Antibody Responses to MAP 1B and Other Cowdria ruminantium Antigens Are Down Regulated in Cattle Challenged with Tick-Transmitted Heartwater

Abstract

Serological diagnosis of heartwater or Cowdria ruminantium infection has been hampered by severe cross-reactions with antibody responses to related ehrlichial agents. A MAP 1B indirect enzyme-linked immunosorbent assay that has an improved specificity and sensitivity for detection of immunoglobulin G (IgG) antibodies has been developed to overcome this constraint (A. H. M. van Vliet, B. A. M. Van der Zeijst, E. Camus, S. M. Mahan, D. Martinez, and F. Jongejan, J. Clin. Microbiol. 33:2405–2410, 1995). When sera were tested from cattle in areas of endemic heartwater infection in Zimbabwe, only 33% of the samples tested positive in this assay despite a high infection pressure (S. M. Mahan, S. M. Samu, T. F. Peter, and F. Jongejan, Ann. N.Y. Acad. Sci 849:85–87, 1998). To determine underlying causes for this observation, the kinetics of MAP 1B-specific IgG antibodies in cattle after tick-transmitted C. ruminantium infection and following recovery were investigated. Sera collected weekly over a period of 52 weeks from 37 cattle, which were naturally or experimentally infected with C. ruminantium via Amblyomma hebraeum ticks, were analyzed. MAP 1B-specific IgG antibody responses developed with similar kinetics in both field- and laboratory-infected cattle. IgG levels peaked at 4 to 9 weeks after tick infestation and declined to baseline levels between 14 and 33 weeks, despite repeated exposure to infected ticks and the establishment of a carrier state as demonstrated by PCR and xenodiagnosis. Some of the serum samples from laboratory, and field-infected cattle were also analyzed by immunoblotting and an indirect fluorescent-antibody test (IFAT) to determine whether this observed seroreversion was specific to the MAP 1B antigen. Reciprocal IFAT and immunoblot MAP 1-specific antibody titres peaked at 5 to 9 weeks after tick infestation but also declined between 30 and 45 weeks. This suggests that MAP 1B-specific IgG antibody responses and antibody responses to other C. ruminantium antigens are down regulated in cattle despite repeated exposure to C. ruminantium via ticks. Significantly, serological responses to the MAP 1B antigen may not be a reliable indicator of C. ruminantium exposure in cattle in areas of endemic heartwater infection.

The rickettsia Cowdria ruminantium is the causative agent of heartwater, an acute, fatal infectious disease of domestic and wild ruminants (5, 40) which is transmitted by ticks of the genus Amblyomma (45). Efforts to study the epidemiology of heartwater and to implement disease control have been hampered by the lack of reliable serodiagnostic tests. Available tests are based on cultured organisms or antigen extracts (7, 9, 13, 15, 23, 26, 34, 36) and on the major antigenic protein of C. ruminantium, MAP 1 (2), which has been targeted as a diagnostic antigen because of its immunodominance (2, 11, 14). These tests are, however, limited by the extensive antigenic similarity between C. ruminantium and closely related agents of the genus Ehrlichia (1, 8, 12, 14, 17, 35, 41), some of which also infect ruminants. Recently, a partial fragment of MAP 1, MAP 1B, which spans amino acids 47 to 92 of the mature protein (42, 43), has been shown to have high specificity for C. ruminantium in an indirect enzyme-linked immunosorbent assay (ELISA) (24, 25, 43). The assay does not detect antibodies to ehrlichial agents infecting domestic ruminants, such as E. bovis, E. ovina, and E. phagocytophila. It does detect antibodies to E. canis (which infects dogs) and antibodies to E. chaffeensis (a pathogen of humans). In the United States, E. chaffeensis infects white-tailed deer (6, 16), a species that is highly susceptible to heartwater. In areas of Zimbabwe (22, 43) and the Caribbean (24, 25) that are designated heartwater free by the absence of Amblyomma ticks and clinical heartwater, the MAP 1B indirect ELISA demonstrated a high specificity with cattle, sheep and goat sera. This assay is also reliable for the detection of experimental infections in small ruminants, and it detects antibodies to geographically diverse C. ruminantium isolates from different countries (24, 43). Hence, its use has been proposed for diagnosis and surveillance of heartwater.

In a preliminary serological survey of heartwater in Zimbabwe using the MAP 1B indirect ELISA, only 33% of cattle sera from areas with endemic heartwater infection tested positive (22). The low seroprevalence was unexpected, given the high infection pressure in these regions and the consequent likely high prevalence of infection (27). Epidemiological studies conducted on some of these farms over several years demonstrated a tick infection rate of 10% and a vector attachment rate of between one and four ticks every 2 days (31). At this tick attack rate, it was estimated that cattle were exposed to fresh infections every 5 to 20 days, and it is assumed that immunity to heartwater is maintained by the repeated challenge with infected ticks (27). This inference is supported by the fact that clinical heartwater cases are very rare on these farms where infection is endemic.

To investigate the reasons for the low seropositivity, a study was undertaken to examine the immunoglobulin G (IgG) antibody kinetics to the MAP 1B antigen in cattle infected with tick-transmitted C. ruminantium under natural and laboratory conditions. To determine whether any observed patterns in serological responses were specific to MAP 1B, serum samples from some of the infected cattle were also analyzed by an immunoblotting assay and an indirect fluorescent-antibody test (IFAT) (20, 36) when peak levels of MAP 1B-specific antibody responses were detected and during periods when these antibodies declined significantly or were not detectable.

