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SUMMARY
Heartwater (also known as cowdriosis) is a rickettsial disease of ruminants caused by Ehrlichia ruminantium (formerly Cowdria ruminantium) and transmitted by Amblyomma ticks. It occurs in nearly all sub-Saharan countries of Africa and in Madagascar, and also in the Caribbean, threatening the American mainland.
Clinically, the disease is characterised by a sudden high fever, often hydropericardium, lung oedema and, in acute and peracute forms, by nervous signs and a high mortality. Subacute heartwater also occurs, and has a higher recovery rate.
Wild animals could play a role as reservoir; Rusa deer seem to be the only wild ruminant in which heartwater has an economic impact.
Identification of the agent: The specific diagnosis of heartwater is based on the observation of colonies of E. ruminantium in capillary endothelial cells of the brain. In the absence of adequate tools, a piece of cerebellum can easily be removed with a curette through the foramen magnum after cutting off the head, while a sample of cerebral cortex can be obtained through a hole made in the skull with a hammer and a large nail. Brain smears are prepared by crushing a small piece of cerebral or cerebellar cortex between two microscope slides. The capillaries are spread out in a single cell layer by drawing one slide across the other. The smears are air-dried, fixed with methanol and stained with Giemsa. With fast stains, the smears can be fixed and stained in under 1 minute. The colonies (clusters) are reddish-purple to blue, and very often close to the nucleus of the infected endothelial cell. They can be scanty and difficult to find, particularly in peracute cases, but they are always present in the brain of a ruminant that died from heartwater, if not treated with drugs. The colonies are still visible 2 days after death in a brain that has been stored at room temperature and up to 34 days in a brain that has been stored in a refrigerator.
Fresh whole blood collected from suspect animals can be inoculated intravenously into a susceptible sheep or goat. The development of clinical signs and the demonstration of Ehrlichia in the brain of the inoculated animal are diagnostic for heartwater.
Ehrlichia ruminantium can be isolated from the blood of an infected host using cultivation on ruminant endothelial cells. When a cytopathic effect consisting of plaques of cell lysis appears, the presence of characteristic morulae is confirmed by staining the cell monolayer with eosin-methylene blue or by immunofluorescence or immunoperoxidase techniques using a specific antiserum.
DNA probes and especially the more sensitive polymerase chain reaction (PCR) techniques are available to reveal the presence of E. ruminantium in the blood of actively infected animals, to a lesser extent in the blood or bone marrow of carrier animals, and in the tick vectors. Apart from diagnosis, PCR is widely used for research on the Ehrlichia genome and for epidemiological studies.
Serological tests: Serological tests include indirect fluorescent antibody tests, enzyme-linked immunosorbent assays (ELISAs) and Western blotting. However, when the whole Ehrlichia is used as antigen, cross-reactions with Ehrlichia spp. occur in all of these tests.
Two recently developed ELISAs that use recombinant major antigenic protein 1 (MAP1) antigens -the MAP1-B ELISA and the MAP1 competitive ELISA - have shown a dramatic improvement in specificity compared with previous tests, making the interpretation of serological results more reliable in regions where Ehrlichia infections occur in ruminants. These tests can help to monitor experimental infections, to check the immune status of immunised animals, and to screen animals prior to importation.
Requirements for vaccines and diagnostic biologicals: The immunisation against heartwater by the 'infection and treatment' method using infected blood or homogenised, pre-fed Amblyomma ticks is still in use in some countries. A first generation vaccine consisting of inactivated purified elementary bodies of E. ruminantium emulsified in Montanide ISA 50 adjuvant has given promising results in experimentally controlled conditions, and is being evaluated in the field in endemic areas. In the near future, it could replace the infection and treatment method, which has practical difficulties.
A. INTRODUCTION
Heartwater (cowdriosis) is a rickettsial disease of domestic and wild ruminants caused by Ehrlichia ruminantium (formerly Cowdria ruminantium) and transmitted by Amblyomma ticks (2, 6, 23). It is also known by the synonyms malkopsiekte (Afrikaans), péricardite exsudative infectieuse (French), hidrocarditis infecciosa (Portuguese), idropericardite dei ruminanti (Italian), and a variety of names in different African languages (5). Ehrlichia ruminantium is classified in the order Rickettsiales and in the Tribe Ehrlichieae, together with the genera Anaplasma.
