Development of inactivated heartwater (Ehrlichia ruminantium) vaccine in South Africa.

Heartwater, Ehrlichia ruminantium infection in cattle, sheep, goats, and some wild ruminants, is an economically important disease in Africa characterized by high mortality rates in susceptible populations. In South Africa, the current commercial heartwater vaccine is an infection and treatment type of immunization using virulent live E. ruminantium organisms generated from blood of infected sheep with subsequent treatment of the animals with antibiotics at specific times during the course of infection. This vaccine has several inherent problems preventing its wide use as the vaccine must be administered intravenously and it does not protect against all the South African field isolates. A vaccine based on inactivation of Zimbabwean E. ruminantium Mbizi strain organisms produced in endothelial cell cultures can be a sustainable option because it will not require antibiotic treatment and will be safe as there is no potential for reversion to virulence. Previous data generated in laboratory trials and under natural field setting provides support for this vaccine approach. Four inactivated vaccine formulations using the E. ruminantium Mbizi strain were tested for their efficacy in Merino sheep compared to an unvaccinated control group (11 sheep per group). Two vaccines were prepared by beta-propiolactone (BPL) inactivation, and two were inactivated with binary ethylenimine (BEI) while purification was done with both percoll and polyethylene glycol (PEG). The four vaccine preparations were formulated with Montanide ISA 50V2 adjuvant and administered twice subcutaneously (2 ml per dose) at an interval of 4 weeks. All groups were challenged with a virulent homologous cell-cultured E. ruminantium inoculated via the intra-venous route on day 56. The primary variable of efficacy was measured by the percentage survival rate or mortality between the Controls and Vaccine Groups. Three vaccine formulations (BEI/Percoll (Group 3), BEI/PEG (Group 4), BPL/Percoll, (Group 1) had a significantly higher percent of animal surviving challenge compared to the unvaccinated control (p-values 0.001, 0.035, 0.030, respectively). The highest number of survivors was obtained in Group 3 BEI/Percoll; 10/11 (91%). Groups 4 (BEI/PEG) and Group 1 (BPL/Percoll) produced similar percentage of survivals of 64%. In contrast, the lowest survival rate of 50% was observed in Group 2 (BPL/PEG) which was numerically different but not significantly different from the unvaccinated control which had an 18% survival rate (2/11). The inactivated vaccine using BEI or BPL as inactivating agents blended with ISA 50 adjuvant induced protective immunity against challenge. The BEI/Percoll (Group 3) vaccination regimen was most efficacious against a lethal heartwater challenge as it significantly protected sheep against mortality which is the most important aspect of heartwater infections. Future work should be directed towards improvement of this vaccine formulation especially from the down-stream processing point of view as the percoll method is not scalable for commercialization purposes.

Prospective evaluation of parental anxiety related to newborn foot disorder.

PURPOSE: The presence of a clubfoot is often found prenatally and some families seek counselling with a specialist. The purpose of this study was to compare the parental anxiety levels in families that: a) knew prenatally and had prenatal counselling; b) knew prenatally but did not seek prenatal counselling; and c) did not know until after delivery. METHODS: This prospective cohort study evaluated the anxiety of parents as they presented to the paediatric orthopaedic clinic with their newborn with a foot disorder (prior to the diagnostic confirmation of clubfoot). Each family filled out the 'Pre-visit orthopaedic surgeon questionnaire' and then after the initial visit with the orthopaedic surgeon (confirming the clubfoot diagnosis) the family filled out the 'Immediately post-visit orthopaedic surgeon questionnaire'. Through these questionnaires, anxiety level was assessed prior to meeting postnatally with the paediatric orthopaedic specialist, as well as after the meeting and compared across groups. RESULTS: A total of 121 parents completed questionnaires: 71% (86/121) confirmed clubfoot; 69% of families (59/86) received prenatal counselling (Group A); 16% (14/86) knew prenatally but had no counselling (Group B); and 15% (13/86) found out at birth (Group C). There was no difference in anxiety levels across groups before (p = 0.78) or after (p = 0.57) meeting with the paediatric orthopaedic surgeon; however, overall anxiety reduced significantly (p < 0.001). CONCLUSION: We found no difference in the anxiety levels of across the three groups. Prenatal counselling for parents of children with likely clubfoot may not decrease parental anxiety, but nonetheless is very appreciated by the families who receive it. LEVEL OF EVIDENCE: Prognostic Level II.