MATERIALS AND METHODS

Initiation of C. ruminantium infections in cattle.

C. ruminantium infections were initiated in cattle either by natural exposure to field ticks on a heartwater-endemic farm (Vlakfontain Farm, 18°47′S 30°40′E) in the highveld of Zimbabwe or under controlled laboratory conditions via Amblyomma hebraeum tick transmission. Six-month-old Mashona (Bos indicus × Bos taurus) cattle were obtained from a heartwater- and Amblyomma-free area of Zimbabwe (29, 30) and were seronegative for C. ruminantium-reactive antibodies by immunoblotting, IFAT, and MAP 1B ELISA. Mashona, an indigenous Zimbabwean cattle breed, was used as it forms a high proportion of the cattle population in commercial and communal farm herds in heartwater-endemic regions of Zimbabwe. These cattle were divided into five groups (groups 1 to 5). Groups 1 and 2 were exposed to natural field infection on Vlakfontain Farm, while laboratory-infected A. hebraeum ticks were fed on group 3, 4, and 5 cattle at different frequencies. All cattle were monitored for febrile reactions for the first 3 months postinfection. Sequential serum samples were collected every 5 to 7 days from all cattle for testing by the indirect MAP 1B ELISA, IFAT, and immunoblotting assay.

Field infection of cattle. (i) Group 1.

Ten cattle were introduced onto Vlakfontain Farm, which has a high infection pressure as determined in detailed epidemiological studies (27, 31). On this farm, tick control has been minimal for over 10 years and C. ruminantium is transmitted by A. hebraeum ticks. Tick infestation levels in the resident cattle were high (See Table 1), and high infection rates with C. ruminantium were present in A. hebraeum ticks (31). Clinical heartwater was rarely observed, and the farm was considered to be endemically stable for the disease (27). The experimental cattle were introduced onto this farm during peak tick season (February) and allowed to become naturally infested with A. hebraeum ticks. Infestation with other ticks (Rhipicephalus appendiculatus, R. evertsi evertsi, Boophilus decoloratus, and Hyalomma spp.) which occur naturally on this farm could not be avoided. This also meant that other tick-borne diseases were also transmitted at the same time as heartwater. Tick control was not practiced on the cattle, except at week 14, when a synthetic pyrethroid pour-on (flumethrin [Bayticol]; Bayer, Leverkusen, Germany) was used to reduce the tick burden. Due to high tick infestation and babesiosis, death occurred in 8 of the 10 cattle.

TABLE 1.

Periodic counts of A. hebreaum tick infestation on group 2 cattle introduced onto a heartwater-endemic farm

Time (wk) in field	No. of ticks counted on 7 cattle (mean ± SD)
	Males	Females	Nymphs
0	0 ± 0	0 ± 0	0 ± 0
4	6 ± 2	2 ± 1	16 ± 3
10	42 ± 8	16 ± 5	22 ± 3
20	63 ± 23	10 ± 2	24 ± 5
30	22 ± 11	9 ± 5	20 ± 7
40	38 ± 16	11 ± 5	28 ± 5
52	36 ± 14	14 ± 4	18 ± 4

Weekly counts of adults and nymphs of Amblyomma ticks were done. Full-body examinations for ticks were conducted, with emphasis on the preferred sites for tick attachment such as the perianal region, base of the tail, legs, hooves, udder, scrotum, belly, head, and ears. Serum and whole-blood samples were collected weekly from each animal.

(ii) Group 2.

To repeat the analysis due to losses of cattle in group 1, seven new Mashona cattle were purchased from the same heartwater-free areas and vaccinated against anaplasmosis (Anaplasma marginale infection) and babesiosis (Babesia bigemina infection) using a bivalent live blood vaccine 6 weeks prior to translocation onto Vlakfontain Farm. For anaplasmosis, an A. centrale blood vaccine was used, and for babesiosis, a B. bigemina vaccine G strain (isolated near Gayndah, Queensland, Australia) was used. The vaccine was inoculated intramuscularly. These cattle were introduced onto the farm during the low tick season (August) to avoid an overwhelming tick challenge and to prevent the deaths observed in group 1 cattle. The cattle were allowed to become naturally infested with A. hebraeum ticks and other ticks. Weekly tick counts of Amblyomma ticks were done for 52 weeks as described above. The cattle remained on the farm for a further 56 weeks. Serum and whole-blood samples were collected from these cattle weekly for 1 year and again at the end of the study during week 120.

Laboratory infections of cattle (groups 3 to 5).

In contrast to the field infections described above, infections in cattle were also initiated in the laboratory for comparison and to control for transmission of any cross-reacting ehrlichial agents by field ticks. Laboratory infections were transmitted by the A. hebraeum ticks from a colony which was established at least 10 years previously. For this, infected A. hebraeum ticks were produced in the laboratory as described below.

