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.     

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.     

A quantitative real-time PCR assay for Ehrlichia ruminantium using pCS20

Heartwater is a tick borne disease that affects ruminants and wild animals in Africa south of the Sahara. It is caused by Ehrlichia ruminantium and transmitted by the tick Amblyomma hebraeum. The protocols currently used to detect heartwater take several days to complete. Here, we describe the development of a pCS20 quantitative real-time PCR TaqMan probe assay to detect E. ruminantium in livestock blood and ticks from the field. The assay is based on the conserved pCS20 gene region of E. ruminantium that contains two overlapping genes, rnc and ctaG [Collins, N.E., Liebenberg, J., De Villiers, E.P., Brayton, K.A., Louw, E., Pretorius, A., Faber, F.E., Van Heerden, H., Josemans, A., Van Kleef, M., Steyn, H.C., Van Strijp, M.F., Zweygarth, E., Jongejan, F., Maillard, J.C., Berthier, D., Botha, M., Joubert, F., Corton, C.H., Thomson, N.R., Allsopp, M.T., Allsopp, B.A., 2005. The genome of the heartwater agent Ehrlichia ruminantium contains multiple tandem repeats of actively variable copy number. PNAS 102, 838-843]. The pCS20 quantitative real-time PCR TaqMan probe was compared to the currently used pCS20 PCR and PCR/(32)P-probe test with regards to sensitivity, specificity and the ability to detect DNA in field samples and in blood from experimentally infected sheep. This investigation showed that the pCS20 quantitative real-time PCR TaqMan probe was the most sensitive assay detecting seven copies of DNA/mul of cell culture. All three assays, however, cross react with Ehrlichia canis and Ehrlichia chaffeensis. The pCS20 real-time PCR detected significantly more positive field samples. Both the PCR and pCS20 real-time PCR could only detect E. ruminantium parasites in the blood of experimentally infected sheep during the febrile reaction. The PCR/(32)P-probe assay, however, detected the parasite DNA 1 day before and during the febrile reaction. Thus, because this new quantitative pCS20 real-time PCR TaqMan probe assay was the most sensitive and can be performed within 2h it is an effective assay for epidemiological surveillance and monitoring of infected animals.     

Heartwater. An overview of the clinical signs, susceptibility and differential diagnoses of the disease in domestic ruminants

Heartwater is a frequently fatal tick-borne disease of ruminants caused by Cowdria ruminantium. In domestic ruminants the incubation period varies considerably and depends on the route of infection, virulence of the isolate and amount of infective material administered. Adult cattle of all breeds appear to be equally susceptible to heartwater. It is generally accepted that calves up to the age of 3 weeks have a high degree of natural resistance which is not related to the immune status of the dam. Nervous symptoms are frequently seen in animals affected by the peracute and acute forms of heartwater and can easily be confused with similar signs caused by infectious conditions, toxic plants, acaricide and heavy metal poisonings.     

Simultaneous detection of Anaplasma and Ehrlichia species in ruminants and detection of Ehrlichia ruminantium in Amblyomma variegatum ticks by reverse line blot hybridization

The detection of Anaplasma and Ehrlichia species is usually based on species-specific PCR assays, since no assay is yet available which can detect and identify these species simultaneously. To this end, we developed a reverse line blot (RLB) assay for simultaneous detection and identification of Anaplasma and Ehrlichia species in domestic ruminants and ticks. In a PCR the hypervariable V1 region of the 16S ribosomal RNA (rRNA) gene was amplified with a set of primers unique for members of the genera Anaplasma and Ehrlichia [Int. J. Syst. Evol. Microbiol. 51 (2001) 2145]. Amplified PCR products from blood of domestic ruminants or Amblyomma variegatum tick samples were hybridized onto a membrane to which eight species-specific oligonucleotide probes and one Ehrlichia and Anaplasma catch-all oligonucleotide probe were covalently linked. No DNA was amplified from uninfected blood, nor from other hemoparasites such as Theileria annulata, or Babesia bigemina. The species-specific probes did not cross-react with DNA amplified from other species. E. ruminantium, A. ovis and another Ehrlichia were identified by RLB in blood samples collected from small ruminants in Mozambique. Finally, A. variegatum ticks were tested after feeding on E. ruminantium infected sheep. E. ruminantium could be detected in adult ticks even if feeding of nymphs was carried out 3.5 years post-infection. In conclusion, the developed species-specific oligonucleotide probes used in an RLB assay can simultaneously detect and identify several Ehrlichia and Anaplasma species. However, as no quantitative data for the detection limit are available yet, only positive results are interpretable at this stage.     

Problems with the interpretation of epidemiological data in heartwater: a study on 23 farms

In an epidemiological study undertaken on 23 farms where heartwater occurs endemically, it was found that on an overall average, antibodies to Cowdria ruminantium were detected in 64.3% of the cattle, 6 adult Amblyomma hebraeum ticks were counted per animal and 7.0% of ticks were infected with the heartwater agent. It was found that the seropositivity of the animals was determined largely by the tick loads to which they were subjected and that the influence of the tick C. ruminantium infection rate was less evident. There was no parallel between the prevalence of heartwater on the farms and the immune status of the animals. In general, higher tick counts were recorded in herds where strategic tick control is practised than on farms with a total tick control programme. The method of tick control did not, however, appear to influence the immune status of the cattle, the tick infection rate, or the prevalence of heartwater.     