A comparison of screw versus drill and curettage epiphysiodesis to correct leg-length discrepancy.

PURPOSE: To compare two common surgical techniques of epiphysiodesis: drill/curettage epiphysiodesis (PDED) versus cross screw epiphysiodesis (PETS). The hypothesis is that the two techniques have similar efficacy but demonstrate differences in length of hospital stay (LOS), time to return to activity and complication rates. METHODS: A retrospective review of growing children and adolescents less than 18 years old who required an epiphysiodesis with leg-length discrepancy (LLD) of 2 cm to 6 cm with minimum two years of follow-up was conducted. Characteristics including age at surgery, gender, epiphysiodesis location, side, operative time, LOS and hardware removal were compared across treatment groups. LLD, expected growth remaining (EGR) and bone age were determined preoperatively and at most-recent visit. The correction ratio (change in EGR) was calculated along with a 95% confidence interval (CI) to assess if correction in leg length was achieved. RESULTS: A total of 115 patients underwent epiphysiodesis in the femur (53%), tibia (24%) or a combination (24%). The cohort was 47% male, with a mean age of 12.6 years (7.7 to 17.7) at surgery. Median follow-up was 3.7 years (2.0 to 12.7). In all, 23 patients underwent PETS and 92 patients had PDED. Both treatment groups achieved expected LLD correction. There was no significant difference in median operative time, complication rates or LOS. PETS patients returned to activity at a mean 1.4 months (interquartile range (IQR) 0.7 to 2.1) while PDED patients returned at a mean 2.4 months (IQR 1.7 to 3) (p < 0.001). CONCLUSION: Effectiveness in achieving expected correction, LOS and operative time are similar between screw and drill/curettage epiphysiodesis. Patients undergoing PETS demonstrated a faster return to baseline activity than patients with PDED. LEVEL OF EVIDENCE: III.

Clubfoot relapse: does presentation differ based on age at initial relapse?

PURPOSE: Treatment of idiopathic clubfoot with the Ponseti method is now standard, but predicting relapse can be difficult. Most experts recommend bracing to the age of four years, but this can be challenging for families, and may not be necessary in all patients. The purpose of this study is to compare patterns of bracing and age of relapse to help determine if predictable patterns exist. METHODS: The 70 patients with idiopathic clubfoot treated initially with the Ponseti technique who had relapse of their clubfoot were identified. Relapse was defined as a return to casting or surgery due to recurrent deformity. Data collected included demographics, treatment and brace adherence. Patients who sustained initial relapse before the age of two years were compared with those who sustained initial relapse after the age of two years. RESULTS: In total 56% (39/70) had their initial relapse prior to age two years while 44% (31/70) were after age two years. Of the patients who relapsed prior to the age of two years, 28% (11/39) were adherent with bracing while 72% were non--adherent. For patients who initially relapsed after age two, 74% (23/31) were adherent with bracing while 26% were non-adherent (p < 0.001). Of those who had initial -relapse -prior to age two, a subsequent relapse was seen in 69% (27/39). CONCLUSION: Patients with idiopathic clubfoot who experienced recurrence prior to age two years are significantly more likely to be non-adherent with bracing than those who sustain recurrence after age two. After initial relapse prior to age two, bracing adherence does not affect likelihood of subsequent recurrence.

Development of a Quantitative PCR Assay for Differentiating the Agent of Heartwater Disease, Ehrlichia ruminantium, from the Panola Mountain Ehrlichia.

Panola Mountain Ehrlichia (PME) is an emerging Ehrlichia sp. reported in ten US states. Based on the sequence homology of all known genes, PME is closely related to Ehrlichia ruminantium (ER), the causative agent of heartwater. Heartwater is an economically important tick-borne disease of cattle, sheep and goats responsible for stock losses in sub-Saharan Africa. Unfortunately, ER was imported to the Caribbean islands in the 19th century, and the presence of this foreign animal disease in the Caribbean poses a threat to the US mainland. If introduced, a heartwater outbreak would cause massive losses of naïve livestock. The serologic assay of choice to diagnose heartwater is cross-reactive with Ehrlichia spp., including PME, as we demonstrate here, which would confound disease surveillance in the event of a heartwater outbreak. The purpose of this study was to develop a diagnostic assay capable of rapidly distinguishing between these pathogens. Using synthetic MAP-1B peptides for ER and PME, we tested the cross-reactivity of this assay using sera from infected livestock. The MAP-1B ELISA cannot distinguish between animals infected with PME and ER. Therefore, a dual-plex Taqman™ qPCR assay targeting the groEL gene of PME and ER was developed and validated. Primers were designed that are conserved among all known strains of ER, allowing for the amplification of strains from the Caribbean and Africa. The assay is highly sensitive (10 copies of DNA) and specific. This assay distinguishes between infection with PME and ER and will be a valuable tool in the event of heartwater outbreak on the US mainland, or for epidemiological studies involving either disease-causing organism.

