Molecular and serological prevalence of corridor disease (buffalo associated Theileria parva infection) in cattle populations at the livestock/game interface of KwaZulu-Natal province, South Africa.

Theileria parva are intracellular protozoal parasites responsible for three disease syndromes in cattle, namely East Coast fever (ECF), Corridor disease (CD) and Zimbabwean theileriosis. The increase in reports of CD outbreaks in recent years has raised questions about the probability of adaptation of buffalo-derived T. parva strains in cattle herds adjacent to game reserves. A cross-sectional study was conducted from March 2016 to December 2018 to investigate the extent of occurrence of T. parva infections in cattle in the CD-controlled area of KwaZulu-Natal Province. Blood samples were collected from 1137 cattle from 14 herds and analysed by quantitative real-time PCR (qPCR) and indirect fluorescent antibody test (IFAT) to determine the prevalence of T. parva. A total of 484 samples from 4 of the 14 herds were further tested on qPCR for the presence of T. taurotragi infections. The data were analysed using descriptive statistics and a chi-square test was used to assess association between variables. The overall prevalence of T. parva was 1.3% (95%CI:1-2%) and 19.9% (95%CI:17-22%) on qPCR and IFAT, respectively. The qPCR positive samples were detected in March and May while IFAT positive samples were detected in all seasons sampled, with higher numbers during summer months. The Pearson Chi-squared test showed that T. parva prevalence rates based on both qPCR and IFAT were positively associated with herds with previous history of CD outbreaks (χ2 = 8.594, p = 0.003; χ2 = 69.513, p < 0.001, respectively). The overall prevalence of T. taurotragi was 39.4% (95% CI: 35-44%) with the herd-level prevalence ranging between 35.0% and 43.4%. Possible cross-reaction of T. parva IFAT to T. taurotragi was detected on few samples, however, there was no significant association between T. taurotragi infections and IFAT positivity (χ2 = 0.829, p = 0.363). Results from this study demonstrated the extent of occurrence of subclinical carriers and the level of exposure to T. parva infections in cattle populations at a livestock/game interface area of KwaZulu-Natal Province. The molecular and seroprevalence rates were low when compared with other areas where cattle-adapted T. parva infections are endemic. The adaptation of buffalo-derived T. parva in cattle population resulting in cattle-cattle transmissions seem to be unlikely under the current epidemiological state.

Polyclonal antibody-based immunohistochemical detection of intraleukocytic Theileria parasites in roan and sable antelopes.

Theileria parasites commonly infect African wild artiodactyls. In rare roan (Hippotragus equinus) and sable (H. niger) antelopes, Theileria sp. (sable)-associated calf mortalities constrain breeding programs. The pathogenicity of most leukocyte-transforming Theileria spp. originates in their invasion of and multiplication in various mononuclear leukocytes, the transformation of both infected and uninfected leukocytes, and their infiltration of multiple organs. Understanding the pathogenesis of theileriosis can be improved by the use of immunohistochemistry (IHC) to identify the localization of the parasites in tissue sections. Our aim was to develop a reproducible IHC assay to detect leukocyte-associated Theileria parasites in formalin-fixed, paraffin-embedded roan and sable tissues. Polyclonal antibodies were purified from the sera of 5 roans from an area endemic for Theileria sp. (sable) and tested for IHC reactivity in 55 infected and 39 control roan and sable antelopes, and for antigen and species cross-reactivity in an additional 58 cases. The 3 strongest antibodies consistently detected intraleukocytic theilerial antigens in known positive cases in roan and sable antelopes, and also detected other Theileria spp. in non-hippotraginid wild artiodactyl tissues. The antibodies did not cross-react with other apicomplexan protozoa, with the exception of Cryptosporidium. Given that PCR on its own cannot determine the significance of theilerial infection in wild ruminants, IHC is a useful laboratory test with which to confirm the diagnosis in these species.

South African Buffalo-Derived Theileria parva Is Distinct From Other Buffalo and Cattle-Derived T. parva.