Heartwater occurs in nearly all the sub-Saharan countries of Africa where Amblyomma ticks are present and in the surrounding islands: Madagascar, Reunion, Mauritius, Zanzibar, the Comoros Islands and Sao Tomé. The disease is also reported in the Caribbean (Guadeloupe, Marie-Galante and Antigua) (21), from where it threatens the American mainland. All domestic and wild ruminants can be infected, but the former appear to be the most susceptible. Indigenous domestic ruminants are usually more resistant to the disease. Wild animals could play a role as reservoir, but Rusa deer seem to be the only wild ruminant in which heartwater has an economic impact.
The average natural incubation period is 2 weeks, but can vary from 10 days to 1 month. In most cases, heartwater is an acute febrile disease, with a sudden rise in body temperature, which may exceed 41°C within 1-2 days after the onset of fever. It remains high with small fluctuations and drops shortly before death.
Fever is followed by inappetence, sometimes listlessness, diarrhoea, particularly in cattle (3), and dyspnoea indicative of lung oedema. Nervous signs develop gradually. The animal is restless, walks in circles, makes sucking movements and stands rigidly with tremors of the superficial muscles. Cattle may push their head against a wall or present aggressive or anxious behaviour. Finally, the animal falls to the ground, pedalling and exhibiting opisthotonos, nystagmus and chewing movements. The animal usually dies during or following such a nervous attack.
Subacute heartwater with less pronounced signs, and peracute heartwater with sudden death, can also occur, according to the breed of ruminant and the strain of Ehrlichia.
The most common macroscopic lesions are hydropericardium, hydrothorax, pulmonary oedema, intestinal congestion, oedema of the mediastinal and bronchial lymph nodes (3), petechiae on the epicardium and endocardium, congestion of the brain, and moderate splenomegaly.
A tentative diagnosis of heartwater is based on the presence of Amblyomma vectors, of clinical nervous signs, and of transudates in the pericardium and thorax at post-mortem examination. Differential clinical diagnosis should be made with bovine cerebral babesiosis and theileriosis, anaplasmosis, botulism, and small ruminant haemonchosis.
B. DIAGNOSTIC TECHNIQUES
| 1. | Identification of the agent
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| | Typical colonies of E. ruminantium can be observed in brain smears made after death.
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| | The tedious task of opening the cranium is not necessary. One alternative method (26) is to cut off the head in front of the first cervical vertebra. Then, a curette is introduced through the foramen magnum, between the medulla and the meninges. The curette is turned over towards the brain and removed with a piece of cerebellum. Another simple method consists of making a hole in the skull with a hammer and a large nail and aspirating a sample of brain cortex with a needle attached to a syringe. These methods also lessen the danger to the operator in cases where the nervous signs have been caused by rabies.
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| | In the live animal, a brain biopsy may be obtained aseptically and harmlessly after local anaesthesia, although with difficulty and appropriate restraint with large and especially with horned animals. Colonies of Ehrlichia are observed during the febrile period. This method is useful for experimental studies, but not suitable for routine diagnosis.
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| | Colonies of Ehrlichia are still present 48 hours after death in a brain that has been stored at room temperature, and for up to 34 days in a brain that has been stored in a refrigerator (4).
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| | A small fragment of grey matter (approximately the size of a match head) is placed on a microscope slide, crushed by another slide and, while maintaining pressure, the slides are drawn over each other lengthwise to produce a single layer of cells. The slides are air-dried, fixed in methanol, stained with Giemsa diluted with Sörensen buffer (2.54 g KH2PO4; 8.55 g Na2HPO4.H2O; q.s. to 5 litres with distilled water), pH 7.2, and washed with tap water. Fast Giemsa stains (DiffQuick, RAL555, Field's stain, CAM's Quick stain) give quicker results, but the colour contrast is usually poorer. Some 'fast' stains do provide excellent contrast, e.g. Hema 3 stain.
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| | The slides are examined under a microscope at a low magnification (x10 objective) to locate the cerebral capillaries. An oil-immersion lens with a magnification of at least x50 is useful for identifying the colonies of rickettsias. Experience is required to identify Ehrlichia colonies and to differentiate them from other haemoparasites (Babesia bovis), certain blood cells (thrombocytes, granulocytes), normal subcellular structures (mitochondria, mast cell granules), or stain artefacts (stain precipitates), etc. The specificity of the reading can be improved by staining formalin-fixed brain sections using immunoperoxidase techniques.
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| | Ehrlichia colonies are formed from clusters of granules (0.2-0.5 µm), sometimes arranged in the shape of a ring or a horseshoe (1-3 µm), that are placed close to the nucleus inside the endothelial cell. The granules can be scanty, particularly in peracute cases, but they are always present in the brain of an animal that died from heartwater. However, if the animal has been drug-treated 48 hours before, the granules of Ehrlichia tend to fuse making the diagnosis very difficult, and sometimes impossible.