Ehrlichia ruminantium in Amblyomma variegatum and domestic ruminants in the Caribbean.

The highly sensitive nested pCS20 polymerase chain reaction assay for Ehrlichia ruminantium was negative on 506 Amblyomma variegatum from Caribbean islands where clinical heartwater has not been reported, mainly the United States Virgin Islands (18), Dominica (170), Montserrat (5), Nevis (34), St. Kitts (262), and St. Lucia (17). Positive results were obtained with positive controls (Crystal Springs strain) and A. variegatum from countries in Africa where infections are endemic, mainly Tanzania (1/37) and Burkino Faso (2/29). Positive major antigenic protein-1 enzyme-linked immunosorbent assays for E. ruminantium were obtained on convenience samples of sera from apparently healthy cattle, sheep, and goats on Dominica (0/95, 0%; 3/135, 2%; 2/57, 4%), Grenada (0/4, 0%; 1/98, 1%; 1/86, 1%), Montserrat (0/12, 0%; 0/28, 2%; 5/139, 4%), Nevis (0/45, 0%; 0/157, 0%; 0/90, 0%), Puerto Rico (0/422, 0%; 0, 0%), St. Kitts (3/86, 4%; 1/25, 0%; 0/26, 0%), and St. Lucia (0/184, 0%; 0/15, 0%; 0, 0%), respectively. The pCS20 polymerase chain reaction results indicate E. ruminantium is not present on islands where clinical heartwater does not occur. The occasional positive major antigenic protein-1B enzyme-linked immunosorbent assay results appear, then, to be false-positive reactions, and serology appears to be of limited use in testing for E. ruminantium in the Caribbean, as is the case in Africa.

A preliminary study to evaluate the immune responses induced by immunization of dogs with inactivated Ehrlichia canis organisms.

Ehrlichia canis is an intracellular pathogen that causes canine monocytic ehrlichiosis. Although the role of antibody responses cannot be discounted, control of this intracellular pathogen is expected to be by cell mediated immune responses. The immune responses in dogs immunized with inactivated E. canis organisms in combination with Quil A were evaluated. Immunization provoked strong humoral and cellular immune responses, which were demonstrable by Western blotting and lymphocyte proliferation assays. By Western blotting antibodies to several immunodominant E. canis proteins were detected in serum from immunized dogs and antibody titres increased after each immunization. The complement of immunogenic proteins recognized by the antisera were similar to those recognized in serum from infected dogs. Upon challenge with live E. canis, rapid anamnestic humoral responses were detected in the serum of immunized dogs and primary antibody responses were detected in the serum from control dogs. Following immunization, a lymphocyte proliferative response (cellular immunity) was detected in peripheral blood mononuclear cells (PBMNs) of immunized dogs upon stimulation with E. canis antigens. These responses were absent from non-immunized control dogs until after infection with live E. canis, when antigen specific-lymphocyte proliferation responses were also detected in the PBMNs of the control dogs. It can be thus concluded that immunization against canine monocytic ehrlichiosis may be feasible. However, the immunization regimen needs to be optimized and a detailed investigation needs to be done to determine if this regimen can prevent development of acute and chronic disease.

The pCS20 PCR assay for Ehrlichia ruminantium does not cross-react with the novel deer ehrlichial agent found in white-tailed deer in the United States of America.