(i) Production of infected A. hebraeum ticks for laboratory infection of cattle:

Six C. ruminantium-naive 6-month-old merino sheep were inoculated intravenously with 2 ml of supernatant from bovine endothelial-cell cultures (3) which were heavily infected with C. ruminantium (Zimbabwean Plumtree isolate) (37). The Plumtree isolate was used to initiate infections in cattle because it has been previously shown to cause a subacute form of heartwater which does not usually result in death of the affected cattle. Laboratory-reared, unfed, uninfected A. hebraeum nymphs (Zimbabwean Sengwe strain) were fed by placing them in bags attached to the backs of these sheep during the febrile reaction to allow acquisition of C. ruminantium during the period of high rickettsemia (21). Engorged nymphs were collected, incubated at 27°C and 75% relative humidity, and allowed to molt to the adult stage. The prevalence and intensity of C. ruminantium infection were assessed in 24 adult ticks (12 male and 12 female) from each sheep by using the C. ruminantium-specific pCS20 DNA probe (19, 21, 44, 46). Infection intensity was estimated semiquantitatively by comparison of probe hybridization signals with those of quantitated C. ruminantium DNA standards, as described previously (31). Specimens of ticks from each batch were shown to be highly infected with C. ruminantium by DNA probe analysis, with infection rates of 88 to 100%. The intensity of detectable infections ranged from 10⁵ to 10⁸ organisms per tick (Fig. 1). The viability of infection in the ticks was confirmed by transmission of C. ruminantium from 30 feeding ticks from each batch on uninfected susceptible sheep. These ticks transmitted acute lethal heartwater (data not shown). Heartwater was confirmed as a cause of death in these sheep at postmortem examination by identifying C. ruminantium colonies in brain crush smears (32). The adult male and female ticks from the batches of infected ticks which hybridized with the DNA probe and transmitted heartwater were utilized for laboratory infection of cattle as described below.

FIG. 1.

DNA from adult A. hebraeum ticks hybridized with the pCS20 C. ruminantium-specific DNA probe. The ticks were infected as nymphs by feeding on sheep infected with C. ruminantium and were then used for laboratory infections of cattle. DNA from serial dilutions of C. ruminantium organisms (10⁹ to 10² and 100 ng of C. ruminantium DNA) was used for positive control samples. DNA from uninfected adult A. hebraeum ticks was used for negative control samples.

(ii) Laboratory infection of group 3.

C. ruminantium (Plumtree isolate)-infected adult A. hebraeum ticks (10 males and 5 females) were placed in bags attached to the dorsum of each of 20 cattle and allowed to feed continuously. Male ticks were removed forcibly after all female ticks had engorged and detached (after approximately 30 days). The cattle were bled for serum collection at 5 day intervals, commencing on day 15 after tick infestation. On day 110 (approximately 16 weeks) after primary tick infestation, the animals were reexposed to C. ruminantium (Plumtree isolate) by feeding 15 infected adult ticks (10 males and 5 females) as before. Thereafter, sera were collected from these cattle at 2-week intervals for 8 weeks.

(iii) Laboratory infection of groups 4 and 5.

Groups 4 and 5, containing five cattle each were initially infested with 20 infected adult A. hebraeum ticks (10 females and 10 males). At 2 weeks (for group 4) and 4 weeks (for group 5) after the primary tick infestation and at 2-week intervals thereafter, for a total of 52 weeks, a new batch of 10 C. ruminantium-infected adult ticks (5 females and 5 males) were fed on each of the cattle. The ticks were allowed to feed continually in order to mimic field exposure to C. ruminantium under minimal tick control. Sera and whole blood were collected weekly from all cattle.

Confirmation of carrier status of cattle by xenodiagnosis and PCR.

Xenodiagnostic methods and the pCS20 PCR (28) assay were used at various stages of the study to demonstrate that the infected cattle remain carriers of infection. This was important for correlation with the MAP 1B ELISA results.

(i) Xenodiagnosis.

Demostration of a carrier state in group 3 cattle was done by feeding nymphal ticks at 2-week intervals from weeks 3 to 12 postinfection. The molted adult ticks from group 3 cattle were then tested for acquisition of C. ruminantium infection by using the pCS20 DNA probe and PCR (28). Demonstration of a carrier state in group 2, 4, and 5 cattle was attempted by feeding 400 uninfected A. hebraeum nymphs on four group 2 field-infected cattle and each of the group 4 and 5 laboratory-infected cattle during weeks 116, 49, and 111 after primary infection, respectively. This period was 12 weeks after the last exposure to field ticks in group 2 cattle and 4 and 59 weeks after the last tick feeds of experimentally infected group 4 and 5 cattle, respectively. Engorged nymphs were collected from all cattle and allowed to molt into adults as described above; they were then fed on heartwater-naive sheep which were free of other hemoparasites. The sheep were monitored for the development of clinical signs of heartwater (fever, anorexia, nervous system signs resulting in recovery or death). Brain biopsies were done on sheep that exhibited febrile reactions on day 3 of the reaction as described previously (39). In addition, plasma was prepared from the blood of all clinically reacting sheep on days 2 and 3 of the febrile reaction for isolation of C. ruminantium in bovine endothelial-cell culture to demonstrate transmission of heartwater by feeding ticks (3, 4). A postmortem examination was done on sheep that died to confirm that death was due to heartwater, and brain crush smears were prepared and examined for C. ruminantium colonies in endothelial cells of brain capillaries (32). Any sheep that survived following tick transmission were challenged intravenously with a predetermined lethal dose of Plumtree isolate from infected bovine endothelial-cell culture supernatants, to determine their clinical response to this infection. Usually, if cattle, goats, or sheep are experimentally or naturally infected with C. ruminantium, following recovery from infection (either naturally or after treatment) they become resistant to rechallenge with the homologous C. ruminantium isolate and survive the challenge (40). Hence, a lack of response to challenge would mean that they have been exposed previously and that they are immune and a clinical response would mean that the animals are heartwater naive.