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| | Fresh whole blood collected from suspect animals can be inoculated intravenously into a susceptible sheep or goat. The development of clinical signs and the demonstration of Ehrlichia in the brain of the inoculated ruminant are diagnostic for heartwater.
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| | Transmission electron microscopy has been used to demonstrate that the Ehrlichia organisms develop inside a vacuole-like structure, which is surrounded by a membrane in the endothelial cell's cytoplasm (25). Each organism is enclosed by a double membrane. Within the vacuole-like structure, Ehrlichia electron-dense forms (elementary bodies), as well as intermediate reticulate forms, are identified.
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| | a) | Isolation of Ehrlichia ruminantium using in-vitro culture
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| | | Isolation of E. ruminantium in cell culture is not the first choice of a test to confirm the diagnosis of cowdriosis, as it is a time-consuming laboratory procedure, although numerous cell lines support growth. However, Ehrlichia isolation is necessary for typing the strains present in a region for the purpose of vaccination programmes. Ehrlichia can be isolated from the blood of reacting animals by cultivation on ruminant endothelial cells. Endothelial cells from umbilical cord, aorta, or the pulmonary artery of different ruminant species (cattle, goat, sheep) are used most often for isolation, although other endothelial cell types (brain capillaries, circulating endothelial cells, etc.) have been described for the routine culture of the microorganism. Endothelial cell lines from sable, eland, buffalo, kudo and bush pig can also be used to grow E. ruminantium. No standard cell line has yet been designated for isolation.
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Isolation procedure
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| | | i) | The blood of the reacting animal is collected in anticoagulant (heparin or sodium citrate) and diluted 1/2 in the culture medium consisting of Glasgow minimal essential medium (MEM) supplemented with 10% inactivated fetal bovine serum, 2.95 mg/ml tryptose phosphate broth, 200 mM L-glutamine, and antibiotics if necessary (penicillin 100 international units/ml, streptomycin 100 µg/ml).
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| | | ii) | The culture medium is poured off the endothelial cell monolayer, and infective blood (approximately 2 ml for a 25 cm2 flask) is added. The flask is incubated at 37°C on a rocking platform for 2 hours.
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| | | iii) | After incubation, the blood is poured off and the monolayer is gently washed three times with prewarmed culture medium. Fresh culture medium is added and the flask is incubated at 37°C. The medium is changed twice weekly.
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| | | | (The use of plasma instead of blood is more efficient when taken from an animal with a febrile reaction >41°C. In this case, steps ii and iii above may be replaced with the following:
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Seed 4 ml of plasma on to a susceptible endothelial cell culture and incubate for 1 hour at 37°C on a rocking platform.
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Wash off plasma with growth medium and then add 5 ml of growth medium (per 25 cm2 flask) and observe for development of cytopathic effect.)
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| | | iv) | The monolayer is inspected regularly for the appearance of small plaques of cell lysis. The first plaques generally appear after about 2 weeks. Passaging on uninfected cell monolayers is performed when the lysis reaches 80% of the cell layer. The remaining cells are stained with eosin/methylene blue and examined microscopically for the presence of E. ruminantium morulae. Alternatively, cells can be stained by an indirect fluorescent antibody (IFA) test or an immunoperoxidase test using a Ehrlichia-specific antiserum.
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| | b) | Isolation of Ehrlichia ruminantium using in-vivo culture
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| | | It is feasible to assess the presence of heartwater in a herd, a region or a country, or to isolate a strain of Ehrlichia, by inoculating blood or tick homogenate into a susceptible animal. Blood from individual animals, or pooled blood, is injected slowly at a dose of 10-100 ml intravenously into a susceptible sheep or goat. Another method consists of collecting and homogenising adult Amblyomma ticks, and after centrifuging the homogenate, inoculating the resulting supernatant. The latter method is more sensitive because the concentration of Ehrlichia is higher in the tick than in the blood. However, the tick infection rate is variable and sometimes as low as 1% (5). In this case, to detect an infection, at least 100 ticks are needed and as many as possible should be used. In both cases, the inoculum with 10% dimethyl suphoxide can be stored in liquid nitrogen for several years. Note that inoculation of tick homogenates into susceptible animals may cause anaphylaxis, which can be prevented by the simultaneous administration of adrenaline. The development of clinical signs and the detection of circulating bacteria by molecular methods and/or the demonstration of Ehrlichia in the brain of the inoculated ruminant are diagnostic for heartwater.
