INTRODUCTION

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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

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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.

(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.

View larger version (66K): [in this window] [in a new window]   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.

	RESULTS

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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.

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).

View larger version (23K): [in this window] [in a new window]   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.

(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.

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.

View larger version (91K): [in this window] [in a new window]   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.

	DISCUSSION

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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.