(ii) PCR analysis.

Buffy coat cells were collected weekly from 10 ml of whole uncoagulated blood from all cattle. DNA was extracted using QIA-DNA extraction kits (Qiagen, Hilden, Germany). Then 10 μl of the DNA sample (5% of total) was used as the template in 50-μl PCR assay mixtures. For each animal, the tests were performed on weeks 0 and 1 to 12 and then monthly through to week 52. The PCR assay was performed as previously described (28), based on primers AB 128 (5′-ACTAGTAGAAATTGCACAATCAT-3′), and AB 129 (5′-TGATAACTTGGTGCGGGAAATCCTT-3′), which amplify a 279-bp fragment from within open reading frame 2 of the 1,306-bp pCS20 DNA sequence (28). To increase the sensitivity of detection of C. ruminantium DNA for samples collected from carriers, DNA from buffy coat cells collected from cattle when antibodies had declined to base levels were tested by a nested PCR. Primers U24 (5′-TTTCCCTATGATACAGAAGGTAAC-3′) and L24 (5′-AAAGCAAGGATTGTGATCTGGACC-3′) were used for the first amplification, followed by AB 128 and AB 129 as the nested primers. Then 30 μl of each completed PCR mixture was analyzed on 1.5% agarose gels stained with ethidium bromide, and the gels were viewed under UV light and photographed. The electrophoresed products were transferred to nylon membranes (GeneScreen Plus; Du Pont) and hybridized with pCS 20 DNA probe, random-primer labeled (Boehringer Mannheim) with [³²P]dCTP (Amersham, Little Chalfont, United kingdom) as a probe (46). The hybridized blots were exposed to X-ray film (Kodak Biomax) for 24 to 48 h to determine the results of these analysis.

Immunoassays. (i) Indirect MAP 1B ELISA.

The indirect MAP 1B ELISA for detection of total IgG responses was performed using a recombinant MAP 1B protein of the Senegal isolate of C. ruminantium as described previously (43). The partial fragment of the mature MAP 1 protein containing amino acids 47 to 152 was expressed in Escherichia coli as a fusion protein in expression vector pQE9 and purified with Ni²⁺-nitrolotriacetic acid agarose under denaturing conditions as described previously (43). For the development and validation of the test, the MAP 1B antigen was used as coating antigen at a concentration of 0.5 μg/ml in 0.5 M carbonate buffer (43). However, in initial trials of validating the test for use in Zimbabwe, it was found that an antigen concentration of 2 μg/ml reduced background noise and improved specificity (data not shown). The test was adapted and optimized to detect the various IgG isotypes in cattle sera. Anti-bovine IgG (H+L) conjugated to horseradish peroxidase was obtained from Kirkegaard and Perry Laboratories (Gaithersburg, Md.), and horseradish peroxidase-conjugated anti-bovine IgG1 and IgG2 antibodies were kindly donated by J. Katende of the International Livestock Research Institute (Nairobi, Kenya). Optical densities of the completed ELISAs were measured with a Titertek Multiskan ELISA reader (Titertek, Flow Laboratories Inc.) using dual wavelengths, 405 and 492 nm. Each serum sample was tested in duplicate, and duplicate samples of negative and positive control serum samples were included on each plate. The negative control serum was from noninfected control animals, while the positive serum was from experimentally infected cattle. For each plate, the cutoff value was calculated as two times the percent positivity of the negative control serum relative to the positive control serum (43). The cutoff points for the various MAP 1B ELISA averaged at 15.6 ± 4.4%.

(ii) Immunoblotting and IFAT analysis.

To determine whether patterns in serological reactivity observed against MAP 1B antigen were also displayed against native MAP 1 antigen and other C. ruminantium antigens, selected sera from the laboratory and field-infected cattle were also tested by the immunoblot assay and IFAT. The sera were selected on the basis of their reaction with MAP 1B antigen and represented peak periods of antibody responses (week 9) and periods when MAP 1B-specific antibodies were declining (week 30) and when seronegativity was detected (week 45). A protein G-based immunoblotting assay, optimized for detection of total IgG antibodies, was performed as described previously (20). Briefly, C. ruminantium organisms (Plumtree isolate) were harvested from supernatants of infected bovine endothelial cells. Host cell debris was removed by low-speed centrifugation (1,000 × g) for 10 min. The organisms were then pelleted by centrifugation at 30,000 × g for 30 min at 4°C and given three washes in phosphate-buffered saline (PBS). The organisms were then resuspended in 1 ml of PBS, freeze-thawed, and sonicated three times. The protein concentration was estimated using the assay of Lowry et al. (18). For sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 20 μg of antigen was loaded per lane and separated on 12% polyacrylamide gels. The proteins were electrotransfered onto nitrocellulose membranes, and the membranes were blocked with 5% low-fat milk in Tris-buffered saline (0.1 M Tris-HCl, 0.9% NaCl [pH 8.0]) and then probed with titrated sera from two field-infected cattle (group 2) and two laboratory-infected cattle (group 5). The sera were titrated, at 10-fold dilutions, to the end point based on the reaction to the immunodominat MAP 1 antigen.

