Prevalence of serum antibodies of tick-borne diseases and the presence of Rhipicephalus microplus in communal grazing cattle in the north-eastern region of the Eastern Cape Province of South Africa.

A survey to determine the prevalence of tick-borne diseases (TBDs) and ticks infesting cattle was conducted in the communal areas of the north-eastern region of the Eastern Cape Province (ECP) between January 2019 and October 2019. Tick counts, packed cell volume (PCV), body condition scores (BCS), and serological test for TBDs were seasonally conducted in communally grazed cattle (n = 240) in Joe Gqabi district in two local municipalities (Elundini and Senqu). A standard indirect fluorescent antibody test (IFAT) was used to determine antibodies to Babesia bigemina, Babesia bovis, and Ehrlichia ruminantium and indirect enzyme-linked immunosorbent assay (ELISA) was employed for antibodies to Anaplasma marginale detection. The highest tick loads were observed on cattle during the hot-wet and post-rainy seasons and lowest during the cool-dry season. The E. ruminantium prevalence in Elundini was 16% and 14% in post-rainy and hot-dry seasons respectively and 15% at Senqu during the post-rainy season. B. bigemina prevalence was highest at Elundini (18%) and Senqu (16%) during the post-rainy season and hot-wet season respectively. Cattle BCS was negatively correlated with E. ruminantium (P < 0.01; r = - 0.203), B. bovis (P < 0.01; r = - 0.125), and A. marginale (P < 0.01; r = - 0.122) seroprevalence. The PCV was negatively correlated with B. bigemina (P < 0.01; r = - 0.138) seroprevalence. On the other hand, E. ruminantium was positively correlated with Amblyomma hebraeum (P < 0.05; r = 0.112) infestation, while B. bovis (P < 0.05; r = 0.134) and B. bigemina (P < 0.05; r = 0.188) were positively correlated with Rhipicephalus microplus infestation, and B. bigemina (P < 0.05; r = 0.077) was positively correlated with Rhipicephalus decoloratus infestation. Our study reports for the first time the presence of R. microplus in the study area. Further research is, however, needed to better understand seroprevalence and the transmission mode of TBDs to cattle so that effective disease control measures can be developed.

A multi-epitope DNA vaccine co-administered with monophosphoryl lipid A adjuvant provides protection against tick transmitted Ehrlichia ruminantium in sheep.

Previously, a heartwater experimental DNA vaccine provided 100% protection following laboratory challenge with Ehrlichia ruminantium administered by needle but not against an E. ruminantium tick challenge in the field. A multi-epitope DNA vaccine incorporating both CD4+ and CD8+ cytotoxic T lymphocytes epitopes could provide a better alternative. In this study, we investigated the use of multi-epitope DNA vaccines against an E. ruminantium experimental tick challenge in sheep. The multi-epitope DNA vaccines were delivered via the intramuscular route and intradermal route using the gene gun in the presence of monophosphoryl lipid A (MPL) adjuvant, which was either applied topically to the gene gun inoculation site or co-administered with the vaccine via the intramuscular route. Initially two constructs namely, pSignal plus and pLamp were tested with MPL applied topically only and no protection was obtained in this formulation. However, when pLamp was co-administered with MPL via the intramuscular route in addition to topical application, its protective efficiency improved to protect 60% of the sheep against tick challenge. In this formulation, the vaccine induced enhanced activation of memory T cell responses both before and after challenge with variations amongst the different sheep possibly due to their different genetic backgrounds. In conclusion, this study showed that a heartwater multi-epitope DNA vaccine, co-administered with MPL adjuvant can protect sheep following a laboratory E. ruminantium tick challenge.

Identification and characterisation of conserved epitopes of E. ruminantium that activate Th1 CD4+ T cells: Towards the development of a multi-epitope vaccine.

