Haemonchus contortus poses a major threat to small ruminant production in subtropical regions worldwide. Unfortunately, there is growing anthelmintic resistance, and the only licensed vaccine has limitations. This paper aimsto review the use of RNA-sequencing in understanding the immune responses of small ruminants to H. contortus infection, focusing on identifying differentially expressed genes and elucidating key immune pathways associated with resistance and susceptibility. This review was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Statement guidelines and Population, Intervention, Control, and Outcome framework covering publications from January 2015 to December 2023. RNA-seq identified the activation of key immune pathways, such as Th1/Th2, NK cell, B cell receptor signalling, MAPK, CAMs, and TNF signalling. There was upregulation of a range of PRRs, including TLRs and CLECs, in the resistant sheep, suggesting a crucial role for trained innate immune cells in resistance. However, there are no direct comparisons of TLR and CLEC expression between resistant and susceptible goats. This shows that there is a gap in understanding of the immune response mechanisms in goats. Addressing these knowledge gaps will lead to the development of more effective and sustainable control strategies.

Heartwater is one of the most economically important tick-borne fatal diseases of livestock. The disease is caused by the bacteria Ehrlichia ruminantium transmitted by Amblyomma ticks. Although there is evidence that interferon-gamma controls E. ruminantium growth and that cellular immune responses are protective, an effective recombinant vaccine for this disease is lacking. Analyses of markers associated with infection as well as protection will lead to a better understanding of the E. ruminantium immune response and corresponding pathways induced in sheep peripheral blood mononuclear cells (PBMC) will assist in development of such a vaccine. In this study, Biomarkers of infection (BMI) were identified as uniquely expressed genes during primary infection and biomarkers of protection (BMP) associated with immune to heartwater were identified post challenge. Sheep were experimentally infected and challenged with E. ruminantium infected ticks. The immune phenotypic and transcriptome profile of their PBMC were compared to their own naïve PBMC collected before infection. The study revealed 305 differentially expressed genes (DEGs) as BMI, of these 17 were upregulated at all three time-points investigated. These DEGs, form part of the bacterial invasion of epithelial cells Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway, and others detected from day 1 post infection and are considered predictive markers for early heartwater infection in ruminants. Similarly, a total of 332 DEGs were identified as BMP, of these 100 were upregulated and 75 were downregulated at all three time-points investigated. However, at D1PC most DEGs were downregulated (n = 1312) that correlated with a reduction in the % CD4 and CD8 T cells detected with flow cytometry. KEGG pathway analyses showed complete down regulation of T cell specific pathways possibly due to homing of immune cells to the site of infection after acquired immunity developed. At D4PC, expression levels of most of these downregulated genes increased and by D6PC they were upregulated. This indicates that the sampling time-point for biomarker analyses is important when results for acquired immune responses are inferred. This data identified DEGs that could be considered as biomarkers of protective immunity that can be used for identification of vaccine antigens and provides a strong foundation to further development of heartwater recombinant vaccines.

The viral proteins VP1-1, VP2, VP4, VP7 and NS3, of African horse sickness virus serotype 4 (AHSV4), have previously been identified to contain CD8+ T cell epitopes. In this study, overlapping peptides spanning the entire sequences of these AHSV4 proteins were synthesized and used to map epitopes. Peripheral blood mononuclear cells (PBMC) isolated from five horses immunized with an attenuated AHSV4 were stimulated in vitro with the synthesized peptides. Various memory immune assays were used to identify the individual peptides that contain CD8+ T cell epitopes, CD4+ T cell epitopes and linear B cell epitopes. The newly discovered individual peptides of AHSV4 proteins VP1-1, VP4, VP7 and/or NS3 that contain CD8+ T cell, CD4+ T cell or linear B cell epitopes could contribute to the design and development of new generation AHS peptide-based vaccines and therapeutics.