White-tailed deer are susceptible to heartwater (Ehrlichia [Cowdria] ruminantium infection) and are likely to suffer high mortality if the disease spreads to the United States. It is vital, therefore, to validate a highly specific and sensitive detection method for E. ruminantium infection that can be reliably used in testing white-tailed deer, which are reservoirs of antigenically or genetically related agents such as Ehrlichia chaffeensis, Anaplasma (Ehrlichia) phagocytophilum (HGE agent) and Ehrlichia ewingii. Recently, a novel but as yet unnamed ehrlichial species, the white-tailed deer ehrlichia (WTDE), has been discovered in deer populations in the United States. Although the significance of WTDE as a pathogen is unknown at present, it can be distinguished from other Ehrlichia spp. based on 16S rRNA gene sequence analysis. In this study it was differentiated from E. ruminantium by the use of the pCS20 PCR assay which has high specificity and sensitivity for the detection of E. ruminantium. This assay did not amplify DNA from the WTDE DNA samples isolated from deer resident in Florida, Georgia and Missouri, but amplified the specific 279 bp fragment from E. ruminantium DNA. The specificity of the pCS20 PCR assay for E. ruminantium was confirmed by Southern hybridization. Similarly, the 16S PCR primers (nested) that amplify a specific 405-412 bp fragment from the WTDE DNA samples, did not amplify any product from E. ruminantium DNA. This result demonstrates that it would be possible to differentiate between E. ruminantium and the novel WTDE agent found in white tailed deer by applying the two respective PCR assays followed by Southern hybridizations. Since the pCS20 PCR assay also does not amplify any DNA products from E. chaffeensis or Ehrlichia canis DNA, it is therefore the method of choice for the detection of E. ruminantium in these deer and other animal hosts.

Some observations on the sero-prevalence of heartwater and tick infestation in Zambian goats.

A survey was carried out to define the distribution of heartwater in goats that originated from six districts in communal grazing semi-arid areas of Zambia. A total of 181 samples (40.1%) out of 451 serum samples from adult goats were positive for Ehrlichia ruminantium antibodies after screening using indirect MAP-1B antigen ELISA technique with statistically significant differences (P < 0.01) between the six districts. Out of 1 036 adult goats examined for tick infestation, 105 were infested by ticks, with Amblyomma species being the most dominant tick encountered. Amblyomma variegatum, which is the vector for heartwater transmission in Zambia constituted 42.4% of the tick species, identified. The overall tick infestation rate was 10% while the tick:goat ratio was 2.1:1. Amblyomma variegatum appears to be widespread throughout the study area, as are antibodies to E. ruminantium.

Development of improved vaccines for heartwater.

Heartwater is controlled by frequent application of acaricides, which is costly, creates endemic instability and has the potential of contaminating the environment. The live blood vaccine currently available has limitations because it is laborious and inconvenient to use, difficult to standardise and can transmit other blood-borne pathogens. The UF/USAID/SADC Heartwater Research Project has conducted research on the development of two types of vaccine for heartwater. The first-generation inactivated vaccine has been intensively tested in the laboratory and subsequently field tested in four southern African countries. It protects cattle, sheep and goats against mortality from heartwater challenge. It can be modified to incorporate any Ehrlichia ruminantium strain to provide protection from field challenge. The second-generation DNA vaccine containing genes encoding immunogenic E. ruminantium proteins has been developed and evaluated in the mouse model as well as in cattle and sheep. The use of improved vaccines against heartwater would have a positive impact on livestock farming in sub-Saharan Africa and the Caribbean and could be used to control the spread of heartwater if it were to be introduced into regions such as the United States.

Comparing the detection of exposure to Ehrlichia ruminantium infection on a heartwater-endemic farm by the pCS20 polymerase chain reaction assay and an indirect MAP1-B enzyme linked immunosorbent assay.

Detection of heartwater is not always easy especially because all the serological assays so far available either have poor sensitivity or specificity. The indirect MAP-1B ELISA has been reported to be the most specific test for heartwater, although it does also detect antibodies to some closely related ehrlichial agents. This study was undertaken to compare two methods for the detection of heartwater infection caused by the ehrlichial agent Ehrlichia (Cowdria) ruminantium. Fifteen cattle on a heartwater-endemic farm infested with high numbers of Amblyomma hebraeum ticks, and hence exposure to E. ruminantium infection were monitored over an 8-week period by pCS20 PCR and an indirect MAP-1B ELISA. Infection was detected by pCS20 PCR in most animals with the highest number of positives (60%) in week 6 of the study. Similarly, exposure to E. ruminantium was detected by indirect MAP-1B ELISA in some animals, with the highest number of seropositives (27%) at weeks 2-6 of the study. The data demonstrated a fluctuating rickettsaemia in cattle in a heartwater-endemic area. Comparison of the two tests indicated that the pCS20 PCR assay was more reliable because it detected more infections than the indirect MAP-1B ELISA and would therefore be the method of choice for detection of E. ruminantium infection.

Detection of antibodies to the Ehrlichia ruminantium MAP1-B antigen in goat sera from three communal land areas of Zimbabwe by an indirect enzyme-linked immunosorbent assay.