Several studies have shown that cytotoxic T lymphocytes (CTL) require CD4 + Th1 epitopes to generate strong immune responses to intracellular pathogens. However, not much is known about Ehrlichia ruminantium epitopes, particularly those that can be considered potential candidates for inclusion in a multi-epitope vaccine. In order to identify CD4+ Th1 epitopes that induce IFNγ, a number of proteins previously identified as immunogenic were first screened to determine if they induce cellular immunity in tick infected immune sheep PBMC. Significant IFN-γ production and other Th1 cytokines were evident for 10 recombinant proteins in all sheep tested. Secondly, peptides (n = 246) derived from the top 10 E. ruminantium vaccine candidate proteins were assayed using enzyme linked immunospot (ELISPOT) assay, quantitative real-time PCR and flow cytometry. Of the 246 peptides, 23 peptides, Erum0660 (p0660-42), Erum1150 (p1150-18, p1150-19), Erum2540 (p2540-6, p2540-16, p2540-19, p2540-20, p2540-21), Erum5420 (p5420-13, p5420-14), Erum7140 (p7140-6, p7140-7, p7140-12, p7140-13, p7140-20), Erum7320 (p7320-8, p7320-9, p7320-21), Erum7350 (p7350-9), Erum7360 (p7360-8), Erum7620 (p7620-2, p7620-12) and Erum8010 (p8010-8) were identified that stimulate the best and different cell mediated immune responses. Amino acid sequences of these peptides except for p7140-12, p7140-13, p7140-20, and p7350-9 were conserved between 13 different local strains. These peptides could efficiently induce memory CD4+ T cells to rapidly proliferate and significantly increase IFN-γ production in immune sheep PBMC. The upregulation of pro-inflammatory cytokines, which include, IL-1α, IL-2, IL-12p40, TNF-α, IFN-γ, inducible nitric oxide synthase (iNOS) and granulocyte-macrophage colony stimulating factor (GM-CSF) was also detected. Our results show that these peptides could serve as promising candidates for a multi-epitope vaccine against E. ruminantium.

Ehrlichia ruminantium antigens and peptides induce cytotoxic T cell responses in vitro.

Since CD8+ T cells play an important role in resistance to infection with heartwater, effective vaccines against this disease will likely require identification of antigens that contain CD8+ T cell epitopes responsible for cytotoxic T lymphocyte (CTL) responses. With the use of the fluorescent antigen-transfected target cell (FATT)-CTL assay, IFN-γ ELISPOT and flow cytometry, peptides that induce CTL, proliferation of CD8 + T cells and IFN-γ production were identified as possible target antigens for vaccine development. Of particular relevance was the finding that different peptides from different antigens were able to elicit varied cytotoxic activities by immune peripheral blood mononuclear cells (PBMC) from heartwater immune tick-infected sheep. Several peptides derived from Erum0660, Erum2330, Erum2540, Erum2580 and Erum5000 induced CTL in immune sheep PBMC. Peptide Erum2540-6 was the only peptide that induced significant CTL, CD8+CD45RO+ and CD8+IFN-γ+ by PBMC from all three sheep, and Erum2540 and p2540-20 induced the highest % CTL response in all three outbred sheep. These results suggest that these epitopes may be of major importance in heartwater recombinant vaccine development.

Innate immune transcriptomic evaluation of PBMC isolated from sheep after infection with E. ruminantium Welgevonden strain.