Heartwater, one of the major tick-borne diseases of some domestic and wild ruminants in Africa, is caused by Ehrlichia ruminantium. The genetic diversity of E. ruminantium isolates renders the available vaccine ineffective against certain virulent isolates. To better understand the E. ruminantium genotypes in South Africa, a total of 1004 Amblyomma hebraeum tick deoxyribonucleic acid (DNA) samples from cattle in three South African provinces were tested by pCS20 Sol1 real-time polymerase chain reaction (qPCR) and characterised by multilocus sequence typing (MLST) using five housekeeping genes. Out of 1004 samples tested, 222 (22%) were positive for E. ruminantium. The occurrence of E. ruminantium in Mpumalanga, KwaZulu-Natal and Limpopo provinces was 19%, 22% and 27%, respectively. The E. ruminantium positive samples were screened for housekeeping genes and sequenced. Phylogenetic analysis revealed three main lineages: clade 1 made up of worldwide isolates (eastern, southern Africa, and Caribbean isolates), clade 2 comprised only West African isolates and clade 3 consisted of Omatjenne, Kümm2 and Riverside. Some study sample sequences were not identical to any of the reference isolates. However, they could all be grouped into the worldwide clade. Genetic variation in the sequenced regions was observed in the form of single nucleotide polymorphisms (SNPs). Using MLST to characterise E. ruminantium field isolates allowed the South African genotypes to be clearly distinguished from the distinct West African isolates. Contribution: Characterisation of E. ruminantium field isolates is important for the control of heartwater and contributes to preliminary knowledge required for the development of a more practical vaccine against heartwater.

Rift Valley Fever virus (RVFV) causes the zoonotic RVF disease, which results in substantial economic losses in livestock industries. Regular vaccination of livestock against RVF is necessary to generate long-term immunity and avoid the loss of livestock. The live attenuated vaccine based on Clone 13 virus strain has been used to reduce the negative impact of RVF disease. The vaccine strain is heat labile and requires stringent conditions for storage and handling. This research evaluated lactose and sucrose-based stabilizers coupled with lyophilisation to enhance stability of the RVF Clone 13 vaccine strain. The glass transition temperature (Tg) of the sucrose-RVF vaccine was 97.0 degrees C with average residual moisture of below 2 %. The lactose formulation was characterised with Tg of 83.5 degrees C and residual moisture of above 2 %. The RVF Clone 13 sucrose-based formulation maintained higher antigen titres during lyophilisation compared to the lactose-formulated vaccine. Cellular-mediated and humoral immunity was evaluated and compared for the two newly formulated vaccines. Pheroid (R) technology was also investigated as a potential adjuvant and its ability to further enhance the immunogenicity conferred by the RVF Clone 13 vaccine formulations in Merino sheep. No adverse reactions were observed following injection of the vaccine formulations in mice, guinea pigs and Merino sheep. Comparable protective humoral immune responses against RVF were obtained for all animals vaccinated with the lactose and sucrose-based sta-bilisers with and without the Pheroid (R) adjuvant. No proliferation of CD8+ and CD4+ T-cells as well as expression of IFN-c was observed for all animals group vaccinated with Pheroid (R) only. Specific CD8+ IFN-c+T-cells were expressed at higher levels compared to the CD4+ IFN-c+T-cells in the RVF Clone 13 vaccines, suggesting that cellular immunity against RVF is through the Class I antigen presentation pathway. (c) 2022 Elsevier Ltd. All rights reserved.

EDITORIAL article Front. Vet. Sci., 30 May 2023Sec. Veterinary Infectious Diseases Volume 10 - 2023 | https://doi.org/10.3389/fvets.2023.1208088

Heartwater is one of the major tick-borne diseases of both domestic and some wild ruminants in Africa and some Caribbean islands. The disease is caused by Ehrlichia ruminantium, a member of the class Proteobacteria and order Rickettsiales. The diversity of E. ruminantium isolates render the commercially available vaccine ineffective against certain virulent isolates. The different genotypes may be caused by recombination that occurs between different genotypes. To better understand the E. ruminantium genotypes circulating in South Africa, a total of 1004 A. hebraeum tick DNA samples from cattle in three South African provinces were tested by pCS20 Sol1 qPCR and characterized by Multi-Locus Sequence Typing (MLST) using five housekeeping genes. Out of 1004 samples tested, 222 (22%) were positive for E. ruminantium. The prevalence of E. ruminantium in the Mpumalanga, KwaZulu Natal and Limpopo provinces was 19%, 22% and 27%, respectively. The E. ruminantium positive samples were screened for five housekeeping genes and sequenced. From the five housekeeping genes investigated, lipB and sodB were the most conserved; all analysed isolates were 100% identical to the previously identified E. ruminantium isolates. Phylogenetic analysis revealed three main lineages: clade 1 made up of worldwide isolates (Eastern, Southern Africa, and Caribbean isolates); clade 2 comprised only West African isolates; and clade 3 consisted of the unique isolates, Omatjenne, Kümm2, and Riverside. Some study sample sequences for the housekeeping genes were not identical to any of the reference isolates. However, they could all be grouped into the worldwide clade. Genetic variation in the sequenced regions was observed in the form of single nucleotide polymorphisms (SNPs). These variations did not alter the function of the proteins encoded by the housekeeping genes. Using MLST to characterize E. ruminantium field isolates allowed the South African genotypes to be clearly distinguished from the distinct West African isolates.

