.

Prior to testing of the samples in the current study, the IFAT was evaluated using bovine serum samples from an earlier A. marginale transmission study.¹⁶The sera were collected from splenectomized calves before (0 DPI) and after (12, 20, 26, 31, 35, 41, 51, 61, 71, 82, 121, 191, and 281 DPI) experimental infection with the St. Maries strain of A. marginale. Serum samples were diluted at 1:80, 1:160, and 1:320 dilutions in PBS, applied to squares delineated on slides at 25 µl/square, and then incubated with the FITC conjugate. Samples were graded as negative (–) or positive (+) after being examined by fluorescence microscopy. Negative (0 DPI) with no fluorescence, and positive (20 and 191 DPI) sera representing weak and strong positive fluorescence, respectively, were chosen as negative and positive controls on each slide of test samples in the current study. Also, these same samples were used as reference sera to assess the fitness of the antigen blood smears prepared using the Manitoba isolate and Brazil strain of A. marginale. Based on this, a 1:160 antibody titer was established as the cut-off value using antigen smears from either source.

Blood analysis

Fresh blood films stained with a Romanowsky stain variant^b were prepared routinely and examined under oil immersion at 100× magnification for evidence of parasitemia in animals sampled in groups 1 and 3. DNA extraction, purification, and molecular analysis have been described previously.⁸ Briefly, to confirm A. marginale infection and/or infection with other species of the Anaplasmataceae family in the animals sampled in the current study, nested PCR assays were performed on available blood or extracted DNA samples for animals in groups 1 and 3, and PCR-positive products were cloned and sequenced to verify the infection status of the animals (data not shown). For animals in group 2, confirmation of A. marginale infection by PCR analysis was reported previously.¹¹

Statistical analysis

Agreement between cELISA and IFAT was estimated by calculating the kappa value. Kappa values of ≥0.81, 0.61–0.80, 0.40–60, and ≤0.40 represent excellent, substantial, moderate to good, and slight to poor agreement, respectively.²⁹

Results

Serum samples collected from the 9 British Columbia cattle naturally infected with Ehrlichia sp. BOV2010 (group 1) were seropositive by cELISA and IFAT. However, microscopic examination of blood smears from these animals did not reveal any intra-erythrocytic bacteria. Serological testing of all samples from Manitoba cattle infected with A. marginale (group 2) also gave positive results by both cELISA and IFAT (Table 1). Serology results for all tested samples were in agreement with the results of molecular analysis.

When banked serum samples from calves experimentally infected with the St. Maries strain of A. marginale were tested using antigen blood smear slides from both Manitoba and Brazil sources, positive results were obtained as early as 20 DPI. Both antigen sources gave consistent IFAT results when used to test serum samples from all groups included in the present study.

The cELISA and IFAT results for serially collected samples from the inoculated calves in group 3 are shown in Table 2. In 2 Ehrlichia sp. BOV2010–inoculated calves from this group, antibodies were first detected at 28 and 33 DPI by cELISA, and reached maximum inhibition values at 33 DPI and 84 DPI for animal A and C, respectively (Fig. 1). The IFAT results supported those of the cELISA with seropositivity first detected at 33 and 42 DPI for animal A and C, respectively (Table 2). The other 3 Ehrlichia sp. BOV2010–inoculated calves (E–G) remained seronegative with both tests until their euthanasia at 28, 28, and 33 DPI, respectively. Animals E–G were euthanized early due to clinical disease of unknown etiology. Seroconversion in both A. marginale–positive control calves (D, H) was first detected at 42 DPI by both cELISA and IFAT. Blood smears from these animals revealed a few erythrocytes with A. marginale starting at 30 DPI for animal D and 28 DPI for animal H. Serum and blood samples from the negative control calf I were consistently negative for A. marginale and Ehrlichia sp. by cELISA, IFAT, and blood smear examination. Serum samples from calf C, inoculated with Ehrlichia sp. BOV2010 and calf D, inoculated with a Manitoba isolate of A. marginale showed positive staining patterns when tested by IFAT. Serum from the control calf I was IFAT negative (Fig. 2). A comparison of all results obtained using the 2 serologic assays is presented in Figure 3. The overall agreement between cELISA and IFAT was excellent with a kappa value of 0.941.