Heartwater is a tick-borne non-infectious fatal disease of wild and domestic ruminants caused by the bacterium Ehrlichia ruminantium, transmitted by Amblyomma ticks. Although there is evidence that interferon-gamma (IFN-γ) controls E. ruminantium growth and that cellular immune responses could be protective, an effective recombinant vaccine for this disease is lacking. An overall analysis of which immune pathways are up- or down-regulated in sheep peripheral blood mononuclear cells is expected to lead to a better understanding of the global immune response of sheep to E. ruminantium infection. Therefore, a systems biology oriented approach following the infection with E. ruminantium was investigated from peripheral blood mononuclear cells to aid recombinant vaccine development. In this study, heartwater naïve sheep were infected and challenged by allowing E. ruminantium infected ticks to feed on them. After primary infection, all the animals were treated with antibiotic during the resulting febrile response. Blood was collected daily for E. ruminantium detection by qPCR (pCS20 assay). The pCS20 assay only detected the pathogen in the blood one day prior to and during the febrile stage of infection confirming infection of the sheep. IFN-γ real-time PCR indicated that this cytokine was expressed at specific time points: post infection, during the febrile stage of the disease and after challenge. These were used as a guide to select samples for transcriptome sequencing. This paper focuses on transcripts that are associated with innate activating pathways that were identified to be up- and down-regulated after primary infection and the subsequent challenge. These included the CD14 monocyte marker, toll-like receptor (TLR), nod-like receptor, chemokine, cytosolic and cytokine-cytokine interaction receptor pathways. In particular, TLR4, TLR9 and CD14 were activated together with DNA detection pathways, suggesting that vaccine formulations may be improved if CpG motifs and lipopolysaccharides are included. This data indicates that innate immune activation, perhaps by using adjuvants, should be an important component for consideration during future heartwater recombinant vaccine development.

A glycosylated recombinant subunit candidate vaccine consisting of Ehrlichia ruminantium major antigenic protein1 induces specific humoral and Th1 type cell responses in sheep.

Heartwater, or cowdriosis, is a tick-borne disease of domestic and wild ruminants that is endemic in the Caribbean and sub-Saharan Africa. The disease is caused by an intracellular pathogen, Ehrlichia ruminantium and may be fatal within days of the onset of clinical signs with mortality rates of up to 90% in susceptible hosts. Due to the presence of competent tick vectors in North America, there is substantial risk of introduction of heartwater with potentially devastating consequences to the domestic livestock industry. There is currently no reliable or safe vaccine for use globally. To develop a protective DIVA (differentiate infected from vaccinated animals) subunit vaccine for heartwater, we targeted the E. ruminantium immunodominant major antigenic protein1 (MAP1) with the hypothesis that MAP1 is a glycosylated protein and glycans contained in the antigenic protein are important epitope determinants. Using a eukaryotic recombinant baculovirus expression system, we expressed and characterized, for the first time, a glycoform profile of MAP1 of two Caribbean E. ruminantium isolates, Antigua and Gardel. We have shown that the 37-38 kDa protein corresponded to a glycosylated form of the MAP1 protein, whereas the 31-32 kDa molecular weight band represented the non-glycosylated form of the protein frequently reported in scientific literature. Three groups of sheep (n = 3-6) were vaccinated with increasing doses of a bivalent (Antigua and Gardel MAP1) rMAP1 vaccine cocktail formulation with montanide ISA25 as an adjuvant. The glycosylated recombinant subunit vaccine induced E. ruminantium-specific humoral and Th1 type T cell responses, which are critical for controlling intracellular pathogens, including E. ruminantium, in infected hosts. These results provide an important basis for development of a subunit vaccine as a novel strategy to protect susceptible livestock against heartwater in non-endemic and endemic areas.

Cellular immune responses induced in vitro by Ehrlichia ruminantium secreted proteins and identification of vaccine candidate peptides.

Secreted proteins are reported to induce cell-mediated immunity characterised by the production of interferon-gamma (IFN)-γ. In this study three open reading frames (ORFs) (Erum8060, Erum7760, Erum5000) encoding secreted proteins were selected from the Ehrlichia ruminantium (Welgevonden) genome sequence using bioinformatics tools to determine whether they induce a cellular immune response in vitro with mononuclear cells from needle and tick infected animals. The whole recombinant protein of the three ORFs as well as four adjacent fragments of the Erum5000 protein (Erum5000A, Erum5000B, Erum5000C, Erum5000D) were successfully expressed in a bacterial expression system which was confirmed by immunoblots using anti-His antibodies and sheep sera. These recombinant proteins were assayed with immune sheep and cattle peripheral blood mononuclear cells (PBMCs), spleen and lymph node (LN) cells to determine whether they induce recall cellular immune responses in vitro. Significant proliferative responses and IFN-γ production were evident for all recombinant proteins, especially Erum5000A, in both ruminant species tested. Thus overlapping peptides spanning Erum5000A were synthesised and peptides that induce proliferation of memory CD4+ and CD8+ T cells and production of IFN-γ were identified. These results illustrate that a Th1 type immune response was elicited and these recombinant proteins and peptides may therefore be promising candidates for development of a heartwater vaccine.