Theileria parva is a protozoan parasite transmitted by the brown-eared ticks, Rhipicephalus appendiculatus and Rhipicephalus zambeziensis. Buffaloes are the parasite's ancestral host, with cattle being the most recent host. The parasite has two transmission modes namely, cattle-cattle and buffalo-cattle transmission. Cattle-cattle T. parva transmission causes East Coast fever (ECF) and January disease syndromes. Buffalo to cattle transmission causes Corridor disease. Knowledge on the genetic diversity of South African T. parva populations will assist in determining its origin, evolution and identify any cattle-cattle transmitted strains. To achieve this, genomic DNA of blood and in vitro culture material infected with South African isolates (8160, 8301, 8200, 9620, 9656, 9679, Johnston, KNP2, HL3, KNP102, 9574, and 9581) were extracted and paired-end whole genome sequencing using Illumina HiSeq 2500 was performed. East and southern African sample data (Chitongo Z2, Katete B2, Kiambu Z464/C12, Mandali Z22H10, Entebbe, Nyakizu, Katumba, Buffalo LAWR, and Buffalo Z5E5) was also added for comparative purposes. Data was analyzed using BWA and SAMtools variant calling with the T. parva Muguga genome sequence used as a reference. Buffalo-derived strains had higher genetic diversity, with twice the number of variants compared to cattle-derived strains, confirming that buffaloes are ancestral reservoir hosts of T. parva. Host specific SNPs, however, could not be identified among the selected 74 gene sequences. Phylogenetically, strains tended to cluster by host with South African buffalo-derived strains clustering with buffalo-derived strains. Among the buffalo-derived strains, South African strains were genetically divergent from other buffalo-derived strains indicating possible geographic sub-structuring. Geographic sub- structuring was also observed within South Africa strains. The knowledge generated from this study indicates that to date, ECF is not circulating in buffalo from South Africa. It also shows that T. parva has historically been present in buffalo from South Africa before the introduction of ECF and was not introduced into buffalo during the ECF epidemic.

Safety and efficacy of an attenuated heartwater (Ehrlichia ruminantium) vaccine administered by the intramuscular route in cattle, sheep and Angora goats.

Heartwater is an economically important tick-borne disease of ruminants in Africa. The current commercial vaccine uses live Ehrlichia ruminantium from blood of infected sheep, requires antibiotic treatment during infection, needs to be administered intravenously and does not protect against all South African isolates. An attenuated tissue culture vaccine not requiring antibiotic treatment and effective against different field strains in small groups of goats and sheep was reported previously. The objective of the present study was to test safety and efficacy of this vaccine administered by intramuscular (i.m.) inoculation in larger groups of sheep, Angora goats and cattle. Animals were vaccinated via intravenous (i.v.) and i.m. routes and received E. ruminantium homologous challenge by feeding of infected ticks or by i.v. inoculation of infected blood. For vaccine titration in sheep and goats, the optimum safe and efficacious dose was determined using 2 ml equivalent of 102-105 culture-derived live elementary bodies (EBs). Similarly, the vaccine was titrated in cattle using 5 ml containing 105-107 EBs. Seventy percent of i.v. vaccinated and 9.7% of i.m. vaccinated Angora goats receiving 105 EBs, developed severe reactions to vaccination and were treated. These treated animals and the remaining 90.3% of i.m.- vaccinated goats showed 100% protection against i.v. or tick challenge. Sheep and Angora goats vaccinated i.m. with 104 EBs had no vaccination reactions and were fully protected against i.v. or tick challenge. Similarly, vaccinated cattle (dose 106 EBs) did not react to vaccine inoculation and were fully protected against i.v. or tick homologous challenge. Control non-vaccinated animals reacted severely to challenge and required oxytetracycline treatment. This successfully demonstrated that Angora goats, sheep and cattle can be safely vaccinated with the attenuated E. ruminantium Welgevonden vaccine via the i.m. route, with no clinical reactions to vaccination and 100% protection against virulent i.v. and homologous tick challenge.

In vitro propagation and genome sequencing of three 'atypical' Ehrlichia ruminantium isolates.