Table 2.

Results of competitive enzyme-linked immunosorbent assay (cELISA) and indirect fluorescent antibody test (IFAT) for Anaplasma marginale performed on serum samples serially collected from cattle experimentally infected with Ehrlichia sp. BOV2010 or A. marginale or from uninfected control.*

	Days post-inoculation
	0		14		28		33		42		56		70		84
ID	cELISA	IFAT	cELISA	IFAT	cELISA	IFAT	cELISA	IFAT	cELISA	IFAT	cELISA	IFAT	cELISA	IFAT	cELISA	IFAT
A	−	−	−	−	+	−	+	+	+	+	+	+	+	+	+	+ Euthanized at 194 DPI
C	−	−	−	−	−	−	+	−	+	+	+	+	+	+	+	+ Euthanized at 196 DPI
E	−	−	−	−	−	−	Euthanized at 28 DPI
F	−	−	−	−	−	−	Euthanized at 28 DPI
G	−	−	−	−	−	−	−	−	Euthanized at 33 DPI
D	−	−	−	−	−	−	−	−	+	+	Euthanized at 43 DPI
H	−	−	−	−	−	−	−	−	+	+	Euthanized at 49 DPI
I	−	−	ND	ND	−	−	−	−	ND	ND	Euthanized at 55 DPI

Molecular analysis results for 7 animals (A, C–G, I) were reported in a previous study.⁸ Molecular analysis of samples from animal H was conducted during the current study. Animals A, C, E–G were infected with Ehrlichia sp. BOV2010. Animals D and H were infected with A. marginale. Animal I served as a control (uninfected) for 42 days. Subsequently this animal was used as a recipient of a Manitoba isolate of A. marginale from donor animal D to produce antigen blood slides for the IFAT and then euthanized 13 days after inoculation to prepare antigen blood slides. + = positive test; – = negative test; DPI = days post-inoculation; ND = not determined.

Figure 1.

Competitive enzyme-linked immunosorbent assay (cELISA) percent inhibition mean values obtained for serum samples from splenectomized calves inoculated with blood from cattle naturally infected with Ehrlichia sp. BOV2010 (calves A, C, E–G) or with stabilates of Anaplasma marginale (calves D, H). Calf I was a noninfected negative control. Calves were euthanized at various times starting at 28 DPI.

Figure 2.

Photomicrograph of indirect fluorescent antibody test (IFAT) results for serum samples tested using antigen from a Manitoba isolate of Anaplasma marginale: 1, calf C, inoculated with Ehrlichia sp. BOV2010; 2, calf D, inoculated with a Manitoba isolate of A. marginale; and 3, calf I, noninfected negative control. Animal I was subsequently inoculated with parasitemic blood from calf D and served as source calf of A. marginale antigen used in IFAT. Bar = 10 µm.

Figure 3.

Comparison of competitive enzyme-linked immunosorbent assay (cELISA) and indirect fluorescent antibody test (IFAT) results for the detection of antibodies to Anaplasma and Ehrlichia sp. BOV2010. The dotted line represents the cut-off value for the cELISA. The cELISA was considered positive at percent inhibition ≥30%. Total number of serum samples tested = 76. IFAT+ and IFAT– indicate positive and negative results, respectively.

Discussion

A recent finding of Ehrlichia sp. BOV2010 in North American cattle has been confirmed by molecular and bioassay studies and is the first report of ehrlichiosis in cattle in North America.⁸ The only other Ehrlichia species known to naturally infect cattle is E. ruminantium (formerly Cowdria ruminantium).¹ The present study provides evidence of serological cross-reactivity between Ehrlichia sp. BOV2010 and A. marginale using the commercially available cELISA test kit for Anaplasma and an IFAT using A. marginale antigen–coated slides.