Economic Game Theory to Model the Attenuation of Virulence of an Obligate Intracellular Bacterium.

Diseases induced by obligate intracellular pathogens have a large burden on global human and animal health. Understanding the factors involved in the virulence and fitness of these pathogens contributes to the development of control strategies against these diseases. Based on biological observations, a theoretical model using game theory is proposed to explain how obligate intracellular bacteria interact with their host. The equilibrium in such a game shows that the virulence and fitness of the bacterium is host-triggered and by changing the host's defense system to which the bacterium is confronted, an evolutionary process leads to an attenuated strain. Although, the attenuation procedure has already been conducted in practice in order to develop an attenuated vaccine (e.g., with Ehrlichia ruminantium), there was a lack of understanding of the theoretical basis behind this process. Our work provides a model to better comprehend the existence of different phenotypes and some underlying evolutionary mechanisms for the virulence of obligate intracellular bacteria.

A user-friendly and scalable process to prepare a ready-to-use inactivated vaccine: the example of heartwater in ruminants under tropical conditions.

The use of cheap and thermoresistant vaccines in poor tropical countries for the control of animal diseases is a key issue. Our work aimed at designing and validating a process for the large-scale production of a ready-to-use inactivated vaccine for ruminants. Our model was heartwater caused by the obligate intracellular bacterium Ehrlichia ruminantium (ER). The conventional inactivated vaccine against heartwater (based on whole bacteria inactivated with sodium azide) is prepared immediately before injection, using a syringe-extrusion method with Montanide ISA50. This is a fastidious time-consuming process and it limits the number of vaccine doses available. To overcome these issues, we tested three different techniques (syringe, vortex and homogenizer) and three Montanide ISA adjuvants (50, 70 and 70M). High-speed homogenizer was the optimal method to emulsify ER antigens with both ISA70 and 70M adjuvants. The emulsions displayed a good homogeneity (particle size below 1 µm and low phase separation), conductivity below 10 µS/cm and low antigen degradation at 4 °C for up to 1 year. The efficacy of the different formulations was then evaluated during vaccination trials on goats. The inactivated ER antigens emulsified with ISA70 and ISA70M in a homogenizer resulted in 80% and 100% survival rates, respectively. A cold-chain rupture assay using ISA70M+ER was performed to mimic possible field conditions exposing the vaccine at 37 °C for 4 days before delivery. Surprisingly, the animal survival rate was still high (80%). We also observed that the MAP-1B antibody response was very similar between animals vaccinated with ISA70+ER and ISA70M+ER emulsions, suggesting a more homogenous antigen distribution and presentation in these emulsions. Our work demonstrated that the combination of ISA70 or ISA70M and homogenizer is optimal for the production of an effective ready-to-use inactivated vaccine against heartwater, which could easily be produced on an industrial scale.

Identification of Ehrlichia ruminantium proteins that activate cellular immune responses using a reverse vaccinology strategy.

Ehrlichia ruminantium is an obligate intracellular bacterial pathogen which causes heartwater, a serious tick-borne disease of ruminants throughout sub-Saharan Africa. The development of promising recombinant vaccines has been reported previously, but none has been as effective as immunisation with live organisms. In this study we have used reverse vaccinology to identify proteins that elicit an in vitro cellular immune response similar to that induced by intact E. ruminantium. The experimental strategy involved four successive steps: (i) in silico selection of the most likely vaccine candidate genes from the annotated genome; (ii) cloning and expression of the selected genes; (iii) in vitro screening of the expressed proteins for their ability to induce interferon-gamma (IFN-γ) production in E. ruminantium-immune lymphocytes; and (iv) further examination of the cytokine response profiles of those lymphocytes which tested positive for IFN-γ induction. Based on their overall cytokine induction profiles the recombinant proteins were divided into four distinct groups. Eleven recombinant proteins induced a cytokine profile that was similar to the recall immune response induced by immune peripheral blood mononuclear cells (PBMC) stimulated with intact E. ruminantium. This response comprised the upregulation of cytokines associated with adaptive cellular immune responses as well as innate immunity. A successful vaccine may therefore need to contain a combination of recombinant proteins which induce both immune pathways to ensure protection against heartwater.