Three isolates of Ehrlichia ruminantium (Kümm 2, Omatjenne and Riverside), the causative agent of heartwater in domestic ruminants, were isolated in Ixodes scapularis (IDE8) tick cell cultures using the leukocyte fraction of infected sheep blood. All stocks were successfully propagated in IDE8 cells, whereas initiation attempts using endothelial cell cultures were unsuccessful. Therefore, the new technique should be included in any attempt to isolate field strains of E. ruminantium to enhance the probability of getting E. ruminantium isolates which might not be initiated in endothelial cells. Draft genome sequences of all three isolates were generated and compared with published genomes. The data confirmed previous phylogenetic studies that these three isolates are genetically very close to each other, but distinct from previously characterised E. ruminantium isolates. Genome comparisons indicated that the gene content and genomic synteny were highly conserved, with the exception of the membrane protein families. These findings expand our understanding of the genetic diversity of E. ruminantium and confirm the distinct phenotypic and genetic characteristics shared by these three isolates.

Serum-free in vitro cultivation of Theileria annulata and Theileria parva schizont-infected lymphocytes.

Theileriosis is a tick-borne disease caused by intracellular protozoa of the genus Theileria. The most important species in cattle are Theileria annulata and Theileria parva. Both species transform leucocyte host cells, resulting in their uncontrolled proliferation and immortalization. Vaccination with attenuated T. annulata-infected cell lines is currently the only practical means of inducing immunity in cattle. Culture media for Theileria spp. typically contain 10%-20% foetal bovine serum (FBS). The use of FBS is associated with several disadvantages, such as batch-to-batch variation, safety and ethical concerns. In this study, the suitability of serum-free media for the cultivation of Theileria-transformed cell lines was examined. Three commercial serum-free media (HL-1, ISF-1 and Hybridomed DIF 1000) were evaluated for their ability to support growth of the T. annulata A288 cell line. The generation doubling times were recorded for each medium and compared with those obtained with conventional FBS-containing RPMI-1640 medium. ISF-1 gave the shortest generation doubling time, averaging 35.4 ± 2.8 hr, significantly shorter than the 52.2 ± 14.9 hr recorded for the conventional medium (p = .0011). ISF-1 was subsequently tested with additional T. annulata strains. The doubling time of a Moroccan strain was significantly increased (65.4 ± 15.9 hr) compared with the control (47.7 ± 7.5 hr, p = .0004), whereas an Egyptian strain grew significantly faster in ISF-1 medium (43.4 ± 6.5 hr vs. 89.3 ± 24.8 hr, p = .0001). The latter strain also showed an improved generation doubling time of 73.7 ± 21.9 hr in an animal origin-free, serum-free, protein-free medium (PFHM II) compared with the control. Out of four South African T. parva strains and a Theileria strain isolated from roan antelope (Hippotragus equinus), only one T. parva strain could be propagated in ISF-1 medium. The use of serum-free medium may thus be suitable for some Theileria cell cultures and needs to be evaluated on a case-by-case basis. The relevance of Theileria cultivation in serum-free media for applications such as vaccine development requires further examination.

Investigations into the carrier-state of Theileria sp. (buffalo) in cattle.

The Theileria are apicomplexan parasites transmitted by ticks to vertebrate hosts. Most Theileria species exhibit some form of host or vector specificity, since under endemic conditions only a limited number of tick species act as vectors and not all vertebrate hosts are able to maintain a persistent carrier state. Data for Theileria sp. (buffalo) suggest host specificity for African buffalo (Syncerus caffer). However, T. sp. (buffalo) infections in cattle co-grazing with African buffalo have been reported in Kenya and schizonts were cultured from these infected cattle, raising questions regarding host specificity. A Corridor disease outbreak in 2013 on a ranch in South Africa where cattle co-grazed with Theileria parva and T. sp. (buffalo) infected buffalo presented the opportunity to investigate the possible carrier-state of T. sp. (buffalo) in cattle using real-time PCR analysis. Almost all buffalo (n = 19, 95%) were infected with T. sp. (buffalo) and showed CP values (22-20) indicative of high parasitemia similar to that observed for buffalo in endemic areas. Conversely, only ~14-27% cattle (n = 69, 100, 96) were positive with CP values (31-40) suggesting low parasitemia and a carrier state epidemiology different from African buffalo. Long term monitoring of T. sp. (buffalo) positive cattle showed that most cattle lost their parasitemia or presented fluctuating parasitemia around the PCR assay detection limit. A single splenectomized animal showed a persistent carrier state. The general trends and epidemiology observed in cattle infected with T. sp. (buffalo) are similar to that seen for buffalo-adapted T. parva, for which a defined carrier state in cattle has not yet been proven. The study suggests that cattle may be infected by T. sp. (buffalo) but are not definitive hosts that play an important part in the epidemiology of this parasite.

