East Coast Fever Research Articles

Corridor disease (buffalo-associated Theileria parva) outbreak in cattle introduced onto a game ranch and investigations into their carrier-state.

East Coast fever (Theileria parva infection in cattle) was eradicated from South Africa in the mid-1900. However, another form named Corridor disease (CD), associated with T. parva carrier buffaloes exists and outbreaks have increased in endemic areas. The occurrence of a CD carrier state in cattle under field situations has not been demonstrated but remains a subject of controversy. The current study investigated the T. parva carrier state following a severe outbreak in cattle introduced onto a game ranch. Monitoring of the outbreak included clinical signs, mortality, microscopy, serology, real-time PCR and xenodiagnoses. The herd of cattle received block treatment using oxytetracyclines (OTC) by the farmer during the outbreak. Cattle were sampled early during the outbreak and twice within the following 75 days. All buffaloes were tested for a T. parva carrier state. Two batches of questing adult R. appendiculatus were collected at the time of disease occurrence and a year later. These ticks were fed on susceptible cattle under controlled conditions and monitored for disease transmission. Ticks infected with a buffalo-derived stock of T. parva were fed on one bovine under controlled conditions and simultaneously injected with OTC, simulating the infection and treatment method of vaccination and was used as a positive control. Clean R. appendiculatus nymphs were fed on four recovered PCR positive cattle from the outbreak and on the positive control animal. The adult ticks were tested for infectivity by xenodiagnoses on susceptible bovines. For the initial outbreak the CD prevalence was 62.3% with a mortality rate of 29.5%. However, the outbreak was contained by block OTC treatment of the herd since only 3.4% cattle subsequently died until the end of the investigations. Adult ticks fed on one field bovine and the laboratory established T. parva carrier both transmitted fatal infections to susceptible cattle. Ticks fed on two field cattle transmitted T. taurotragi and one failed to transmit any infection. Questing adult R. appendiculatus collected during the outbreak transmitted fatal CD to two bovines while ticks collected a year later transmitted T. taurotragi. These findings demonstrated the effectiveness of disease control either by cattle treatment using OTC simulating the ITM or by intensive cattle dipping following the outbreak or by both interventions. The potential risk of creating carrier cattle by OTC treatment during CD outbreaks should be considered, supporting the continued control measures of segregation of cattle and buffalo herds.

Detection of carrier state and genetic diversity of Theileria parva in ECF-vaccinated and naturally exposed cattle in Tanzania.

Infection and Treatment Method (ITM) has been practiced in Tanzania for over 20 years as a prevention measure against East Coast Fever disease. It is known that ITM, like natural ECF infection, leads to a carrier state, whereby vaccinated cattle become asymptomatic carriers of the parasite. It is expected that ECF vaccination using ITM also leads to generation of combinations of vaccine specific Theileria parva and local strains that circulate in the field what contributes to an unknown level of parasite diversity. Moreover, the long term impact of ITM on carrier state and parasite diversity in cattle are largely unknown. To address this question blood was collected from ECF-vaccinated (n = 239) and unvaccinated (n = 97) cattle from Loiborsoit, Emboreet, Esilalei, Manyara ranch and Mswakini villages in the Maasai steppe of northern Tanzania, as well as Mruazi and Leila farms in Tanga in eastern Tanzania. Screening for T. parva using nested PCR revealed an overall prevalence of T. parva to be 34.5%, with a significant higher prevalence among ECF-vaccinated cattle. Using three VNTR markers (ms2, ms5 and MS7) higher parasite genetic diversity in terms of higher number of alleles and expected heterozygosity was shown in vaccinated than unvaccinated cattle. These parameters were highest in cattle from Manyara ranch. Nevertheless, the principle component analysis (PCoA) showed no distinct clustering patterns as most T. parva alleles clustered together throughout the four quadrants implying parasite homogeneity among the sampled populations. However, some of the parasite alleles closely clustered with Muguga vaccine alleles in two of the quadrants, consistent with closer genetic relatedness between the vaccine strains and the T. parva populations from the Maasai steppe. Likewise analysis of molecular variance (AMOVA) revealed most of the genetic variation (93%) being contained within populations with only 7% being among populations. This study therefore confirms the role of ECF vaccination in enhancing carrier state and T. parva diversity in vaccinated cattle populations. Higher T. parva diversity may play an important role in carrier cattle by way of restricting breakthrough infections from field parasite strains.