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| 2. | Molecular methods
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| | a) | Detection of Ehrlichia ruminantium using DNA probes
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| | | A genomic DNA fragment specific for E. ruminantium has been cloned and used as a nucleic acid probe (29). It recognises all strains of E. ruminantium tested so far. This probe, designated pCS20, readily detects infection in clinically ill animals and experimentally infected Amblyomma ticks. However, it is not sufficiently sensitive to detect most carrier animals and/or low level infections in ticks. The pCS20 probe proved nevertheless to be more sensitive than 16S and MAP1 (major antigenic protein 1) probes for the detection of E. ruminantium in ticks when hybridised on a polymerase chain reaction (PCR)-amplified product of the homologous DNA fragment (1).
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| | b) | Detection of Ehrlichia ruminantium using PCR and nested PCR
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| | | Two primers - AB128 and AB129 - have been designed from the DNA sequence of the pCS20 probe (15) for use in a PCR. A modification of the PCR method consists in blotting the pCS20 probe on the amplification fragment in an additional step, which results in a tenfold increase in the sensitivity (24). This latter technique (PCR/hybridisation) has been shown to be 350-fold more sensitive than the nucleic acid probe alone. Low levels of infection in animals and in ticks fed on carrier animals are detected by PCR, while a hybridisation reaction with the pCS20 probe alone usually remains negative (24). Experimentally, the detection limit of the conventional PCR assay was found to be between 10 and 102 organisms, whereas it was between 1 and 10 organisms after PCR/hybridisation. The PCR/hybridisation has been shown to detect 37 strains from all endemic areas with a specificity of 98%. However, the sensitivity of the PCR assay is variable, ranging from 97 to 88% with tick samples containing 107 to 104 organisms, and dropping to 61% and 28% with samples containing 103 and 102 organisms, respectively (22). Consequently, the rate of 86% of ticks testing positive when fed on a reacting animal dropped to 21% when fed on carrier animals due to a lower rickettsemia in such animals.
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| | | A nested PCR targeting the same pCS20 DNA fragment has also been developed (18). The pair of external primers comprises the AB128 sense primer together with an anti-sense primer called AB130. These amplify a 413 bp fragment used as a matrix in a second round PCR using AB128 and AB129 as internal primers. The use of AB128 and AB129 primers avoids the need to repeat a full evaluation of the test specificity. The nested PCR shows a 2 log 10 improvement in sensitivity compared with a simple PCR, and an average detection limit of 6 organisms. The direct implication of this was an increase in the detection rate in wild ticks of from 1.7% to 36% in an epidemiological study in the Caribbean. The detection limit is comparable to that of the PCR/hybridisation method, which is nevertheless much more complex and time-consuming to perform. The pCS20 nested PCR allowed regular detection of E. ruminantium organisms from ticks, blood, brain and lungs from infected animals, whether the samples were processed fresh, or after freezing or preservation in 70% ethanol.
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| | | A nested PCR targeting the entire map1 polymorphic gene has been developed in parallel in order to type the strains by restriction fragment length polymorphism or sequencing of the amplification fragment directly from the pathological samples testing positive in the pCS20 nested PCR (18). A high genetic diversity of E. ruminantium is observed in the field that influences the formulation of vaccines and needs to be further investigated. The map1 nested PCR performs well although with a slightly lower sensitivity than the pCS20 nested PCR. Its detection limit was evaluated at around 60 organisms and only 91% of samples testing positive in the pCS20 nested PCR also tested positive in the map1 nested PCR; some positives of low intensity found using the pCS20 nested PCR were negative in the map1 PCR.
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| | | Although the PCR methods have proved highly effective in detecting infection in ticks or in animal samples during the clinical phase of the disease or after death, only limited studies have been done to evaluate their value in healthy carrier ruminants. Ehrlichia ruminantium can easily be demonstrated in the blood of infected animals just before the onset of the febrile period and for a few days after recovery, but after that it seems to be absent from the circulation at a detectable level for long periods. In one study in Zimbabwe only between 3.3 and 26.7% of cattle, and 23.3% of goats were found to be positive whereas almost 100% of them had been infected with Ehrlichia (13). Whether the absence of detection in most carrier animals is due to an insufficient sensitivity of the PCR methods for detecting very low rickettsemia, or is due to an intermittent release of organisms in the circulation, is not known. A useful technique for confirming the status of a suspected carrier animal, whose blood is PCR negative, is to feed batches of naive ticks on the animal and then test the ticks by PCR. It is not known whether ticks act simply by concentrating circulating organisms, or also by amplifying their number or even by inducing release of microorganims in the circulation during feeding.