The host-specificity of Theileria sp. (sable) and Theileria sp. (sable-like) in African Bovidae and detection of novel Theileria in antelope and giraffe.

Tick-borne diseases caused by Theileria are of economic importance in domestic and wildlife ruminants. The majority of Theileria infects a limited number of host species, supporting the concept of host specificity. However, some Theileria seem to be generalists challenging the host specificity paradigm, such as Theileria sp. (sable) reported from various vertebrate hosts, including African buffalo, cattle, dogs and different antelope species. We tested the hypothesis that T. sp. (sable) uses Bovidae as hosts in general using a real-time polymerase chain reaction assay specific for T. sp. (sable) and a closely related genotype: T. sp. (sable-like). Various antelope species from the Tragelaphini (black wildebeest, blesbuck, blue wildebeest, gemsbuck, sable and waterbuck) tested positive for either T. sp. (sable) or T. sp. (sable-like). However, no African buffalo (n = 238) or cattle (n = 428) sampled in the current study tested positive, suggesting that these latter species are not carrier hosts. The results were confirmed using next-generation sequencing which also indicated at least 13 new genotypes or species found in various antelope and giraffes. Genotypes were found in single host species or in evolutionarily related hosts, suggesting that host specificity in Theileria may be a lineage specific phenomenon likely associated with tick-host-parasite co-evolution.

Risk of establishment of canine leishmaniasis infection through the import of dogs into South Africa.

Canine leishmaniasis is a vector-borne disease caused by protozoa of the genus Leishmania that affect dogs, humans and wildlife. Sandflies of the genera Phlebotomus and Lutzomyia are the primary vectors. Canine leishmaniasis is an exotic and controlled disease in South Africa. The main purpose of our risk assessment study was to evaluate the likelihood that this exotic disease could enter and be established in South Africa through importation of live dogs. Risk analysis to the spread of the disease follows the World Organization for Animal Health (OIE) formal method of quantitative risk assessment documented as a step-by-step process. We have identified and discussed 11 possible risk factors involved in three steps for final assessment. The annual average number of diagnostic tests performed on imported dogs from 44 countries for 2011-2015 was 1158. Leishmania is reported to occur in 21/44 (47.7%) exporting countries. A total of 71.1% of Leishmania positive dogs were imported from these endemic countries. The yearly percentage of Leishmania positive dogs ranged from 0.2% to 2%. Three confirmed clinical and fatal cases of leishmaniasis in dogs of unidentified origin have been reported by our laboratory and the state veterinarians. The disease has been reported in neighbouring countries as well as the putative sandfly vectors. This study concluded that the risk for the introduction and degree of uncertainty of Leishmania in imported dogs in South Africa are moderate. Risk mitigation and recommendations such as investigations into possible occurrence of autochthonous leishmaniasis in the country, surveillance in its wildlife reservoirs and systematic surveillance of sandfly populations are discussed.

Multiplex hydrolysis-probe assay for the simultaneous detection of Theileria equi and Babesia caballi infections in equids.