The IFAT, based on cell-cultured C. ruminantium whole organisms and detected total IgG antibodies to C. ruminantium, was used as described previously (36). The organisms were harvested from infected bovine endothelial-cell cultures (Plumtree isolate) which were showing maximum cytopathic effects. Following removal of cell debris (400 × g for 10 min), the organisms were washed in PBS at 30 000 × g for 30 min at 4°C and resuspended in PBS. Antigen slides were prepared from suspensions containing Cowdria organims or uninfected cells and fixed in acetone at −20°C overnight as described previously (36). For IFAT analysis, weekly serum samples from 10 field-infected cattle (group 1) as well as from 5 laboratory-infected cattle (group 5) were analyzed at doubling dilutions until peak titers were detected. Thereafter, sera collected at 5-week intervals from the five laboratory-infected cattle and the surviving field-infected cattle were titrated to the end point. A cutoff reciprocal titer of 40 (2 × 1:20, the background titer) was decided after testing preinfection samples from each animal.

RESULTS

Clinical responses of cattle to field and laboratory tick challenge.

Eight cattle in group 1 exhibited mild clinical signs of heartwater with low-grade fever between weeks 3 and 5 after exposure in the field, but two of the cattle remained unreactive in response to field tick challenge. In this group, four of the cattle died at week 6, one died at week 9, and three died at week 14 of anemia caused by babesiosis and a massive tick infestation by A. hebraeum and other tick species (data not shown). Tick control was not practiced on the cattle, except at week 14, when a synthetic pyrethroid pour-on (flumethrin) was used once to reduce the tick burden after eight of the cattle succumbed to anemia due to high tick burdens. One animal died at week 36 from non-tick-related causes. The tenth animal survived the period of study of 52 weeks.

In group 2, four out of the seven cattle exhibited mild febrile reactions of 39.5 to 40.5°C after 10 to 12 weeks in the field. The other three remained nonreactive. Tick counts recorded weekly indicate that A. hebraeum adult males, females, and nymphs infested these cattle throughout the 52 weeks of study (Table 1). Ambylomma tick infestation levels on cattle increased over the first 10 weeks following their introduction to the field, but thereafter their levels fluctuated. No deaths occurred in this group of cattle during the 52 weeks of study. These cattle remained on the farm for a further 56 weeks after the end of the 52-week study. During this period, three of the cattle died from non-heartwater-related conditions.

In laboratory containment, A. hebraeum ticks transmitted C. ruminantium to all cattle in group 3, 4, and 5, which resulted in a mild form of clinical heartwater. This was consistent with the susceptibility of this breed of cattle and with the virulence of the Plumtree isolate. Of the 10 laboratory-infected cattle, 4 exhibited mild febrile reactions of 39.5 to 40.2°C between weeks 3 and 4 and 2 exhibited the reactions between weeks 8 and 9. The remaining cattle did not show any febrile reactions. No deaths were recorded in these three groups.

Cattle persistently challenged with C. ruminantium become seronegative to MAP 1B antigen. (i) Field-infected cattle.

MAP 1B-specific IgG antibodies were first detected at weeks 3 to 4 in all cattle after exposure to field ticks, except for one animal in group 2, which became positive during week 6. Peak IgG levels were attained at weeks 4 to 9 in both groups 1 and 2 (Table 2). In four group 1 cattle that survived for at least 14 weeks, antibodies had declined to baseline levels by weeks 10 to 12 after exposure to field ticks (Fig. 2a). This decline to baseline levels occured despite high levels of A. hebraeum tick challenge and hence C. ruminantium exposure (31). The antibodies in the fifth animal (which exhibited the highest levels of IgG antibodies) showed a slower decline and had not reached baseline by 14 weeks (Fig. 2a).

TABLE 2.

Detection of MAP 1B-specific IgG responses in sera of C. ruminantium-infected cattle^a

Group	MAP 1B-specific IgG1 antibodies		MAP 1B-specific total IgG
	Time [wk] of peak detection	Duration [wk] of detection	Time [wk] of peak detection	Duration [wk] of detection
Field-infected cattle
Group 1 (10)	4–7	3–14	4–7	3–14
Group 2 (7)	6–9a	3–18 (5)	4–9b	3–33 (5)
		3–38 (2)		3–52 (2)
Experimentally infected  cattle
Group 3 (20)	4–6	4–24	4–6	3–28
Group 4 (5)	5–7	4–18	5–7	4–18, 21–24
Group 5 (5)	5–7	5–14	5–9	4–18, 21–25

^aNumber of animals are indicated in parentheses. 

^bSome cattle exhibited lower multiple-antibody peaks. 

FIG. 2.

Kinetics of total IgG antibodies detected by MAP 1B indirect ELISA in cattle exposed to C. ruminantium by field- or laboratory-infected A. hebraeum ticks. (a and b) Group 1 and 2 cattle, respectively, naturally infected on a heartwater-endemic farm; (c) cattle experimentally infected by repeated infestation with laboratory-infected A. hebraeum ticks.