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| | | Primers 32F1 and 32R1 designed from the sequence of the MAP1 gene of E. ruminantium were successfully used in a PCR to detect the pathogen in blood and bone marrow of carrier sheep and wild African ungulates, but the method has not been widely evaluated and used.
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| | c) | Detection of Ehrlichia ruminantium using the reverse line blot technique
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| | | The reverse line blot technique (RLB) has been used for the simultaneous detection and identification of Anaplasma and Ehrlichia species known to occur in ruminants on the basis of differences in the small subunit rRNA gene (2). Primers 16S8FE and B-GA1B-new were designed from conserved domains and used to amplify a 492-498 bp fragment of the 16S rRNA gene spanning the variable V1 region. Species-specific oligonucleotide probes were designed in this V1 loop to allow species-specific detection of E. ruminantium, E. ovina, E. sp. strain Omatjenne, Anaplasma marginale, A. centrale, A. bovis, A. ovis and A. phagocytophilum. One oligonucleotide probe cross-reactive with all species (catch-all probe) was also designed to serve as control in case a PCR product does not hybridise to any of the species-specific probes. In the method, the species-specific probes are covalently linked to the hybridisation membrane, which is hybridised with the PCR product obtained using primers 16S8FE and B-GA1B-new. PCR products obtained from all above-mentioned microorganisms were shown to bind with specific oligonucleotide probes only. No PCR product was detected and no hybridisation occurred when the PCR-RLB was applied to Theileria annulata, Babesia bigemina or mammalian DNA. Similarly, negative control ticks were always negative in the RLB assay whereas it was possible to detect Ehrlichia ruminantium infection in 15-70% of ticks fed on experimentally infected or long-term carrier sheep. In Mozambique, E. ruminantium could also be detected in the blood of 12 sentinel small ruminants placed in the field with the infected animals; mixed infection was detected in five of the infected sentinel animals, thus demonstrating the usefulness of the method for detecting multiple infections. However, the sensitivity of the assay has not yet been determined and there is a need to further validate the technique in large epidemiological studies.
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Reading the results
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| | | As E. ruminantium in an obligate intra-cellular bacteria that cannot be cultivated in acellular media and its isolation is complex and takes several weeks, molecular detection techniques are the best methods for the diagnosis of cowdriosis. PCR proves to be easier to perform and more sensitive than DNA probes. With all PCRs, however, care must be taken to ensure that no cross-contamination occurs between samples. Negative and positive controls must be included in each test. As heartwater serology has several limitations (see Section B.3.), the PCR could be used to confirm serological results when, for example, seronegative animals originating from an endemic area must be translocated to a heartwater-free area at risk (presence of potential vectors). However, despite interesting experimental results in detecting subclinical carriers, there is not enough available information on the reliability of carrier detection by PCR, and further research is needed before a standard test with known sensitivity can be designed. The current results obtained with the PCR, the nested PCR or the RLB assay show that the direct detection of E. ruminantium in the blood is only reliable during and around the febrile phase of the disease. PCR-based methods appear to be more reliable in detecting infection in ticks, and this could have epidemiological value in determining the geographical distribution of Ehrlichia. In addition, when necessary in endemic areas, the inclusion of testing (originally naive) ticks fed on a suspect animal would greatly improve the sensitivity of carrier detection when serology and PCR on blood have failed. The procedure is nevertheless not convenient for routine diagnostic laboratories as it requires the maintenance of tick colonies and the capacity to experimentally infect animals.
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| 3. | Serological tests
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| | Various serological tests for diagnosing heartwater have been described: an IFA test with E.-ruminantium-infected endothelial cell culture as antigen (CIFA test), indirect ELISA, a competitive ELISA (C-ELISA), and a Western blot. The IFA test using E.-ruminantium-infected mouse peritoneal macrophages (MIFA) is now seldom used.
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| | One drawback of all of these tests is the detection of false-positive reactions due to common antigenic determinants between the E. ruminantium MAP1 (9) and the presence of similar proteins in several Ehrlichia species. Almost all of these tests are no longer used for epidemiology or diagnosis. The CIFA test is still used in some places, but care must be taken when interpreting the results because of the problem of false-positive reactions.
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| | To overcome the problem of cross-reactions with Ehrlichia, two ELISAs based on a recombinant MAP1 antigen have been developed. The first is an indirect ELISA that uses an immunogenic region of the MAP1 protein (called MAP1-B) and gives far fewer cross-reactions with Ehrlichia spp. (MAP1-B ELISA) (28). The second is a competitive ELISA that uses the MAP1 gene cloned in a baculovirus and monoclonal antibodies (MAbs) raised against the MAP1 protein (MAP1 C-ELISA) (10). Both tests have dramatically improved specificity, but they still show some reactivity with high titre sera against E. canis, E. chaffeensis and an unclassified white-tailed deer agent.