Quantitative real-time PCR assays previously developed for the detection of Theileria equi and Babesia caballi, were combined in a single multiplex TaqMan qPCR platform for the simultaneous detection of both heamoprotozoan parasites in equids. The multiplex equine piroplasmosis (M-EP) qPCR assay was shown to be efficient and specific. The detection limit was determined to be 1.4 × 10-4 % parasitized erythrocytes (PE) for T. equi and 2.8 × 10-4 % PE for B. caballi. The effect of differential DNA concentrations on the outcome of the M-EP qPCR for each target species was also investigated. The data demonstrated that the assay could reliably detect both targets, over a range of at least 1000-fold difference in target concentrations, without loss of sensitivity. The assay was subsequently evaluated on 243 field samples collected from areas where limited tick control strategies were implemented. The IFAT detected circulating T. equi and B. caballi antibodies in 100% and 92% of the samples, respectively. The M-EP qPCR assay detected T. equi parasite DNA in 98% of the samples, while B. caballi could only be detected in 6% of the samples tested, confirming that B. caballi infections generally occur at extremely low parasitaemias that rarely exceed 1%. The developed M-EP qPCR assay therefore serves as a reliable tool for the rapid diagnosis and epidemiological survey of equine piroplasmosis.

Transcriptomic analysis of Ehrlichia ruminantium during the developmental stages in bovine and tick cell culture.

The use of bioinformatics tools to search for possible vaccine candidates has been successful in recent years. In an attempt to search for additional vaccine candidates or improve the current heartwater vaccine design, a genome-wide transcriptional profile of E. ruminantium (Welgevonden strain) replicating in bovine endothelial cells (BA886) and Ixodes scapularis embryonic tick cells (IDE8) was performed. The RNA was collected from the infective extracellular form, the elementary bodies (EBs) and vegetative intracellular form, reticulate bodies (RBs) and was used for transcriptome sequencing. Several genes previously implicated with adhesion, attachment and pathogenicity were exclusively up-regulated in the EBs from bovine and tick cells. Similarly, genes involved in adaptation or survival of E. ruminantium in the host cells were up-regulated in the RBs from bovine cells. Thus, it was concluded that those genes expressed in the EBs might be important for infection of mammalian and tick host cells and these may be targets for both cell and humoral mediated immune responses. Alternatively, those exclusively expressed in the RBs may be important for survival in the host cells. Exported or secreted proteins exclusively expressed at this stage are ideal targets for the stimulation of cytotoxic T-lymphocyte (CTL) immune responses in the host.

In vitro culture and structural differences in the major immunoreactive protein gp36 of geographically distant Ehrlichia canis isolates.

Ehrlichia canis, the etiologic agent of canine ehrlichiosis, is an obligate intracytoplasmic Gram-negative tick-borne bacterium belonging to the Anaplasmataceae family. E. canis is distributed worldwide and can cause serious and fatal infections in dogs. Among strains of E. canis, the 16S rRNA gene DNA sequences are highly conserved. Using this gene to genetically differentiate isolates is therefore difficult. As an alternative, the gene gp36, which encodes for a major immunoreactive protein in E. canis, has been successfully used to characterize the genetic diversity of this pathogen. The present study describes the isolation and continuous propagation of a Spanish and 2 South African isolates of E. canis in IDE8 tick cells. Subsequently, canine DH82 cell cultures were infected using initial bodies obtained from infected IDE8 cultures. It was possible to mimic the life cycle of E. canis in vitro by transferring infection from tick cells to canine cells and back again. To characterize these E. canis strains at the molecular level, the 16S rRNA and gp36 genes were amplified by PCR, sequenced, and aligned with corresponding sequences available in GenBank. All 16S rRNA sequences amplified in this study were identical to previously reported E. canis strains. Maximum likelihood analysis based on the gp36 amino acid sequences showed that the South African and Spanish strains fall into 2 well-defined phylogenetic clusters amongst other E. canis strains. The members of these 2 phylogenetic clusters shared 2 unique molecular properties in the gp36 amino acid sequences: (i) deletion of glycine 117 and (ii) the presence of an additional putative N-linked glycosylation site. We further show correlation between the putative secondary structure and the theoretical isoelectric point (pI) of the gp36 amino acid sequences. A putative role of gp36 as an adhesin in E. canis is discussed. Overall, we report the successful in vitro culture of 3 new E. canis strains which present different molecular properties in their gp36 sequences.