Immunity of tick-exposed seronegative and seropositive small stock challenged with two stocks of Cowdria ruminantium

Nineteen per cent and 32% of serologically positive sheep and goats in heartwater endemic areas of the Republic of South Africa were susceptible to challenge with, respectively, the Ball 3 and Welgevonden stocks of Cowdria ruminantium. There was good correlation between the results of the indirect fluorescent antibody test and immunity, since without exception all the seronegative animals reacted when they were challenged. The fact that only 74% of the seropositive sheep and goats were immune to challenge can probably be ascribed to the poor cross-protection between stocks of C. ruminantium and not to false positive serological reactions.     

Veterinary extension on sampling techniques related to heartwater research

Heartwater, a tick-borne disease caused by Ehrlichia ruminantium, is considered to be a significant cause of mortality amongst domestic and wild ruminants in South Africa. The main vector is Amblyomma hebraeum and although previous epidemiological studies have outlined endemic areas based on mortalities, these have been limited by diagnostic methods which relied mainly on positive brain smears. The indirect fluorescent antibody test (IFA) has a low specificity for heartwater organisms as it cross-reacts with some other species. Since the advent of biotechnology and genomics, molecular epidemiology has evolved using the methodology of traditional epidemiology coupled with the new molecular techniques. A new quantitative real-time polymerase chain reaction (qPCR) test has been developed for rapid and accurate diagnosis of heartwater in the live animal. This method can also be used to survey populations of A. hebraeum ticks for heartwater. Sampling whole blood and ticks for this qPCR differs from routine serum sampling, which is used for many serological tests. Veterinary field staff, particularly animal health technicians, are involved in surveillance and monitoring of controlled and other diseases of animals in South Africa. However, it was found that the sampling of whole blood was not done correctly, probably because it is a new sampling technique specific for new technology, where the heartwater organism is much more labile than the serum antibodies required for other tests. This qPCR technique is highly sensitive and can diagnose heartwater in the living animal within 2 hours, in time to treat it. Poor sampling techniques that decrease the sensitivity of the test will, however, result in a false negative diagnosis. This paper describes the development of a skills training programme for para-veterinary field staff, to facilitate research into the molecular epidemiology of heartwater in ruminants and eliminate any sampling bias due to collection errors. Humane handling techniques were also included in the training, in line with the current focus on improved livestock welfare.     

Genetic diversity of Ehrlichia ruminantium in Amblyomma variegatum ticks and small ruminants in The Gambia determined by restriction fragment profile analysis

Understanding genetic diversity of Ehrlichia ruminantium in host and vector populations is an important prerequisite to controlling heartwater by vaccination in traditional livestock systems in sub-Saharan Africa. We carried out a study in two phases: (i) evaluating the usefulness of the PCR-RFLP assay based on the map1 coding sequence of E. ruminantium as a discriminatory tool to characterise genetic diversity, (ii) applying the technique to field samples from Amblyomma variegatum ticks and small ruminants to characterise genotypic diversity of the organism in three main agroecological zones of The Gambia, Sudano-Guinean (SG), Western Sudano-Sahelian (WSS) and Eastern Sudano-Sahelian (ESS). Restriction fragment length polymorphisms were observed among different strains of E. ruminantium supporting the usefulness of the PCR-RFLP technique for studying genetic diversity of the organism. Restriction enzyme map1 profile analysis indicated the presence in The Gambia of multiple genotypes (at least 11) of E. ruminantium with sites in the WSS and SG zones showing comparatively high number of diverse genotypes. Profiles similar to the Kerr Seringe genotype (DQ333230) showed the highest distribution frequency, being present at sites in all three agroecological zones, thereby making the strain a suitable candidate for further characterisation in cross-protection studies. An additional three genotypes showed relatively high distribution frequency and were present in all three zones making them equally important for isolation and subsequent characterisation. The study demonstrated the occurrence of mixed infections with E. ruminantium genotypes in ruminants and ticks.     

Heartwater in Nigeria

A study was carried out to determine which materials from animals dying or dead of heartwater could initiate the disease in susceptible goats, using the intravenous and subcutaneous routes. C. ruminantium was consistently isolated by intravenous injection of the whole blood or of lung macrophages and by subcutaneous injection of brain homogenate. In animals dead of heartwater, it appeared that isolation of the organism was achieved only when extensive post-mortem autolysis had not supervened. Experiments with blood fractions showed that leucocytic and plasma fractions of infective blood transmitted heartwater; the erythrocytic fraction consistently failed to induce an infection.     