Cowdria polymorphic gene 1 (cpg1, Erum2510, ERUM_RS01380) has been shown to induce 30% and 100% protection in sheep immunised by deoxyribonucleic acid (DNA) prime combined with DNA boost and DNA prime combined with protein boost, respectively, against heartwater infection via needle challenge. To localise its antigenic regions for inclusion in a multi-epitope DNA vaccine against heartwater, Erum2510 was cleaved into five overlapping subfragments. These subfragments were expressed individually in an Escherichia coli host expression system and evaluated for their ability to induce proliferative responses, Th1 and Th2 cytokines (interferon gamma [IFN-γ] and interleukin 4 [IL-4]) via enzyme-linked immunospot (ELISpot), quantitative real time polymerase chain reaction (qRT-PCR) and flow cytometry. Recombinant (r)proteins 3 and 4 were shown to induce immunodominant Th1 and Th2 immune responses characterised by the secretion of effector cytokines IFN-γ and IL-4 in addition to differential messenger ribonucleic acid (mRNA) expression of tumour necrosis factor (TNF), IL-2, IL-1, IL-18, IL-10, transforming growth factor (TGF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and inducible nitric oxide synthase (iNOS). Thirty-seven overlapping synthetic peptides (16 mer) spanning the lengths of these immunodominant rproteins were synthesised and assayed. A peptide pool comprising p9 and p10 derived from rprotein 3 induced a Th1-biased immune response. A peptide pool comprising p28 and p29 derived from rprotein 4 induced a mixed Th1 and Th2 immune response characterised by secretion of IFN-γ and differential mRNA expression of IL-1, IL-2, IL-10, IL-12, iNOS, TGF, TNF and GM-CSF. Only one of the peptides (p29) induced secretion of IL-4. Phenotypic analysis showed significant activation of cluster of differentiation 8+ (CD8+), cluster of differentiation 4+ (CD4+) and B+ lymphocyte populations. Findings suggest that Erum2510 rproteins and synthetic peptides can induce both cellular and humoral immune responses, thereby implicating their importance in protection against heartwater. Contribution: This study will facilitate the design of an effective multi-epitope DNA vaccine against heartwater that will contribute to control this economically important disease in sub-Saharan Africa and beyond.

Expanding on our previous work, this study used transcriptome analysis of RNA sequences to investigate the various factors that contributed to either inducing apoptosis that resulted in cell death or promoting the survival of African horse sickness virus serotype 4 (AHSV4)-infected horse peripheral blood mononuclear cells (PBMC) after 24 h. Apoptosis is a host defense mechanism that prevents virus replication, accumulation and spread of progeny viruses. AHSV4-infected PBMC were killed via the intrinsic and the perforin/granzyme pathways of apoptosis during the attenuated AHSV4 (attAHSV4) in vivo primary and secondary immune responses. Trained innate immunity played an important role in circumventing viral interference that resulted in the elimination of AHSV4-infected PBMC through the intrinsic and the extrinsic pathways of apoptosis during the virulent AHSV4 (virAHSV4) in vitro secondary immune response. Oxidative stress in conjunction with IRE1 alpha pro-apoptotic signaling played a major role in the induction of the intrinsic pathway of apoptosis and cytotoxic lymphocytes induced the perforin/granzyme or extrinsic pathways of apoptosis. In contrast, AHSV4-infected PBMC survived during the virAHSV4 in vitro primary immune response, which allows unrestrained viral replication. The virAHSV4 interference with the innate immune response resulted in impaired NK cell responses and delayed immune responses, which together with the antioxidant defense system promoted AHSV4-infected PBMC survival.

Transcriptome analysis was used to characterise the in vitro primary and secondary immune responses induced in horse peripheral blood mononuclear cells (PBMC) stimulated for 24 h with the individual recombinant proteins of a virulent AHSV serotype 4 (AHSV4) field isolate (rAHSV4 proteins) that were previously expressed in Escherichia coli (E. coli). The results showed that the E. coli contamination products greatly affected the innate and humoral immune response transcripts. Hence, the impact of E. coli contamination products present in the individual rAHSV4 proteins on the translational immune response was determined. The combined amplification effects of synergistic pattern recognition receptors (PRRs), TNF-α and IL-1β signalling induced potent pro-inflammatory responses that were too overwhelming for the anti-inflammatory cytokines and regulators to control. In addition to inducing robust B cell and antibody-mediated responses, lipopolysaccharide (LPS) activation of the innate-like B cells and subsequent polyreactive (natural) antibody responses could potentially contribute to endotoxin tolerance.