A total of 1,286 caprine serum samples collected from three communal land areas in Zimbabwe from March 1999 to February 2000 were tested for Ehrlichia ruminantium antibodies using the indirect MAP1-B enzyme-linked immunosorbent assay. Of the 480 samples tested from Mudzi, a non-heartwater area, 425 (89.4 %) were positive. In the heartwater endemic areas, of the 441 samples 352 (79.4 %) from Gwanda and 300 of the 365 samples (83.2 %) from Bikita tested positive. The seroprevalence in the Bikita and Gwanda (approaching 90 %) is consistent with reports in related serological surveys that puts the seroprevalence of E. ruminantium in goats from endemic areas of Zimbabwe at 90 %. However, the high seroprevalence in the non-heartwater area of Mudzi is unexpected and can be a result of the presence of a serologically cross-reacting organism, which has to be isolated and characterized. The results need to be confirmed by alternative tests, based on molecular diagnostic tools. There were no significant differences in seroprevalence between the three sampling areas as there were between the three sampling periods. The highest corresponded with the period January to February (peak tick activity) and the lowest with the period July to September (minimal tick activity).

Introduction of the exotic tick Amblyomma hebraeum into Florida on a human host.

A resident of Florida returned from a short visit to southern Africa to find a male Amblyomma hebraeum tick attached to the skin behind her knee. Amblyomma hebraeum is a major vector of 2 pathogens that cause important diseases in southern Africa, heartwater of ruminants and African tick-bite fever of humans. The tick was tested by polymerase chain reaction assay for evidence of infection with Cowdria ruminantium and Rickettsia africae (the causative agents of heart-water and African tick-bite fever, respectively) and was found to be negative for both agents. This is the second record of the exotic tick, A. hebraeum, being introduced into the United States on a human host.

The prevalence of Cowdria ruminantium in free-living adult Amblyomma hebraeum collected at a communal grazing area and in 2 wildlife reserves in South Africa.

In order to detect the prevalence of Cowdria ruminantium in the vector tick, Amblyomma hebraeum, free-living, unfed adult ticks were collected with the aid of pheromone/CO2 traps. Ticks were collected at the Rietgat communal grazing area, as well as in the southwestern Kruger National Park and in the Songimvelo Game Reserve, all located in heartwater-endemic areas of South Africa. The presence of C. ruminantium in these ticks was determined by polymerase chain reaction (PCR) analysis. Ticks from the Rietgat communal grazing area were assayed in 2 batches and 4.7% of the one and 11.3% of the other were positive for infection, while 5.7% of the ticks collected in the Kruger National Park and 25% in the Songimvelo Game Reserve were positive. These results support the contention that a vector-wildlife cycle of transmission of C. ruminantium, the cause of heartwater in domestic ruminants, can be maintained in the absence of the latter animals.

A subset of Cowdria ruminantium genes important for immune recognition and protection.

Cowdria ruminantium causes the tick-borne rickettsial disease of heartwater, which is devastating to livestock production in sub-Saharan Africa. Current diagnosis and control methods are inadequate. We have identified and sequenced a subset of genes encoding recombinant antigens recognized by antibody and peripheral blood mononuclear cells from immune ruminants. The identified genes include many with significant similarity to those of Rickettsia prowazekii, genes predicted to encode different outer membrane proteins and lipoproteins and a gene containing an unusual tandem repeat structure. Evidence is presented for immune protection by recombinant antigens in a mouse model of C. ruminantium infection. These data identify new recombinant antigens for evaluation in vaccines and diagnostic tests to control heartwater.

Reduction in mortality from heartwater in cattle, sheep and goats exposed to field challenge using an inactivated vaccine.

Inactivated vaccines for heartwater prepared with the commercially acceptable Montanide ISA 50 (ISA 50) adjuvant were field tested in Boer goats in Botswana, Angora goats in South Africa, and Merino sheep in Zambia and Zimbabwe. Two vaccines, one made using the Zimbabwean Mbizi isolate and the other using the respective local field isolate (Sunnyside in Botswana; Bathurst in South Africa; Lutale in Zambia), were tested at each site, except in Zimbabwe where only the Mbizi vaccine was tested. Compared with unvaccinated animals, the Mbizi vaccine significantly protected goats and sheep against field Amblyomma tick challenge in Botswana, Zambia and Zimbabwe (P = 0.018, 0.002 and 0.017, respectively), but failed to protect Angora goats in South Africa. However, in South Africa the vaccine prepared using the local field isolate Bathurst, induced significant protection (P=0.008). The vaccines containing the local isolates at all other sites were less protective than the Mbizi vaccine. The Mbizi inactivated vaccine also significantly protected 17 of 21 cattle (P = 0.05) against heartwater challenge from field ticks in Zimbabwe. Against the same challenge only 7 of 21 unvaccinated control cattle survived. This study demonstrates that heartwater is a major constraint to upgrading livestock in endemic areas, and caused an overall mortality of 77.6% in naive sheep and goats (97 of 125 died) and 67% in cattle (14 of 21 died). In contrast, the vaccine had a protective effect by reducing the overall mortality in sheep and goats to 54.3% (113 of 208 died) and to 19% in cattle (4 of 21 died).