In five of the seven cattle in group 2, MAP 1B-specific IgG antibodies remained detectable for 19 to 31 weeks (Fig. 2b), and in two of the cattle they were detectable for 38 to 48 weeks (data not shown). In two of the seven cattle in group 2 which had become seronegative by weeks 22 and 25, a second peak of MAP 1B-specific IgG antibodies was detected between weeks 38 and 42, but these cattle became seronegative between weeks 43 and 52. Generally, the antibody decline was slower in group 2 cattle than in group 1 cattle. These cattle remained on the farm for a further 56 weeks after the end of the 52-week study. During this period, three of the cattle died from non-heartwater-related conditions. The four remaining cattle tested at the end of this period (week 108 after field exposure) were still seronegative for IgG antibodies by the MAP 1B ELISA (data not shown).

IgG1 antibodies were detected in these cattle with similar kinetics to those of total IgG (data not shown), but IgG2 antibodies to MAP 1B antigen were never detected.

(ii) Laboratory-infected cattle.

The patterns of MAP 1B-specific IgG antibody responses in all laboratory-infected cattle were similar to each other and to the responses of field-infected cattle, irrespective of the regimen of exposure to infected ticks. A sample of these responses is shown in Fig. 2c. IgG antibodies were first detected in all cattle at 3 to 5 weeks after exposure to ticks and reached peak levels at weeks 4 to 9 (Table 2). However, regardless of the tick infestation regimen, the antibody levels declined to baseline, with IgG1 levels reaching baseline quicker (by 14 to 24 weeks) than total IgG levels (by 18 to 28 weeks) (Fig. 2c; Table 2). One of the group 5 cattle showed a second peak of MAP 1B-specific IgG antibodies between weeks 26 and 30, after having tested seronegative from week 16. MAP 1B-specific IgG2 antibodies were not detected.

MAP 1B-seronegative cattle remain carriers of C. ruminantium infection.

C. ruminantium infection was consistently detected in all surviving field-infected and laboratory-infected cattle by PCR during weeks 3 to 20 postinfection, the period during and beyond recovery from primary infection. Thereafter, detection became inconsistent, and cattle in the various groups were intermittently positive by PCR. In group 1, in the two animals that survived longer than 14 weeks, C. ruminantium could be detected from weeks 20 to 36 in one and at weeks 26 to 28 and 32 in the other (Table 3). In three of the seven cattle in group 2, C. ruminantium was detectable in different animals at weeks 24, 32, and 40 (Table 3). In groups 4 and 5, C. ruminantium was detectable in 5 of the 10 cattle at various intervals between weeks 32 and 52 (Table 3). Group 3 cattle were not tested by PCR.

TABLE 3.

Duration of PCR detection of C. ruminantium in blood of cattle infected by A. hebraeum ticks

Field-infected cattle				Laboratory-infected cattle
Group 1 animal no.a	Duration (wk) when Cowdria DNA detected	Group 2 animal no.	Duration (wk) when Cowdria DNA detected	Group 4 animal no.	Duration (wk) when Cowdria DNA detected	Group 5 animal no.	Duration (wk) when Cowdria DNA detected
102	3–8, 12, 14	130	2–5, 7, 20	103	3–5, 8, 9, 16, 20, 52	101	3–4, 8, 16
107	4, 6–9	131	4–8, 10, 20	108	4–5	106	3–5
111	4–6, 8–9, 20–36	132b	4, 7–8, 12, 20	110	3–4, 8, 9, 10, 40	109	3–5, 10, 16
115	3–6	136	6–7, 40	125	4, 8, 10	112	3–4, 40
116	4–6	137	5–7, 10, 12	126b	4, 8, 40, 44	114	3–4, 32
117	4, 6, 8, 20–21, 26–28, 32	138b	3, 5–9, 20, 32, 40
120	4–5, 8–10, 14	139	3–4, 6–7, 9, 11, 16, 24, 40
122	3–6
123	3–6
127	3–7

^aAnimals 115, 116, 122, and 123 died at week 6, 107 died at week 9, and 102, 120, and 127 died at week 14 from anemia and babesiosis after exposure to field ticks; 111 died at week 36 from non-tick-related causes. 

^bTicks fed on animals transmitted C. ruminantium infection to sheep. 

Adult ticks fed as nymphs on two of the group 2 field-infected cattle transmitted C. ruminantium to a heartwater-naive sheep. The sheep developed a febrile reaction, was brain biopsy positive, and also was confirmed to have died of heartwater by postmortem observations of brain crush smears, although isolation of C. ruminantium from plasma of this sheep in tissue cultures was not successful. Ticks fed on the other two cattle did not transmit heartwater to sheep. These four field-infected cattle were seronegative by MAP 1B ELISA during the period when xenodiagnosis was conducted (data not shown).

A carrier state in group 3 cattle was demonstrated by conducting PCR on ticks fed on these cattle. These cattle infected at least 10% of the ticks fed on them for up to weeks 11 to 12 postinfection. During this period, they were positive for MAP 1B antibodies. Adult ticks from one of five cattle in group 4 (animal 126), fed during week 49 of study, transmitted C. ruminantium to a heartwater-naive sheep, which developed a febrile reaction and was positive for heartwater infections by brain biopsy and by C. ruminantium isolation in bovine endothelial cell cultures from the plasma prepared during the febrile reaction. This sheep survived the infection and was protected against challenge with a lethal predetermined dose of Plumtree C. ruminantium tissue culture supernatant. MAP 1B-specific IgG antibodies had not been detectable in this animal (animal 126) from weeks 23 to 52 postinfection. C. ruminantium was also detected by PCR in buffy coat cells of this animal at weeks 40 and 44, demonstrating persistent infection (Table 3). Adult ticks that had been fed as nymphs on group 5 cattle 59 weeks after last infected tick feed did not transmit C. ruminantium to any of the sheep. Collectively, the xenodiagnosis and PCR analysis demonstrated carrier status in some of these cattle despite their being MAP 1B negative.