Evaluation of a TaqMan real-time PCR for the detection of Theileria parva in buffalo and cattle.

A real-time PCR assay based on TaqMan probe chemistry was developed for the detection of Theileria parva DNA in blood samples. It uses a Theileria genus-specific PCR primer set and a T. parva-specific probe to amplify and hybridize with a species-specific part of the 18S rRNA gene of the parasite. The test was evaluated using positive and negative reference blood samples and shown to be specific for T. parva. Analytical sensitivity was determined by testing a dilution series of T. parva positive blood. It was shown to be able to detect parasitaemia as low as 2 × 10(-6)%. The Taqman assay results were also compared with that obtained with the real-time hybridization probe PCR assay, which is currently employed as the official test for the diagnosis of T. parva infections in buffalo and cattle and was shown to be equally sensitive. A panel of 1164 field samples was screened using both assays and 164 samples tested positive in both tests, indicating a good correlation.

In vitro cultivation of a Theileria species from a roan antelope (Hippotragus equinus).

Here we describe the in vitro isolation, propagation, and characterization of a Theileria species from roan antelope (Hippotragus equinus). Cultures were initiated using parts of a prescapular lymph node of an infected roan antelope. After 16 days of culture propagation, the first subculture was carried out; thereafter, subcultures were carried out twice a week. Standard methods for the cultivation of Theileria macroschizonts were applied. DNA was extracted from culture material and a partial polymerase chain reaction amplification of the 18S ribosomal RNA (rRNA) gene was carried out using Theileria genus-specific primers. It has been shown that Theileria sp. (roan) had high levels of nucleic acid identity with sequence data of the 18S rRNA gene of a Theileria sp. previously isolated from a sable antelope. The phylogenetic analysis showed that this isolate is closely related to several undescribed Theileria spp. which have previously been identified from a dog and some other antelope species in South Africa.

Theileria-infected cell line from an African buffalo (Syncerus caffer).

Mononuclear cells were isolated from the peripheral blood of a buffalo infected with a Theileria sp. using density gradient centrifugation, and the cells were put into culture flasks covered by a monolayer of bovine endothelial cells. Twenty days after culture initiation, cells containing macroschizonts were detected in Giemsa-stained smears. The first subculture was carried out on day 45 of culture propagation. Subsequently, infected cells were subcultured twice a week, and each time 1 to 2 x 10(6) per milliliter cells were harvested. DNA was extracted from culture material and a partial polymerase chain reaction amplification of the 18S ribosomal RNA (rRNA) gene was carried out using Theileria genus-specific primers. Sequence data and phylogenetic analysis using the 18S rRNA gene indicated a close relationship to Theileria sp. buffalo, previously described in literature. Here, the first successful attempt to establish a macroschizont-infected lymphoblastoid cell line of Theileria sp. (buffalo) from an African buffalo is described.

Experimental use of the attenuated Ehrlichia ruminantium (Welgevonden) vaccine in Merino sheep and Angora goats.

The attenuated Ehrlichia ruminantium (Welgevonden) stock provides protection against a virulent homologous needle challenge in Merino sheep and Boer goats against heartwater. In this study, cryopreserved stabilates were tested for their suitability as a vaccine in Merino sheep. Vaccination did not produce disease and upon challenge with the virulent homologous stock all animals were fully protected. The vaccination protected all except one animal out of 5 challenged 12 months after immunization. The intramuscular route gave better protection than the subcutaneously applied vaccine. The attenuated vaccine was further evaluated in highly susceptible Angora goats. Although the attenuated vaccine showed an unexpectedly high degree of virulence, animals were fully protected against a lethal needle challenge.

An attenuated Ehrlichia ruminantium (Welgevonden stock) vaccine protects small ruminants against virulent heartwater challenge.