Characterization of the pCS20 region of different Ehrlichia ruminantium isolates

Heartwater is a serious tick-borne disease of ruminants caused by the rickettsial organism Ehrlichia (Cowdria) ruminantium. A diagnostic test, targeting the pCS20 genomic region and using PCR amplification and probe hybridization, detects E. ruminantium infection in ticks and animals. However, only the pCS20 sequence of the Crystal Springs E. ruminantium isolate is available and the existence of sequence variation amongst different E. ruminantium isolates has not been determined. Primers were designed from the published pCS20 sequence to obtain sequences of the pCS20 region of various E. ruminantium isolates. These primers were unable to amplify the pCS20 region from genomic Welgevonden DNA and genome walking was used to characterize the pCS20 region. This technique showed that the published pCS20 sequence is from a chimeric clone. Sequences of the pCS20 region of 14 different E. ruminantium isolates were determined after amplification with newly designed primers. Sequencing data indicated that West African E. ruminantium isolates are highly conserved, whereas more variation occurs amongst the southern African isolates. These results facilitated the design of a short pCS20 probe and a large PCR target that improved the sensitivity of the E. ruminantium detection assay.     

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.     

Identification of drugs effective against Cowdria ruminantium using a mouse screening system

Heartwater is a tick-transmitted rickettsial infection of ruminants, caused by Cowdria ruminantium. The tetracyclines are the only compounds available for therapy of the disease. A screen, using mice infected with C. ruminantium, was developed and used to identify new compounds with potential for the control of heartwater. A series of di-4-methyl-thiosemicarbazones was shown to be highly active in the mouse model and their efficacy was confirmed in further trials in sheep infected with C. ruminantium. The mouse screen was shown to be simple to operate and reliably predictive of activity against heartwater. Ways in which the screen may be improved are suggested.     

The application of the indirect fluorescent antibody test in research on heartwater

The preparation of the antigen, details of the reagents, the titration of the antispecies conjugates and the execution of the indirect fluorescent antibody test are described. The sensitivity and specificity of the test and its applicability to the detection of antibodies to Cowdria ruminantium are recorded. The test is both highly specific and sensitive and can be applied to a wide range of studies on heartwater, including epidemiology, determination of the C. ruminantium infection rate of Amblyomma ticks and the evaluation of immunization against heartwater. The test can also be used to detect antibodies to the heartwater agent in the sera of game.     

Heartwater. The artificial transmission of Cowdria ruminantium in domestic ruminants and mice

The artificial transmission of Cowdria ruminantium with infected blood, organ homogenates, peritoneal macrophages, tick stabilate and tissue culture cells is discussed. Organ homogenates prepared from the myocardium, spleen, kidneys and liver of diseased animals are commonly used to infect mice. The efficacy of organ homogenates as a source of C. ruminantium depends on factors such as the route of inoculation and the heartwater isolate used. Heartwater is artificially transmitted with infected tick stabilate, haemocytes, rectal ampules and hypodermal homogenates. The infectivity of saliva collected from Amblyomma hebraeum female ticks was very low compared to the ground-up suspensions prepared from the same group of ticks.     

Asymptomatic carrier state in Creole goats and cattle after recovery from Cowdria infection in Guadeloupe]

Creole goats and cattle in Guadeloupe can be carriers of cowdriosis (heartwater: Cowdria ruminantium) after recovery for a period as long as 11 months in goats and 2 months in cattle. The carrier status was demonstrated by feeding Amblyomma variegatum nymphs on recovered animals and the resulting adult ticks on susceptible goats. Cowdria ruminantium was not detected permanently during the carrier status.     

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.     

Epidemiology of heartwater in Guadeloupe and in the Caribbean

At present, heartwater in the Caribbean is known with certainty only on Guadeloupe, Marie Galante and Antigua; the first 2 islands are widely infected. The most important factors responsible for particular aspects of heartwater in Guadeloupe are: Cowdria ruminantium of high virulence. A very resistant cattle population (Creole), not normally clinically affected. A fairly susceptible goat population (Creole) (22% goats born in endemic areas die after experimental inoculation) which, fortunately, includes breeding lines with inherited resistance characteristics. Amblyomma variegatum which is present all over the island and all through the year, but with a low infection rate (1-2% of adult ticks are infected) because of the short period of rickettsemia in infected animals. The low rate of tick infection results in a low endemicity of the disease. For goats, the epidemiologic situation can be regarded as unstable because the low rate of infection in ticks does not allow a natural immunization of the majority of young kids when they still have a non-specific resistance. The possible evolution of heartwater in the Caribbean and in the United States in considered.     

A method for determining the Cowdria ruminantium infection rate of Amblyomma hebraeum: effects in mice injected with tick homogenates

Amblyomma hebraeum ticks, collected in the field and individually homogenized, were injected into mice. Thirteen out of 240 ticks were shown to be infected with the heartwater agent. Antibodies against Cowdria ruminantium were detected in the sera of the mice by means of the indirect fluorescent antibody test. Giemsa-stained smears, prepared from the haemocytes of the ticks, revealed morphologically different forms of the heartwater agent. A strain of C. ruminantium, designated the Welgevonden strain, was isolated in mice from one of the infected ticks and passaged in mice for 8 generations. When inoculated intravenously, it was highly infective to mice, sheep and cattle. The murinotropism of the Welgevonden strain is compared with that of other strains previously described.