Diversity of two Theileria parva CD8+ antigens in cattle and buffalo-derived parasites in Tanzania

Theileria parva is a tick-transmitted protozoan parasite that causes a disease called East Coast fever (ECF) in cattle. This important tick borne-disease (TBD) causes significant economic losses in cattle in many sub-Saharan countries, including Tanzania. Cattle immunization using Muguga cocktail has been recommended as an effective method for controlling ECF in pastoral farming systems in Tanzania. However, immunity provided through immunization is partially strain-specific. Therefore, the control of ECF in Tanzania is still a challenge due to inadequate epidemiological information. This study was conducted to assess genetic diversity of Tp1 and Tp2 genes from T. parva isolates that are recognized by CD8 + T-cells in cattle and buffalo. The Tp1 and Tp2 genes are currently under evaluation as candidates for inclusion in a subunit vaccine. A total of 130 blood samples collected from cattle which do not interact with buffalo (98), cattle co-grazing with buffalo (19) and buffalo (13) in Mara, Mbeya, Morogoro, Tanga, and Coast regions in Tanzania were used in this study. Genomic DNA was extracted from the blood samples, Tp1 and Tp2 genes were amplified using nested PCR and the PCR products were purified and sequenced. The partial sequencing of the Tp1 and Tp2 genes from T. parva isolates exhibited polymorphisms in both loci, including the epitope-containing regions. Results for sequence analysis showed that the overall nucleotide polymorphism (π) was 0.7% and 13.5% for Tp1 and Tp2, respectively. The Tajima’s D and Fu’s Fs test showed a negative value for both Tp1 and Tp2 genes, indicating deviations from neutrality due to a recent population expansion. The study further revealed a low to high level of genetic differentiations between populations and high genetic variability within populations. The study also revealed that most samples from the seven populations possessed several epitopes in antigens that were identical to those in the T. parva Muguga reference stock, which is the main component of the widely used live vaccine cocktail. Therefore, different strategic planning and cost-effective control measures should be implemented in order to reduce losses caused by ECF in the study areas.

Molecular and Antigenic Properties of Mammalian Cell-Expressed Theileria parva Antigen Tp9.

East Coast Fever (ECF), caused by the tick-borne apicomplexan parasite Theileria parva, is a leading cause of morbidity and mortality in cattle of sub-Saharan Africa. The infection and treatment method (ITM) is currently the only vaccine available to control T. parva. Although ITM elicits levels of protection, its widespread adoption is limited by costs, laborious production process, and antibiotic co-treatment requirement, necessitating the development of a more sustainable vaccine. To this end, efforts have been concentrated in the identification of new T. parva vaccine antigens and in the development of suitable platforms for antigen expression. In this study, we investigated the molecular and antigenic properties of T. parva antigen Tp9 expressed by mammalian cells. Data indicate that Tp9 contains a signal peptide that is weakly functional in mammalian cells. Thus, Tp9 secretion from mammalian cells increased 10-fold after the native signal peptide was replaced with the human tissue plasminogen activator signal peptide (tPA). Sera from all T. parva-immune cattle recognized this recombinant, secreted Tp9. Additionally, PBMC from ITM-immunized cattle produced significant (p < 0.05) amounts of IFNγ following ex vivo exposure to Tp9, but this response varied between cattle of different MHC class I and class II genotypes. In addition, depletion experiments demonstrated that IFNγ to Tp9 was primarily produced by CD4+ T cells. Molecular analysis demonstrated that Tp9 presents a signal peptide that is weakly functional in mammalian cells, suggesting that it remains within lymphocytes during infection. Tp9 secretion from mammalian cells was substantially increased when the tPA secretion signal sequence was substituted for the native secretion signal sequence. Using full-length, recombinant Tp9 secreted from mammalian cells, we demonstrated that T. parva-immune cattle develop both humoral and cellular immune responses to this antigen. Collectively, these results provide rationale for further evaluation of Tp9 as a component of a T. parva subunit vaccine.

Infection and treatment method (ITM) vaccine against East Coast fever: reducing the number of doses per straw for use in smallholder dairy herds by thawing, diluting and refreezing already packaged vaccine

BackgroundThe Infection and Treatment Method (ITM) of vaccination is the only immunization procedure currently available to protect cattle against East Coast fever (ECF), a tick-transmitted disease responsible for losses of several hundreds of millions of dollars per year in sub-Saharan Africa. The vaccine comprises a homogenized preparation of infected ticks packaged in straws and stored in liquid nitrogen. The current manufacturing protocol results in straws containing 30–40 doses (ILRI 0804), which is impractical for immunizing small herds as found in dairy and smallholder farming systems. The ILRI 0804 SD stabilate was prepared as a 1:5 dilution of the parent stabilate, with the aim of producing vaccine stabilate straws containing between four to eight doses and thus suitable for smallholder farming systems. Infectivity of the diluted stabilate was assessed and the protective efficacy of the diluted stabilate was determined by performing experimental and field immunizations.ResultsTwo groups of six cattle were inoculated with 1 ml of the diluted stabilate at 1:20 (equivalent to the recommended field dose for ILRI 0804, assuming no loss of sporozoite viability during thawing and refreezing) and 1:14 (assuming 30–35% loss of sporozoite viability). Schizonts were detected in all 12 animals, showing viability of sporozoites. Ten animals from the infectivity study and two control animals not previously exposed to T. parva were challenged with the parental ILRI 0804 stabilate. The results show that the two control animals displayed severe ECF reactions and were treated 14 days after challenge. Of the previously infected animals, only one underwent a severe reaction following challenge, a result in accord with the challenge experiments performed previously with the parent stabilate [Ticks Tick-Borne Dis 7:306-314, 2016]. The animal that displayed a severe reaction had no detectable schizonts and did not seroconvert following the initial inoculation with ILRI 0804 SD. In addition, 62 animals immunized under field conditions showed a mean seroconversion rate of 82%.ConclusionThe results presented in this article demonstrate that it is possible to prepare straws suitable for use in smallholder herds by thawing, diluting and refreezing already packaged vaccine.