Resistance of leopard tortoises and helmeted guineafowl to Cowdria ruminantium infection (heartwater).

Experimental infection trials were conducted to investigate susceptibility of leopard tortoises (Geochelone pardalis) and helmeted guineafowl (Numida meleagris) to infection with Cowdria ruminantium, the causative agent of heartwater, a tickborne disease of domestic and wild ruminants. Ten guineafowl were inoculated intravenously with a virulent dose of C. ruminantium derived from bovine endothelial cell cultures, and four leopard tortoises were exposed to C. ruminantium infection by the feeding of infected Amblyomma hebraeum ticks. Uninfected A. hebraeum ticks (on both tortoises and guineafowl) and Amblyomma marmoreum ticks (on tortoises only) were fed on the animals during weeks 2 and 3 post-exposure in an attempt to detect infection. These ticks were analyzed for C. ruminantium infection by xenodiagnosis and with the C. ruminantium-specific pCS20 polymerase chain reaction (PCR) assay. Attempts to detect infection in ticks fed on either species were negative by both tests. These results suggest that leopard tortoises and helmeted guineafowl are refractory to C. ruminantium infection and, therefore, are unlikely to be capable of introducing heartwater directly into new areas. However, leopard tortoises are efficient hosts of A. marmoreum and A. hebraeum and are likely to be important epidemiologically in the transport and maintenance of these tick vector species.

Antibody responses to MAP 1B and other Cowdria ruminantium antigens are down regulated in cattle challenged with tick-transmitted heartwater.

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.

Evidence of Cowdria ruminantium infection (heartwater) in Amblyomma sparsum ticks found on tortoises imported into Florida.

Amblyomma marmoreum and A. sparsum ticks were collected from tortoises imported into Florida from Africa and were tested for Cowdria ruminantium infection using a C. ruminantium-specific pCS20 polymerase chain reaction assay. In I shipment imported from Zambia, 15 of the 38 A. sparsum male ticks collected from the leopard tortoises (Geochelone pardalis) were found to be positive for infection with C. ruminantium. In contrast, all 148 A. marmoreum tested were negative for C. ruminantium infection. This is the first reported evidence of the introduction of heartwater-infected ticks into the United States, but there were no opportunities to confirm isolation of C. ruminantium from the ticks by either culture or transmission studies.

A polarized Th1 type immune response to Cowdria ruminantium infection is detected in immune DBA/2 mice.

Immune responses to Cowdria ruminantium, an intracellular organism that causes heartwater in domestic ruminants, were characterized in a DBA/2 mouse model. Immunity induced by infection and treatment was adoptively transferable by splenocytes and could be abrogated by in vivo depletion of T cells but not by inhibition of nitric oxide synthase using NG-monomethyl-L-arginine. IgG2a and IgG2b C. ruminantium-specific responses were detected in immune mice. Culture supernatants of splenocytes from immune DBA/2 mice, which were stimulated with crude C. ruminantium antigens or recombinant major antigenic proteins 1 or 2, contained significant levels of interferon (IFN)-gamma and interleukin (IL)-6, but insignificant levels of IL-1alpha, IL-2, IL-4, IL-5, IL-10, IL-12, tumor necrosis factor-alpha (TNF), and nitric oxide. A similar response was detected during primary infection, although IFN-gamma levels decreased significantly during clinical illness and then increased following natural or antibiotic-aided recovery. These data support the conclusion that protective immunity to C. ruminantium in DBA/2 mice is mediated by T cells and is associated with a polarized T helper 1 type of immune response. This murine model could be utilized to screen for protective C. ruminantium antigens that provoke Th1 type immune responses and for evaluation of these antigens in recombinant vaccines against heartwater.