Heartwater is a tick-borne disease of ruminants caused by the intracellular rickettsia Ehrlichia ruminantium. The only commercially available immunization procedure involves infecting animals with cryopreserved sheep blood containing virulent E. ruminantium organisms, followed by treatment with tetracyclines when fever develops. The virulent Welgevonden stock of E. ruminantium was attenuated by continuous propagation of the organisms in a canine macrophage-monocyte cell line (DH82), followed by re-adaptation to grow in a bovine endothelial cell line (BA 886). The material used for the present experiments consisted of the attenuated stock between passages 43 and 64 after re-adaptation. When inoculated into sheep or goats the attenuated organisms did not produce disease, and the only symptom observed was a rise in body temperature in most, but not all, animals. All sheep injected with 2 ml of culture suspension were subsequently found to be fully protected against a lethal needle challenge with the virulent homologous stock or with one of four different heterologous stocks (Ball 3, Gardel, Mara 87/7, Blaauwkrans). Titrations of elementary body suspensions showed that 2ml of a 1:10,000 dilution of culture suspension injected into sheep or goats was still sufficient to trigger an immune response which resisted a lethal needle challenge with the virulent Welgevonden stock. Adult Amblyomma hebraeum ticks, fed as nymphs on sheep immunized with DH82-derived organisms of passage 111, were able to transmit the attenuated stock to a naive sheep, which was found to be protected against a subsequent lethal homologous needle challenge.

The genome of the heartwater agent Ehrlichia ruminantium contains multiple tandem repeats of actively variable copy number.

Heartwater, a tick-borne disease of domestic and wild ruminants, is caused by the intracellular rickettsia Ehrlichia ruminantium (previously known as Cowdria ruminantium). It is a major constraint to livestock production throughout subSaharan Africa, and it threatens to invade the Americas, yet there is no immediate prospect of an effective vaccine. A shotgun genome sequencing project was undertaken in the expectation that access to the complete protein coding repertoire of the organism will facilitate the search for vaccine candidate genes. We report here the complete 1,516,355-bp sequence of the type strain, the stock derived from the South African Welgevonden isolate. Only 62% of the genome is predicted to be coding sequence, encoding 888 proteins and 41 stable RNA species. The most striking feature is the large number of tandemly repeated and duplicated sequences, some of continuously variable copy number, which contributes to the low proportion of coding sequence. These repeats have mediated numerous translocation and inversion events that have resulted in the duplication and truncation of some genes and have also given rise to new genes. There are 32 predicted pseudogenes, most of which are truncated fragments of genes associated with repeats. Rather then being the result of the reductive evolution seen in other intracellular bacteria, these pseudogenes appear to be the product of ongoing sequence duplication events.

The Kümm isolate of Ehrlichia ruminantium: in vitro isolation, propagation and characterization.

An effective culture system for Ehrlichia (Cowdria) ruminantium comb. nov. was first established in 1985 and many stocks were subsequently isolated and propagated in vitro. A notable exception, however, was the Kümm isolate that resisted all attempts at in vitro culture until the successful experiment described here. In one experiment white blood cells were harvested from heparinized blood derived from a sheep infected with the Kümm isolate. The cells were added to DH 82 cells and incubated at 37 degrees C. The high metabolic activity of the DH 82 cells necessitated that cell growth be retarded by the addition of cycloheximide. Colonies were first detected 19 days after culture initiation and, once the cultures were established, they could be passaged every 3 days. Bovine and sheep endothelial cells were readily infected with culture supernatant obtained from the infected DH 82 cells. In a further experiment another sheep was infected, using a higher dose of the same batch of Kümm stabilate, and we attempted to infect several different cell lines: these were DH 82 cells, bovine aorta (BA 886) cells, sheep brain endothelial (SBE 189) cells and sheep fibroblastoid cells (E2). Ten days after culture initiation only the E2 cells had become positive for E. ruminantium. Culture supernatant from the first cultured isolate (Kümm-1) was less virulent for mice than that of the second cultured isolate (Kümm-2) which killed all mice. Upon molecular characterization with E. ruminantium 16S probes we found that Kümm-1 hybridized with a Senegal 16S genotype probe, whereas Kümm-2 hybridized only with an Omatjenne 16S genotype probe. The original stabilate used to infect the sheep hybridized with both probes. These results clearly indicate that two different stocks had been isolated in culture.