In vitro and in vivo evaluation of five low molecular weight proteins of Ehrlichia ruminantium as potential vaccine components.

Low molecular weight (LMW) proteins of E. ruminantium can induce proliferation of immune peripheral blood mononuclear cells (PBMCs) and the production of interferon-gamma (IFN-gamma) by CD4+-enriched T-cells. In this study, a reverse vaccinology approach was applied to identify additional vaccine candidates focusing on genes that encode LMW proteins smaller than 20 kDa. Five open reading frames (ORFs) were selected from the E. ruminantium genome and their corresponding recombinant (r) proteins were produced in a bacterial expression system. Their ability to induce proliferative responses and IFN-gamma production was evaluated in vitro using lymphocyte proliferation and ELISPOT assays. All five recombinant proteins induced proliferation of immune PBMCs and IFN-gamma production by these cells. The corresponding five genes were each individually incorporated into pCMViUBs, a mammalian expression vector and tested as a potential vaccine in sheep using a DNA prime-protein boost immunisation regimen. A cocktail of these DNA constructs protected one out of five sheep against a virulent E. ruminantium (Welgevonden) needle challenge. Three of the five vaccinated sheep showed an increase in their proliferative responses and production of IFN-gamma before challenge. This response decreased after challenge in the sheep that succumbed to the challenge and increased in the sheep that survived. This finding indicates that sustained IFN-gamma production is likely to be involved in conferring protective immunity against heartwater.

Efficiency of inactivated vaccines against heartwater in Burkina Faso: impact of Ehrlichia ruminantium genetic diversity.

In order to identify the appropriate strains to use in vaccination trials against heartwater in Burkina Faso, the protective effect of Gardel and Welgevonden strains was assessed against local strains on sheep vaccinated by infection-and-treatment method: Gardel protected significantly against Burkina Faso strains tested (survival rate 59% for immunised sheep vs 13% for control sheep) while Welgevonden did not (survival rate 45% for immunised sheep vs 25% for control sheep). The efficacy of the ISA50 inactivated vaccine, produced under industrial process, was evaluated in sheep during field challenges in two successive years. During year 1, there was a limited protective effect of the Gardel vaccine with 65% of survival rate for the vaccinated group compared to 49% for the control group (N=153, p=0.053). During year 2, the vaccine containing Gardel and a local strain gave an increased protective effect compared to the first trial: 72% of the vaccinated animals survived compared to 47% of the naïve animals (N=173, p<0.001). There was an important genetic diversity of strains in the field with detection of 11 different map1 genotypes in brains from control and vaccinated sheep post mortem. Map1 genotyping of strains detected in brains from control sheep showed that genotype distribution varied according to time and study areas, which could explain the difference in efficacy of the vaccine.

Studies of a polymorphic Ehrlichia ruminantium gene for use as a component of a recombinant vaccine against heartwater.

A previously identified polymorphic Ehrlichia ruminantium gene, Erum2510, was investigated to determine its ability to induce protective immunity in ruminants following two different DNA immunisation strategies; DNA-only and a DNA prime/recombinant protein (rprotein) boost immunisation. The DNA-only vaccine was also compared to a cocktail of three polymorphic E. ruminantium (Welgevonden) open reading frames (ORFs) adjacent to Erum2510 in the genome. Weak protection was observed in animals immunised with the pCMViUBs_Erum2510 construct alone, while none of the animals immunised with the DNA cocktail were protected. In contrast, all five animals immunised using a DNA prime/rprotein boost strategy survived challenge, thereby indicating that Erum2510 is a good candidate for inclusion in a recombinant vaccine against heartwater. One drawback of using polymorphic genes is a possible lack of cross-protection between genotypes, therefore the genetic diversity of Erum2510 was investigated to establish the degree of polymorphism among different E. ruminantium stocks. Three distinct genotypes were identified indicating that if this gene is used as a vaccine (prime/boost strategy) the vaccine should include a representative Erum2510 gene from each genotype.