The cELISA used in the current study is based on a mAb (ANAF16C1) that recognizes MSP5 in A. marginale, A. ovis, and A. centrale.³⁰ The present study demonstrates that cattle naturally or experimentally infected with Ehrlichia sp. BOV2010⁸ were seropositive for A. marginale by both cELISA and IFAT (Tables 1, 2). It appears that the epitope recognized by this mAb may also be expressed in Ehrlichia sp. BOV2010. However, it is possible that the cross-reactivity is not restricted to MSP5 but may include epitopes of other proteins of Ehrlichia sp. BOV2010 and A. marginale as suggested by the IFAT results, due to the presence of homologous families of immunodominant outer membrane proteins that are expressed in several Ehrlichia and Anaplasma species.¹

Cattle in regions where A. marginale is endemic could be exposed to infections with other rickettsial pathogens that may induce antibodies cross-reactive with A. marginale proteins, leading to false-positive serological test results. This has been demonstrated for the MAP1 diagnostic antigen of E. ruminantium whereby serological cross-reactions between E. ruminantium and Ehrlichia canis, Ehrlichia chaffeensis, A. bovis, and A. phagocytophilum have been reported using IFAT and indirect ELISA.^{12,13 17,19,28} Similarly, serological cross-reactions between E. chaffeensis and Ehrlichia ewingii infections in human beings have been reported using IFAT.^3,23 However, not all E. ewingii–infected dogs or human beings develop antibodies cross-reactive to E. chaffeensis.^20,23 Ehrlichia canis, which appears to be most closely related to Ehrlichia sp. BOV2010 in cattle,⁸ also serologically cross-reacts with E. chaffeensis.^2,24 Thus, Ehrlichia sp. BOV2010 may also confound serological testing for E. ruminantium as well as A. marginale in cattle.

The commercial cELISA test kit is widely accepted as the most reliable test for screening cattle for A. marginale infection. The IFAT using bovine blood films with A. marginale as a source of antigen, although less preferred due to lower throughput of samples and nonspecific fluorescence, is also recognized as an acceptable test for diagnosing anaplasmosis in cattle.³¹ The cross-reactivity reported in the present study has important implications for the use of these tests in domestic and wild animals that are susceptible to infection with both Anaplasma and Ehrlichia, such as cattle, and in particular in areas where both infections may coexist. In such cases, results obtained by cELISA and IFAT should be interpreted carefully. Since a seropositive animal could be infected with A. marginale, Ehrlichia spp., A. centrale, or A. ovis, differentiation should be based on further testing by PCR analysis using the appropriate specific primers for each pathogen followed by sequencing of amplified products.

Only 2 out of 76 serum samples tested produced negative IFAT and positive cELISA results (Fig. 3). These disparate results were obtained for sera from calves A and C, which were experimentally infected with Ehrlichia sp. BOV2010. The IFAT first detected animals A and C as positive at 33 and 42 DPI, respectively, whereas the cELISA detected them as positive at 28 and 33 DPI, respectively. Differences in detectable seroconversion between the 2 test methods and between animals could be attributed to variations in the sensitivity of the assays or in the immune response of individual animals. However, the overall agreement between the 2 tests was excellent as indicated by kappa value = 0.941. Experimental animals E and G, similarly infected with Ehrlichia sp. BOV2010, remained seronegative by both tests, presumably because animals E and G were euthanized at 28 and 33 DPI, respectively and had not yet generated a detectable immune response. Molecular analysis of blood from experimentally infected cattle confirmed infection with Ehrlichia in all animals except 1 animal (F)⁸ euthanized at 28 DPI that also remained seronegative by both cELISA and IFAT.

Using the commercial cELISA test kit, previous studies have used cut-off inhibition values of both 30% and 42% for cELISA in serological surveys of A. marginale in Canadian cattle.^4,27 In the present study, a cut-off value of 30% inhibition was used as recommended by the manufacturer.^a Seven serum samples from Ehrlichia-infected animals, with cELISA values, which would be interpreted as negative using a 42% instead of 30% cut-off inhibition, were positive by both IFAT and PCR. Thus, when test results of cELISA interpreted using a 42% inhibition cut-off were compared with results interpreted with the recommended 30% inhibition cut-off, the relative sensitivity of the test decreased to 85.7% instead of 96% and the kappa value from 0.94 to 0.80. Although the sample size in the current study was small (76 tested sera), results are consistent with performance of the cELISA in previous anaplasmosis serological studies reported with large populations.^4,27 The cross-reactivity between Ehrlichia sp. BOV2010 and A. marginale demonstrated in the current study indicates that increasing the inhibition cut-off value of cELISA would significantly decrease the sensitivity of this test for both infections.