Persistently challenged cattle show down regulation of antibody responses to all C. ruminantium antigens.

Analysis of sequential serum samples of two field-infected and two laboratory-infected cattle by immunoblotting exhibited MAP 1 reciprocal titers of 8,000 to 16,000 by week 9, but the titers had decreased to 100 to 1,000 by weeks 30 to 45 after infection (Fig. 3). In fact, antibody responses to other C. ruminantium antigens were detectable only at a 1:100 serum dilution (Fig. 3), demonstrating that antibody responses to all C. ruminantium antigens were down regulated.

FIG. 3.

Immunoblot reactions of titrated sequential serum samples from naturally and experimentally infected cattle against C. ruminantium antigens. Serum dilutions were as follows: strip 1, = 1:100; strip 2, = 1:1,000; strip 3–1:2,000; strip 4, 1:4,000; strip 5, 1:8,000; strip 6, 1:16,000; strip 7, 1:32,000; strip 8, 1:64,000. Week 9 sera from experimentally infected cattle 103 and 108 had eight dilutions tested. The rest of the serum bleeds had only five or six dilutions tested. At week 9 postinfestation, reciprocal end-point titers of MAP 1 antibody responses for both naturally and experimentally infected cattle were 8,000 to 16,000, which then decreased to 100 to 1,000 by weeks 30 to 45.

Analysis of antibody responses in sera from field and laboratory-infected cattle by IFAT revealed the same pattern of antibody kinetics as detected by immunoblotting and MAP-1 B ELISA. In the 10 group 1 field-infected cattle, peak titers were observed in sera from weeks 5 and 6, and this was followed by a decline to a reciprocal titers of <80 in four of the surviving five cattle by week 14 (Table 4). In the surviving two cattle, antibody levels did not decline to baseline levels. Peak titers of 80 to 2,560 were detectable in five laboratory-infected cattle (group 5) during weeks 5 to 10 after tick infestation. This was followed by a decline to reciprocal titers of 10 to 40 between weeks 10 and 50 (Table 5). IFAT analysis of sera from other groups was not done.

TABLE 4.

Antibody titers to C. ruminantium by IFAT in cattle (group 1) naturally infested with A. hebraeum ticks on a heartwater-endemic farm

Animal no.a	Preexposure titer	Time of first detection of antibodies	Reciprocal titerc after cattle exposure in the field for following period (wk):
			1	2	5	6	10	14	20	30	40	50
115	<20	Wk 3	<20	<20	640	2,560
116	<20	Wk 4	<20	<20	160	1,280
122	<20	Wk 4	<20	<20	160	320
123	<20	Wk 5	<20	<20	80	640
107	<20	Wk 4	<20	<20	1,280	NDd	640
102	<20	Wk 2	<20	80	1,280	ND	320	80
120	<20	Wk 3	<20	<20	320	ND	320	40
127	<20	Wk 3	<20	<20	640	ND	320	40
111b	<20	Wk 3	<20	<20	80	ND	320	80	160	160	80
117b	<20	Wk 4	<20	<20	40	ND	640	160	160	80	80	80

^aAnimals 115, 116, 122, 123, 107, 102, 120, and 127 died of anaemia and babesiosis between weeks 6–14, and 111 died at week 36 from non-tick-related causes. 

^bTreated with acaricide to reduce tick burden. 

^cA cutoff reciprocal titer of 40 (2 × 1:20, the background titer) was decided after testing preinfection samples from each animal. 

^dND, not done. 

TABLE 5.

Antibody titers to C. ruminantium by IFAT of laboratory-infected cattle (group 5)

Animal no.	Preexposure titer	Time of first detection of antibodies	Reciprocal IFAT antibody titera for following period (wk):
			5	10	15	20	30	40	50
103	<20	Wk 2	2,560	1,280	320	160	80	80	40
108	<20	Wk 5	160	40	40	20	20	10	≤10
110	<20	Wk 4	80	40	20	10	10	≤10	≤10
112	<20	Wk 4	320	640	NDb	20	10	10	10
114	<20	Wk 4	640	160	ND	40	20	10	10

^aA cutoff reciprocal titer of 40 (2 × 1:20, the background titer) was decided after testing preinfection samples from each animal. 

^bND, not done. 