Dual vaccines (n = 6) against both lumpy skin disease (LSD) and bovine ephemeral fever (BEF) were constructed, based on the BEFV glycoprotein (G) gene, with or without the BEFV matrix (M) protein gene, inserted into one of two different LSDV backbones, nLSDV∆SOD-UCT or nLSDVSODis-UCT. The inserted gene cassettes were confirmed by PCR; and BEFV protein was shown to be expressed by immunofluorescence. The candidate dual vaccines were initially tested in a rabbit model; neutralization assays using the South African BEFV vaccine (B-Phemeral) strain showed an African consensus G protein gene (Gb) to give superior neutralization compared to the Australian (Ga) gene. The two LSDV backbones expressing both Gb and M BEFV genes were tested in cattle and shown to elicit neutralizing responses to LSDV as well as BEFV after two inoculations 4 weeks apart. The vaccines were safe in cattle and all vaccinated animals were protected against virulent LSDV challenge, unlike a group of control naïve animals, which developed clinical LSD. Both neutralizing and T cell responses to LSDV were stimulated upon challenge. After two inoculations, all vaccinated animals produced BEFV neutralizing antibodies ≥ 1/20, which is considered protective for BEF.

African horse sickness (AHS) is caused by African horse sickness virus (AHSV), a double stranded RNA (dsRNA) virus of the genus Orbivirus, family Reoviridae. For the development of new generation AHS vaccines or antiviral treatments, it is crucial to understand the host immune response against the virus and the immune evasion strategies the virus employs. To achieve this, the current study used transcriptome analysis of RNA sequences to characterize and compare the innate immune responses activated during the attenuated AHSV serotype 4 (attAHSV4) (in vivo) and the virulent AHSV4 (virAHSV4) (in vitro) primary and secondary immune responses in horse peripheral blood mononuclear cells (PBMC) after 24 h. The pro-inflammatory cytokine and chemokine responses were negatively regulated by anti-inflammatory cytokines, whereas the parallel type I and type III IFN responses were maintained downstream of nucleic acid sensing pattern recognition receptor (PRR) signalling pathways during the attAHSV4 primary and secondary immune responses. It appeared that after translation, virAHSV4 proteins were able to interfere with the C-terminal IRF association domain (IAD)-type 1 (IAD1) containing IRFs, which inhibited the expression of type I and type III IFNs downstream of PRR signalling during the virAHSV4 primary and secondary immune responses. Viral interference resulted in an impaired innate immune response that was not able to eliminate virAHSV4-infected PBMC and gave rise to prolonged expression of pro-inflammatory cytokines and chemokines during the virAHSV4 induced primary immune response. Indicating that virAHSV4 interference with the innate immune response may give rise to an excessive inflammatory response that causes immunopathology, which could be a major contributing factor to the pathogenesis of AHS in a naïve horse. Viral interference was overcome by the fast kinetics and increased effector responses of innate immune cells due to trained innate immunity and memory T cells and B cells during the virAHSV4 secondary immune response.

Heartwater is a non-contagious tick-borne disease of domestic and wild ruminants. Data regarding the complex processes involved during pathogen-vector?host interaction during Ehrlichia ruminantium infection is lacking and could be improved with knowledge associated with gene expression changes in both the pathogen and the host. Thus, in the current study, we aimed to identify E. ruminantium genes that are up-regulated when the pathogen enters the host and before the disease is established. Identification of such genes/proteins may aid in future vaccine development strategies against heartwater. RNA-sequencing was used to identify E. ruminantium genes that were exclusively expressed at the tick bite site in sheep skin biopsies (SB) and in adult tick salivary glands (SG). RNA was extracted from pooled samples of the SB or SG collected at different time points during tick attachment and prior to disease manifestation. Ribosomal RNA (rRNA) was removed and the samples were sequenced. Several E. ruminantium genes were highly expressed in all the samples while others were exclusively expressed in each. It was concluded that E. ruminantium genes that were exclusively expressed in the SB or both SB and SG when compared to the transcriptome datasets from bovine elementary bodies (BovEBs) from cell culture may be considered as early antigenic targets of host immunity. In silico immunogenic epitope prediction analysis and preliminary characterization of selected genes in vitro using ELIspot assay showed that they could possibly be ideal targets for future vaccine development against heartwater, however, further epitope characterization is still required.