The present study indicates that both cELISA and IFAT can detect antibodies to Ehrlichia sp. BOV2010 and A. marginale infections in cattle, but neither assay can differentiate between these infections. Diagnostic specificity of serological tests for Ehrlichia might be improved by using cell culture–derived Ehrlichia sp. BOV2010 antigens for serological testing. Such a step would entail preparation of mAbs against specific surface proteins from Ehrlichia sp. BOV2010 to test antigen blood film slides using IFAT or ELISA plates coated with highly specific proteins. A serological assay specific for Ehrlichia could then be used to differentiate between Anaplasma and Ehrlichia.

The serologic cross-reactivity between A. marginale and Ehrlichia sp. BOV2010 reported in the current study limits the diagnostic accuracy of the cELISA and IFAT for A. marginale in cattle. Therefore, positive results derived from these assays should be interpreted with caution, and whenever possible confirmed by molecular analysis.

Footnotes

Acknowledgements

The authors thank all the CFIA employees who provided assistance directly or indirectly in support of this study. Special thanks to Dr. Betty Golsteyn-Thomas and staff at the Lethbridge Laboratory for providing blood and serum samples from field animals. The authors are greatly indebted to the staff of the Centre for Food-borne and Animal Parasitology for the excellent technical support, especially Jane Benjamin, Robyn Ostrander, and Yunxiu Dai. Dr. Murray Lankester provided helpful laboratory and organizational support. Professor Odilone Vidotto, Department of Preventive Veterinary Medicine, State University of Londrina-Brazil, provided A. marginale antigen blood smear slides (Brazil strain) for IFAT in addition to advice on preparing these slides in our laboratory. The animal care and blood collection were provided by staff at the Animal Care Unit, Western College of Veterinary Medicine, University of Saskatchewan. Special thanks to the various CFIA employees who reviewed the manuscript and provided helpful comments.

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Financial and in-kind support for this research was provided by the Canadian Food Inspection Agency.

Notes

References

Allsopp

: 2010, Natural history of Ehrlichia ruminantium. Vet Parasitol 167:123–135.

Anderson

Dawson

Jones

Wilson

: 1991, Ehrlichia chaffeensis, a new species associated with human ehrlichiosis. J Clin Microbiol 29:2838–2842.

Buller

Arens

Hmiel

.: 1999, Ehrlichia ewingii, a newly recognized agent of human ehrlichiosis. N Engl J Med 341:148–155.

Coetzee

Schmidt

O’Connor

Apley

: 2010, Seroprevalence of Anaplasma marginale in 2 Iowa feedlots and its association with morbidity, mortality, production parameters, and carcass traits. Can Vet J 51:862–868.

Doudier

Olano

Parola

Brouqui

: 2010, Factors contributing to emergence of Ehrlichia and Anaplasma spp. as human pathogens. Vet Parasitol 167:149–154.

Dreher

de la Fuente

Hofmann-Lehmann

.: 2005, Serologic cross-reactivity between Anaplasma marginale and Anaplasma phagocytophilum. Clin Diagn Lab Immunol 12:1177–1183.

Figueroa

Chieves

Johnson

Buening

GMM

: 1993, Multiplex polymerase chain reaction based assay for the detection of Babesia bigemina, Babesia bovis and Anaplasma marginale DNA in bovine blood. Vet Parasitol 50:69–81.

Gajadhar

Lobanov

Scandrett

.: 2010, A novel Ehrlichia genotype detected in naturally infected cattle in North America. Vet Parasitol 173:324–329.

Kocan

Ewing

.: 1997, Use of a nonradioactive DNA probe for detection of Anaplasma marginale infection in field cattle: comparison with complement fixation serology and microscopic examination. J Vet Diagn Invest 9:39–43.

10.

Goff

Stiller

Roeder

.: 1990, Comparison of a DNA probe, complement-fixation and indirect immunofluorescence tests for diagnosing Anaplasma marginale in suspected carrier cattle. Vet Microbiol 24:381–390.

11.

Howden

Geale

Paré

.: 2010, An update on bovine anaplasmosis (Anaplasma marginale) in Canada. Can Vet J 51:837–840.

12.