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| | So far, the MAP 1-B ELISA has been the most extensively used and will be described in more details.
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| | a) | Indirect fluorescent antibody test with infected endothelial cell tissue culture as antigen (CIFA test) (17)
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| | | To prepare the antigen, a E. ruminantium strain is cultivated in ruminant endothelial cell cultures. When most cells are lysed, the remaining adherent cells are scraped and mixed with the supernatant. The cells are centrifuged three times with phosphate buffered saline (PBS) at 200 g for 10 minutes. Of the washed cell suspension, 10 µl is placed in every well of an immunofluorescence slide. The antigen slides are dried, fixed in acetone and stored at -20°C.
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Test procedure
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| | | i) | The sera to be tested are diluted 1/20 in PBS, added to the antigen wells and incubated for 30 minutes in a humid chamber at 37°C.
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| | | ii) | The slides are then washed in buffer for 15 minutes.
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| | | iii) | The appropriate anti-species conjugate, usually diluted 1/60, is added to cover the wells. The slides are incubated again for 30 minutes at 37°C.
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| | | iv) | After a second washing, the slides are mounted in glycerine buffer under a cover-slip and examined under a fluorescence microscope.
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| | | v) | Control positive and negative sera are included on each slide.
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| | b) | Indirect fluorescent antibody test with infected mouse peritoneal macrophages as antigen (MIFA test) (8)
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| | | Mice are injected intraperitoneally with 0.2 ml of Kümm strain stabilate following retrieval from liquid nitrogen storage. Clinical signs - ruffled hair coat and lethargy - appear 12 days later, and several mice may die. The surviving mice are killed. The peritoneal cells containing some macrophages with morula colonies are removed by injecting 2 ml of PBS into the peritoneal cavity and withdrawing the fluid. The pooled peritoneal fluid is centrifuged for 5 minutes at 2000 g and the pellet is resuspended in 0.3 ml of buffer.
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| | | A droplet of the cell suspension is placed in every well of an immunofluorescence slide to form a monolayer of cells. The antigen slides are air-dried, wrapped in tissue and tinfoil and stored: at 4°C for 21 days, at -18°C for 6-9 months, or at or under -70°C for over 1 year.
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| | | Just before use, the antigen slides are plunged into cold methanol for 1-3 seconds. A thick felt pen is used to separate the wells to avoid confluence of sera between wells.
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| | | The IFA procedure is the same as the previous test, but the initial serum dilution is 1/80.
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| | c) | MAP1-B enzyme-linked immunosorbent assay (28)
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| | | Using the vector pQE9, the PCR fragment MAP1-F2R2, which encodes the amino acids 47-152 of the MAP1 protein including the immunogenic region MAP1-B, is expressed in Escherichia coli M15[pREP4] as a fusion protein containing six additional histidine residues. The recombinant MAP1-B is purified using Ni2+-NTA agarose (nitrilotriacetic acid agarose) under denaturing conditions as described by the manufacturer (Qiagen, Max-Volmer-Straße 4, 40724 Hilden, Germany). The antigen is preserved at 4°C and each batch is titrated.
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| | | The antigen is diluted at 0.5 µg/ml in 0.05 M sodium carbonate buffer, pH 9.6, and immobilised on to polystyrene plates by incubation for 1 hour at 37°C, and stored at 4°C until use.
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Test procedure
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| | | i) | Plates are blocked for 30 minutes by adding 100 µl per well of 0.1 M PBS, pH 7.2, supplemented with 0.1% Tween 20 and 3% nonfat dry milk (PBSTM).
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| | | ii) | The plates are washed three times with PBS supplemented with 0.1% Tween 20 (PBST) and twice with distilled water.
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| | | iii) | 100 µl of test serum diluted 1/100 in PBSTM is added in duplicate to wells, which are then incubated for 1 hour at 37°C.
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| | | iv) | Plates are washed three times in PBST and twice in distilled water.
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| | | v) | Horseradish-peroxidase-conjugated anti-species IgG optimally diluted in PBSTM is added at 100 µl per well and the plate is incubated for 1 hour at 37°C.
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| | | vi) | After washing as in step iv, each well is filled with 100 µl of 0.1 M citrate buffer, pH 5.5, containing 0.5 mg/ml orthophenylene-diamine and 3 µl/ml of 9% H2O2.
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| | | vii) | The reaction is stopped after 30 minutes of incubation at room temperature by adding 50 µl of 2 N H2SO4. Absorbance is read at 495 nm. Positive and negative controls are included in each plate.