Lumpy skin disease and Rift Valley fever are two high-priority livestock diseases which have the potential to spread into previously free regions through animal movement and/or vectors, as well as intentional release by bioterrorists. Since the distribution range of both diseases is similar in Africa, it makes sense to use a bivalent vaccine to control them. This may lead to the more consistent and sustainable use of vaccination against Rift Valley fever through a more cost-effective vaccine. In this study, a recombinant lumpy skin disease virus was constructed in which the thymidine kinase gene was used as the insertion site for the Gn and Gc protective glycoprotein genes of Rift Valley fever virus using homologous recombination. Selection markers, the enhanced green fluorescent protein and Escherichia coli guanidine phosphoribosyl transferase (gpt), were used for selection of recombinant virus and in a manner enabling a second recombination event to occur upon removal of the gpt selection-pressure allowing the removal of both marker genes in the final product. This recombinant virus, LSD-RVF.mf, was selected to homogeneity, characterized and evaluated in cattle as a vaccine to show protection against both lumpy skin disease and Rift Valley fever in cattle. The results demonstrate that the LSD-RVF.mf is safe, immunogenic and can protect cattle against both diseases.

Bovine anaplasmosis is a globally economically important tick-borne disease caused by the obligate intraerythrocytic rickettsia, Anaplasma marginale. A live Anaplasma centrale blood-based vaccine is available, but it does not protect against all A. marginale field strains and may also transmit other blood-borne pathogens. Five potential outer membrane protein (OMP) vaccine candidates have been well-characterised in A. marginale strains from the USA, however, their levels of conservation in other countries must be ascertained in order to inform their use in a vaccine with regional or global efficacy. This study assessed the amino acid variation in vaccine candidate OMPs in South African strains of A. marginale, and also compared the immunogenic properties between South African and US strains. OMP genes Am779, Am854, omp7, omp8 and omp9 were amplified and sequenced from a set of genetically diverse South African samples with different msp1 alpha-genotypes. OMPs Am854 and Am779 were highly conserved, with 99-100 % amino acid identity, while Omp7, Omp8 and Omp9 had 79-100 % identity with US strains. As has been shown previously, Omp7-9 possess conserved Nand Ctermini, a central variable region, and a highly conserved CD4 T-cell epitope, FLLVDDA(I/V)V, in the N-terminal region. Western blot analysis of recombinant OMPs indicates strong antigenic conservation between South African and US strains of A. marginale, suggesting that they are good candidates for use in a novel global vaccine cocktail, although further work on the best formulation and delivery methods will be necessary.

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.

Heartwater is a tick-borne disease caused by the intracellular rickettsial parasite Ehrlichia ruminantium and transmitted by Amblyomma hebraeum ticks. Heartwater is problematic in endemic areas because it causes high mortality in ruminants and leads to economic losses that threaten productivity and food security. This may indicate that there is augmented genetic diversity in the field, which may result in isolates that are more virulent than the Ball3 and Welgevonden isolates. The genetic diversity of E. ruminantium was investigated in this study, focussing on the pCS20 gene region and four polymorphic open reading frames (ORFs) identified by subtractive hybridisation. The 16S ribosomal ribonucleic acid gene confirmed E. ruminantium in brain, blood and tick genomic deoxyribonucleic acid samples (n = 3792) collected from 122 farms that were randomly selected from seven provinces of South Africa where heartwater is endemic. The conserved E. ruminantium pCS20 quantitative polymerase chain reaction (qPCR) assay was used to scan all collected field samples. A total of 433 samples tested positive with the qPCR using the pCS20 gene region, of which 167 were sequenced. The known stocks and field samples were analysed, and phylogenetic trees were generated from consensus sequences. A total of 25 new clades were identified; of these, nine isolates from infected blood could be propagated in cell cultures. These clades were not geographically confined to a certain area but were distributed amongst heartwater-endemic areas in South Africa. Thus, the knowledge of strain diversity of E. ruminantium is essential for control of heartwater and provides a basis for further vaccine development.

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 ruminantizim 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 IFN gamma, 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-gamma 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), Enun1150 (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-gamma production in immune sheep PBMC. The upregulation of pro-inflammatory cytokines, which include, IL-1 alpha, IL-2, IL-12p40, TNF-alpha, IFN-gamma, 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.

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.