Jongejan

Wassink

Thielemans

.: 1989, Serotypes of Cowdria ruminantium and their relationship with Ehrlichia phagocytophila determined by immunofluorescence. Vet Microbiol 21:31–40.

13.

Katz

Barbet

Mahan

.: 1996, A recombinant antigen from the heartwater agent (Cowdria ruminantium) reactive with antibodies in some southeastern United States white-tailed deer (Odocoileus virginianus), but not cattle, sera. J Wildl Dis 32:424–430.

14.

Knowles

Torioni de Echaide

Palmer

.: 1996, Antibody against an Anaplasma marginale MSP5 epitope common to tick and erythrocyte stages identifies persistently infected cattle. J Clin Microbiol 34:2225–2230.

15.

Kocan

de la Fuente

Blouin

.: 2010, The natural history of Anaplasma marginale. Vet Parasitol 167:95–107.

16.

Lankester

Scandrett

Golsteyn-Thomas

.: 2007, Experimental transmission of bovine anaplasmosis (caused by Anaplasma marginale) by means of Dermacentor variabilis and D. andersoni (Ixodidae) collected in western Canada. Can J Vet Res 71:271–277.

17.

Logan

Holland

Mebus

Ristic

: 1986, Serological relationship between Cowdria ruminantium and certain ehrlichia. Vet Rec 119:458–489.

18.

Madruga

Marques

APC

Leal

CRB

.: 2000, Evaluation of an enzyme-linked immunosorbent assay to detect antibodies against Anaplasma marginale. Presq Vet Bras 20:109–112.

19.

Mahan

Tebele

Mukwedeya

.: 1993, An immunoblotting diagnostic assay for heartwater based on the immunodominanat 32-kilodalton protein of Cowdria ruminantium detects false positives in field sera. J Clin Microbiol 31:2729–2737.

20.

Murphy

Ewing

Whitworth

.: 1998, A molecular and serologic survey of Ehrlichia canis, E. Chaffeensis, and E. ewingii in dogs and ticks from Oklahoma. Vet Parasitol 79:325–339.

21.

Olfert

Cross

McWilliams

, eds.: 1993, Guide to care and use of experimental animals, vol. 1. Canadian Council on Animal Care, Ottawa, Ontario.

22.

Oliveira

Madruga

Schenk

MAM

.: 2003, Antigenic characterization of Brazilian isolates of Anaplasma marginale. Mem Inst Oswaldo Cruz 98:395–400.

23.

Paddock

Folk

Shore

.: 2001, Infections with Ehrlichia chaffeensis and Ehrlichia ewingii in persons coinfected with human immunodeficiency virus. Clin Infect Dis 33:1586–1594.

24.

Perez

Rikihisa

Wen

: 1996, Ehrlichia canis-like agent isolated from a man in Venezuela: antigenic and genetic characterization. J Clin Microbiol 34:2133–2139.

25.

Strik

Alleman

Barbet

.: 2007, Characterization of Anaplasma phagocytophilum major surface protein 5 and the extent of its cross-reactivity with A. marginale. Clin Vaccine Immunol 14:262–268.

26.

Torioni de Echaide

Knowles

McGuire

.: 1998, 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. J Clin Microbiol 36:777–782.

27.

Van Donkersgoed

Gertonson

Bridges

.: 2004, Prevalence of antibodies to bluetongue virus and Anaplasma marginale in Montana yearling cattle entering Alberta feedlots: fall 2001. Can Vet J 45:486–492.

28.

van Vliet

van der Zeijst

Camus

.: 1995, Use of a specific immunogenic region on the Cowdria ruminantium MAP1 protein in a serological assay. J Clin Microbiol 33:2405–2410.

29.

Viera

Garret

JMM

: 2005, Understanding interobserver agreement: the kappa statistics. Fam Med 37:360–363.

30.

Visser

McGuire

Palmer

.: 1992, The Anaplasma marginale msp5 gene encodes a 19-kilodalton protein conserved in all recognized Anaplasma species. Infect Immun 60:5139–5144.

31.

World Organization for Animal Health (OIE): 2009, Bovine anaplasmosis. In: Manual of diagnostic tests and vaccines for terrestrial animals, 6th ed., pp. 599–610. OIE, Paris, France.