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| | d) | MAP1 competitive enzyme-linked immunosorbent assay (20)
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| | | Recombinant MAP1 antigen is prepared as follows: 8-day-old Trichoplusia ni insect larvae are infected by a baculovirus expressing the map1 gene and moribund larvae are homogenised (10% [w/v]) in PBS supplemented with 0.001% (v/v) Triton X-100.
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| | | Anti-MAP1 MAb is prepared as follows: spleen cells of BALB/C mice previously inoculated with larval homogenate are fused to SP2/0 cells. Supernatant fluids from hybridoma cell cultures are screened for reactivity with MAP1 by immunoblotting and immunoperoxidase methods. A reactive cell culture is subcloned, isotyped and subsequently used for ascites production.
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| | | After a further 1/800 (v/v) dilution in PBS, the antigen is immobilised on to polystyrene plates (Nunc-Immuno Plates PolySorp) by incubation overnight at 4°C, and stored at -70°C
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Test procedure
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| | | i) | Prior to use, the plates are blocked for 30 minutes by adding 100 µl per well of PBS, pH 7.2, supplemented with 0.05% Tween 20 and 5% nonfat dry milk.
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| | | ii) | Plates are washed three times with PBS/Tween, 50 µl/well of test serum diluted 1/50 in PBS supplemented with 0.05% Tween 20 and 1% nonfat dry milk (PBSTM) is added in duplicate and the plates are incubated for 30 minutes at 37°C.
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| | | iii) | Without an intervening washing step, 75 µl/well of the MAb diluted 1/4000 (v/v) in PBSTM is added and the plates are incubated for another 30 minutes at 37°C.
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| | | iv) | Plates are washed three times in PBS/Tween and horseradish-peroxidase-conjugated anti-mouse IgG optimally diluted in PBSTM is added at 50 µl per well. The plate is incubated for 1 hour at 37°C.
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| | | v) | After three washings as before, 100 µl of 0.1 M citrate buffer, pH 5.5, containing 0.5 mg/ml O-phenylene diamine and 3 µl/ml of 9% H2O2 are added to each well. After 30 minutes of incubation at room temperature in the dark, the reaction is stopped by adding 50 µl of 2 N H2SO4 and the absorbance is read at 495 nm. Positive and negative controls are included in each plate.
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Reading the results
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| | | All serological tests based on non-recombinant Ehrlichia antigens, such as CIFA, ELISAs, and Western blotting, are still used for experimental studies but are no longer used for sero-epidemiological studies. The tests have been compared and applied to known positive and negative sera to E. ruminantium (7). No false-positive reactions were observed with any of the tests against known negative sera. There is good correlation among tests, but the specificity of all five tests is low because cross-reactions occur with certain Ehrlichia spp.
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| | | The interpretation of results of the various tests applied to field surveys is thus difficult in areas where Ehrlichia infections occur in ruminants, which is probably the case in most of the heartwater-endemic regions of Africa. This situation has also been demonstrated in farms without Amblyomma but infected with tick species not known to be vectors of E. ruminantium.
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| | | Both the MAP1-B ELISA and the MAP1 C-ELISA have shown a high specificity after evaluation in 3000 ruminant sera (goat, sheep and cattle) collected from 14 A.-variegatum-infested islands of the Lesser Antilles, among which only three are known to be infected by E. ruminantium (20). Overall specificity calculated from the 11 heartwater-free islands was 98.5% and 99.4% for the MAP1 C-ELISA and the MAP1-B ELISA, respectively. Although a few false-positive sera are still found, these tests are likely to solve much of the specificity problems of the earlier serological tests.
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| | | Evaluating the sensitivity of the tests is more problematic as it would require knowledge of the exact status of a high number of animals sampled in the field. As mentioned before there is currently no simple technique available to confirm if an animal is infected. Experimentally, the sensitivity of the C-ELISA in goats was reported to be 91.6-95.4% for the MAP1-B ELISA, and 96.3-96.9% for the MAP1 C-ELISA (20). However, in another study the sensitivity averaged 95% for cut-off values set at 31% and 26.6% of the positive control serum for sheep and goat sera, respectively (19). Indeed, calculations are based on a limited number of experimentally inoculated animals in a period of time soon after inoculation, when almost all the animals are still positive. Sensitivity in cattle is even lower and several reports show that after infection most of the animals become seronegative again in less than 6 months and some animals even never seroconvert. This observation is in line with the difference in antibody prevalence observed between small ruminants and cattle in epidemiological surveys that cannot be explained by a lower risk of infection of the latter. For example, in Zimbabwean farms situated in endemic areas, more than 90% of goats presented antibodies in their serum compared with only 33% of cattle maintained in the same conditions (14). Similar observations were made in the Caribbean.