Seroprevalence of tick-borne diseases in communal cattle reared on sweet and sour rangelands in a semi-arid area of South Africa.

A survey was conducted to determine the seroprevalence of tick-borne diseases (TBD) in Nguni and non-descript cattle on the sweet and sour communal rangelands across seasons. Body condition scores, bodyweights, packed cell volume (PCV) and antibodies to B. bovis, B. bigemina, Ehrlichiaruminatium and Anaplasma marginale were determined seasonally in 144 cattle raised on communal rangelands from August 2007 to April 2008. Approximately 45% of the cattle were seropositive for B. bovis, 46% for B. bigemina and 26% for A. marginale. All animals were seronegative for E. ruminantium. Nguni cattle had lower (P<0.05) seroprevalence for A. marginale and B. bigemina in the cool-dry and hot-wet seasons. Cattle in the sweet rangeland had significantly lower seroprevalence of B. bovis and B. bigemina in all the seasons. Infection with B. bovis and B. bigemina negatively affected (P<0.05) bodyweight and body condition scores whilst B. bovis and A. marginale infections significantly affected PCV. The seroprevalence of TBD was lower in the Nguni breed. More work is required to understand the mechanisms of the adaptation and possible resistance of Nguni cattle to TBD.

Persistence mechanisms in tick-borne diseases.

The use of new, highly sensitive diagnostic methods has revealed persistent infections to be a common feature of different tick-borne diseases, such as babesiosis, anaplasmosis and heartwater. Antigenic variation can contribute to disease persistence through the continual elaboration of new surface structures, and we know in several instances how this is achieved. Known or suspected mechanisms of persistence in babesial parasites include cytoadhesion and rapid variation of the adhesive ligand in Babesia bovis and genetic diversity in several merozoite stage proteins of different Babesia spp. In Anaplasma, extensive variation in the pfam01617 gene family accompanies cycling of organism levels in chronic infection. One result from the pioneering research at Onderstepoort is the definition of a related polymorphic gene family that is likely involved in immunity against heartwater disease. We are beginning to understand the sizes of the antigenic repertoires and full definition is close, with the possibility of applying simultaneous high-throughput sequencing to the order of 1000 small genomes. We also, for the first time, can consider modifying these genomes and looking at effects on persistence and virulence. However, important biological questions remain unanswered; for example, why we are seeing a new emerging Anaplasma infection of humans and is infection of endothelial cells by Anaplasma significant to persistence in vivo.

Trends in the control of heartwater.

Heartwater is an economically serious tick-borne disease of ruminants caused by the intracellular bacterium Ehrlichia ruminantium. The disease has traditionally been controlled by four different approaches: controlling the tick vector by dipping, establishing endemic stability, performing immunisation by infection and treatment, and preventing the disease by regular administration of prophylactic antibiotics. The first three of these methods are subject to failure for various epidemiological reasons, and serious disease outbreaks can occur. Prophylaxis is effective, but very expensive, and the logistics are daunting when large herds of animals are involved. The development of a safe, cheap and effective vaccine is the only likely way in which heartwater can be economically controlled, and over the past 15 years three new types of experimental vaccine have been developed: inactivated, attenuated, and recombinant vaccines. These new vaccines have shown varying degrees of promise, but none is as yet sufficiently successful to be marketable. We describe the experimental products, and the various technical and biological difficulties which are being encountered, and report on ways in which new technologies are being used to improve vaccine effectiveness.

Serological survey of antibodies to Ehrlichia ruminantium in small ruminants in Tanzania.