DISCUSSION

Previous evaluations of antibody kinetics by the MAP 1B indirect ELISA and by other serodiagnostic tests for heartwater have utilized sera from needle infections and in most cases have analyzed sera collected only during the acute phase of infection and a relatively short period after recovery. The unnatural and short-term nature of these studies is likely to have produced misleading results. The MAP 1B indirect ELISA has been described as a specific serological assay for detection of exposure to heartwater infections, since it detects fewer false-positives than other tests do (43). Its use in epidemiology and surveillance of heartwater is also proposed because it has a high sensitivity when primary-infection sera are tested. However, surprisingly, the use of this assay in determining the serological status of cattle in heartwater-endemic areas of Zimbabwe demonstrated low seropositivity (22). In the present study, we examined the persistence of antibodies detected by the MAP 1B indirect ELISA in both laboratory- and field-infected cattle which were infected and repeatedly exposed to C. ruminantium via the natural vector in Zimbabwe. The data demonstrate that this assay is a poor indicator of C. ruminantium exposure in cattle continually challenged by infected ticks. While cattle developed primary IgG responses to MAP 1B, these responses were not detectable from 14 to 28 weeks after experimental infections and 19 to 31 weeks after placement in a heartwater-endemic area. Occasionally, however, a few of the cattle showed short periodic peaks of seropositivity after testing negative in the MAP 1B ELISA. These cattle generally remained consistently negative by this ELISA despite frequent rechallenge with infected ticks and despite the fact that a carrier status was demonstrated. Detection of heartwater infections by PCR correlated well with antibodies detectable by MAP 1B ELISA during early infection (Tables 2 and 3). Later on, the correlation was less predictable. However, once the cattle tested negative by this MAP 1B ELISA, C. ruminantium could still be detected in the blood of some of the cattle by PCR.

These results demonstrate the down regulation of MAP 1B antigen-specific antibody responses in cattle postrecovery. The observed MAP 1B seroreversion would explain the low seroprevalence detected in cattle from heartwater-endemic areas of Zimbabwe (22), where approximately 33% of sera from heartwater-endemic cattle were found to be positive by MAP 1B ELISA. In this study, 29% of group 2 cattle and 20% of group 5 cattle which had become seronegative showed short periods of seropositivity followed again by seroreversion, which might explain the low percentage of field cattle in an endemic setting periodically testing MAP 1B positive. In one of the group 2 field-infected cattle, this second period of seropositivity coincided with the period when C. ruminantium was also detectable by PCR (Tables 2 and 3). These data suggest the possibility of (i) cyclical rickettsemia in carrier animals, (ii) a situation of new variants being expressed, or (iii) infection with a divergent C. ruminantium isolate. The data presented here also demonstrate that the high seronegativity of these cattle was not due to a lack of recognition of the MAP 1B antigen, since all laboratory and field-infected cattle produced IgG antibody responses with similar kinetics following primary infection with C. ruminantium. The reason for the seroreversion is unclear. It is possible that antigenic variation of the MAP 1B region is responsible for this seroreversion. MAP 1 genes of C. ruminantium isolates from different geographical areas contained three hypervariable regions whose sequences differ from each other by 0.6 to 14%, with a predicted protein sequence variation of 0.8 to 10% (33). The MAP 1B fragment of the mature MAP 1 protein contains the first hypervariable region. In addition, the MAP 1 gene of C. ruminantium is a member of a multigene family (38). It is possible that during persistent infection, in addition to antigenic variations, other genes of this MAP 1 family are expressed to produce a MAP 1B antigen fragment which does not have antigenic similarity to the antigen in this assay. Antigenic variants that emerge during persistent rickettsemia have been found in cattle infected with A. marginale (10). Similar work has not been done for C. ruminantium, and so it is not possible with the current available information to determine if this is also occuring with C. ruminantium persistent infections. There is also the possibility that some of the cattle clear the infection. This would explain why we were not able to confirm a carrier state in some cattle by xenodiagnosis and PCR, although very low rickettsemia could be another reason for lack of transmision and negative PCRs.

Serological analysis using MAP 1B ELISA was compared with immunoblotting and IFAT analysis of the same sera because these assays would detect responses to the MAP 1 antigen and other C. ruminantium antigens. By immunoblot and IFAT analysis, a similar phenomenon was observed, and antibody levels against MAP 1 and other C. ruminantium antigens in both laboratory- and field-infected cattle also declined, albeit at a lower rate. This observation indicates that down regulation of the production of antibodies against all C. ruminantium antigens seems to occur in continually challenged cattle. While more detailed studies are required on the factors influencing the fate of antibody responses, these preliminary results suggest that serological analysis based on the currently available tests may be misleading in determining the prevalence of C. ruminantium exposure in cattle populations. Direct detection of the infecting agent, such as PCR-based detection alone or in combination with serology, may provide a more reliable means of assessing C. ruminantium prevalence in all susceptible ruminant species that are affected by heartwater. In addition, the presence of infected tick vectors in areas of surveillance would be a recommended epidemiological parameter to assess heartwater prevalence.

This study also casts doubt on the reliable use of one MAP 1B antigen if antigenic variation of MAP 1B antigens exists, possibly along with other C. ruminantium antigens for the serodiagnosis of heartwater in recovered cattle. Further study is needed to determine whether the phenomenon of generalized down regulation of antibody responses to C. ruminantium in cattle also occurs in other ruminant species such as sheep and goats. Preliminary evaluation of goat populations in the same areas of Zimbabwe (where cattle exhibit a high seronegativity) indicates a high seropositivity by the indirect MAP 1B ELISA. Therefore, the phenomenon of seroreversion may be unique to continually challenged cattle. The results of this study highlight the need to critically assess the use and value of serodiagnostic assays for epidemiological studies of heartwater. Tests may be applied without adequate knowledge of their expected performance, and the results may be used to make erroneous decisions concerning disease management.