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| | | Serological tests are useful for the assessment of heartwater infection in vaccinated animals. The tests can also be used to screen animals prior to importation into heartwater-free areas, bearing in mind that antibodies are maintained at detectable levels in naturally infected domestic ruminants for a few months only, and that circulating antibodies disappear more rapidly in cattle than in small ruminants. It is thus possible that serologically negative animals may be carriers of infection. Serology should therefore be regarded as a diagnostic method to be applied at the herd level, not at the individual level.
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| | | Molecular methods, such as PCR assay, could potentially help in detecting carrier animals without detectable antibodies, but this approach has still significant drawbacks (see Section B.2. Molecular methods).
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C. REQUIREMENTS FOR VACCINES AND DIAGNOSTIC BIOLOGICALS
Although promising research results have been obtained with attenuated and inactivated organisms, no commercial vaccines are available at present. The only method of immunisation against heartwater remains the 'infection and treatment' method using infected blood or homogenised pre-fed infected ticks followed by treatment of reacting animals with tetracycline. This method is still in use in several areas. However, it is likely to be replaced soon by vaccination with preparations of inactivated E. ruminantium elementary bodies emulsified in oil adjuvants, following the demonstration that susceptible goats can be protected by inactivated Ehrlichia in Freund's adjuvant (16). This vaccine also protected against challenge in sheep (11) using different strains of E. ruminantium, and in cattle (27) using the same strain as in goats. A first generation vaccine preparation of inactivated Ehrlichia in Montanide ISA 50 oil adjuvant (adjuvant licensed for animal use) was shown to be similarly effective to the Freund's adjuvant preparation on laboratory challenge of immunised goats.
Animals can be immunised with two subcutaneous injections of 250 µg of antigen emulsified (50/50) in Montanide ISA 50 adjuvant in a volume of 2 ml. Further studies on optimisation of vaccine production, quality control and efficiency in the different target species are currently being conducted. In experimental conditions, it has recently been shown on goats that the vaccine dose can be lowered down to 32 µg of antigen without decreasing the effect on protection.
Evaluation of an inactivated vaccine adjuvanted with ISA 50 has demonstrated protection of sheep against natural field challenge in Zimbabwe (12). In addition, large field evaluation trials are currently being done in Africa in several farming systems. A major challenge remains the characterization of the extent of strain diversity in a region to be covered by an appropriate formulation of the vaccine. This knowledge will also be essential for new generation vaccines that will be developed in the future.
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| 24. | Peter T.F., Deem S.L., Barbet A.F., Norval R.A.I., Simbi B.H., Kelly P.J. & Mahan S.M. (1995). Development and evaluation of PCR assay for detection of low levels of Cowdria ruminantium infection in Amblyomma ticks not detected by DNA probe. J. Clin. Microbiol., 33, 166-172.
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| 25. | Pienaar J.G. (1970). Electron microscopy of Cowdria (Rickettsia) ruminantium (Cowdry, 1926) in the endothelial cells of the vertebrate host. Onderstepoort J. Vet. Res., 37, 67(78.
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| 26. | Schreuder B.E.C. (1980). A simple technique for the collection of brain samples for the diagnosis of heartwater. Trop. Anim. Health Prod., 12, 25-29.
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| 27. | Totte P., McKeever D., Martinez D. & Bensaid D. (1997). Analysis of T-cell responses in cattle immunised against heartwater by vaccination with killed elementary bodies of Cowdria ruminantium. Infect. Immun., 65, 236-241.
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| 28. | Van Vliet A.H.M., Van Der Zeijst B.A.M., Camus E., Mahan S.M., Martinez D. & Jongejan F. (1995). Use of a specific immunogenic region on the Cowdria ruminantium MAP1 protein in a serological assay. J. Clin. Microbiol., 33, 2405-2410.
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| 29. | Waghela S.D., Rurangirwa F.R., Mahan S.M., Yunker C.E., Crawford T.B., Barbet A.F., Burridge M.J. & McGuire T.C. (1991). A cloned DNA probe identifies Cowdria ruminantium in Amblyomma variegatum ticks. J. Clin. Microbiol., 29, 2571-2577.
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NB: There is an OIE Reference Laboratory for Heartwater (please consult the OIE Web site at: http://www.oie.int/eng/OIE/organisation/en_LR.htm).
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Contact : scientific.dept@oie.int
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