Sera from 497 sheep and 555 goats collected in a cross sectional study from different geographical locations in north-eastern Tanzania were examined for antibodies to Ehrlichia ruminantium using MAP 1-B ELISA technique. E. ruminantium antibodies were found in 68.6% (341/497) of sheep and 64.7% (359/555) of goats. Overall seroprevalence was 66.5% (700/1052). Infection rates were higher in sheep than goats (P < 0.05), in pastoral than in agro-pastoral production systems (P < 0.05) and in female sheep than males (P < 0.05). (131/143) 91.6% of the farms/flocks tested revealed sero-positive animals. E.ruminantium infections were found in all the geographical villages and districts tested. The infection rates per administrative district varied from 36.4% (Muheza) to 90% (Mkinga) in goats and from 11.9% (Muheza) to 94.6% (Mkinga) in sheep. The results shows E. ruminantium infection was prevalent and widely but unevenly distributed throughout the eight districts under study. These findings should be taken into consideration when future disease control and livestock upgrading programs are implemented.

The prevalence of serum antibodies to Ehrlichia ruminantium infection in ranch cattle in Tanzania: a cross-sectional study.

Serum samples collected in a cross-sectional survey of grazing cattle on Manyara Ranch, Monduli district, Tanzania, were tested by indirect major antigenic protein 1 fragment B (MAP 1-B) ELISA to determine the seroprevalence of Ehrlichia ruminantium and to assess ranch-level risk factors for heartwater. Heartwater-exposed cattle were widespread on the ranch and overall seroprevalence was 50.3% (95% CI, 44.9-55.6), enough to indicate an endemically unstable situation. Multivariate logistic regression modelling was used to identify risk factors associated with seropositivity. Two factors appeared to increase the herd's risk for contracting heartwater. Seroprevalence increased significantly with age (beta = 0.19 per year of age, P < 0.001) and animals carrying ticks of any species were associated with an increased risk of infection with E. ruminantium (Odds ratio, OR = 3.3, P < 0.001). The force of infection based on the age seroprevalence profile was estimated at 18 per 100 cattle year-risk. The current tick control measures on the ranch were associated with a decreased risk of infection with E. ruminantium (OR = 0.25 for no dipping and OR = 0.31 for low dipping, P < 0.001). Six tick species were identified; in order of frequency these were: Ambylomma variegatum 59.9%, Rhipicephalus evertsi evertsi 13.9%, Rhipicephalus pulchellus 12.5%, Hyalomma truncatum 7.03% and Rhipicephalus appendiculatus 6.07%. The least encountered tick was Rhipicephalus simus, which accounted for 0.38%. The cattle seemed well adapted to their environment and capable of resisting the tick burden under this extensive wildlife/livestock grazing and interaction system.

A heterologous prime/boost immunisation strategy protects against virulent E. ruminantium Welgevonden needle challenge but not against tick challenge.

Heterologous prime/boost immunisation strategies using the Ehrlichia ruminantium 1H12 pCMViUBs_ORFs [Pretorius A, Collins NE, Steyn HC, Van Strijp F, Van Kleef M, Allsopp BA. Protection against heartwater by DNA immunisation with four Ehrlichia ruminantium open reading frames. Vaccine 2007;25(12):2316-24] were investigated in this study. All the animals immunised twice with a recombinant (r) DNA cocktail of four 1H12 pCMViUBs_ORFs followed by a r1H12 protein and those immunised 3x with 1H12 plasmid rDNA showed 100% protection against a virulent E. ruminantium Welgevonden needle challenge. In addition, 90% of the sheep immunised twice with rDNA and boosted with r1H12 lumpy skin disease virus (LSDV) survived. Only the lymphocytes isolated from the r1H12 protein boost group showed specific proliferation and increased interferon (IFN)-gamma expression. In contrast, only 20% protection was obtained in animals immunised with the rDNA prime/r1H12 protein boost when subjected to natural tick challenge in the field. Thus this heterologous prime/boost immunisation strategy had not conferred any significant protection against a field challenge.

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.