A participatory epidemiological study of major cattle diseases amongst Maasai pastoralists living in wildlife-livestock interfaces in Maasai Mara, Kenya

Livestock-wildlife interactions promote the transmission of a wide range of infectious diseases that constraint livestock production. We used a participatory appraisal approach to find out and rank infectious diseases of concern to pastoralists in a zone of intense wildlife-livestock interaction and another zone with limited interactions. Four villages were selected purposively in areas with intensive cattle-wildlife interactions (zone 1), and another two in areas with low to moderate cattle-wildlife interactions (zone 2). Data were collected in focus group discussions (FGDs) using participatory epidemiological methods (PE); each group had 8–13 participants. Results of impact matrix scoring from all sites indicated that malignant catarrhal fever (MCF), anthrax, foot and mouth disease (FMD), contagious bovine pleuropneumonia (CBPP), east coast fever (ECF) and African animal trypanosomiasis (ATT), in decreasing order, had the highest impact on livestock production. A Kruskal–Wallis test revealed a significant difference in FMD annual prevalence between cattle age groups (p < 0.001) and was the highest in animals > 4 years (median score of 32.5, range, 10–50). FMD had the highest impact on milk production, but based on veterinary costs (treatment costs), it was ranked second to CBPP. The study provides information on disease priorities that occur in the target zones in Mara ecosystem and which the local pastoralists must consider when accessing key ecosystem services such as water and pasture.

Leveraging the Medicines for Malaria Venture malaria and pathogen boxes to discover chemical inhibitors of East Coast fever

Chemotherapy of East Coast fever, a lymphoproliferative cancer-like disease of cattle causing significant economic losses in Africa, is largely dependent on the use of buparvaquone, a drug that was developed in the late 1980's. The disease is caused by the tick-borne protozoan pathogen Theileria parva. Buparvaquone can be used prophylactically and it is also active against tropical theileriosis, caused by the related parasite Theileria annulata. Recently, drug resistance was reported in T. annulata, and could occur in T. parva. Using a 3H-thymidine incorporation assay we screened 796 open source compounds from the Medicines for Malaria Venture (MMV) to discover novel chemicals with potential inhibitory activity to T. parva. We identified nine malaria box compounds and eight pathogen box compounds that inhibited the proliferation of F100TpM, a T. parva infected lymphocyte cell line. However, only two compounds, MMV008212 and MMV688372 represent promising leads with IC50 values of 0.78 and 0.61 μM, respectively, and CC50 values > 5 μM. The remaining compounds exhibited a high degree of toxicity (CC50 values < 1.09 μM) on the proliferation of bovine peripheral blood mononuclear cells stimulated with concanavalin A. We also tested the anti-cancer drug, dasatinib, used in the chemotherapy of some leukemias. Dasatinib was as active and safe as buparvaquone in vitro, with an IC50 of 5 and 4.2 nM, respectively, and CC50 > 10 μM. Our preliminary data suggest that it may be possible to repurpose compounds from the cancer field as well as MMV as novel anti-T. parva molecules.

Combining Landscape Genomics and Ecological Modelling to Investigate Local Adaptation of Indigenous Ugandan Cattle to East Coast Fever.

East Coast fever (ECF) is a fatal sickness affecting cattle populations of eastern, central, and southern Africa. The disease is transmitted by the tick Rhipicephalus appendiculatus, and caused by the protozoan Theileria parva parva, which invades host lymphocytes and promotes their clonal expansion. Importantly, indigenous cattle show tolerance to infection in ECF-endemically stable areas. Here, the putative genetic bases underlying ECF-tolerance were investigated using molecular data and epidemiological information from 823 indigenous cattle from Uganda. Vector distribution and host infection risk were estimated over the study area and subsequently tested as triggers of local adaptation by means of landscape genomics analysis. We identified 41 and seven candidate adaptive loci for tick resistance and infection tolerance, respectively. Among the genes associated with the candidate adaptive loci are PRKG1 and SLA2. PRKG1 was already described as associated with tick resistance in indigenous South African cattle, due to its role into inflammatory response. SLA2 is part of the regulatory pathways involved into lymphocytes' proliferation. Additionally, local ancestry analysis suggested the zebuine origin of the genomic region candidate for tick resistance.

An Ad/MVA vectored Theileria parva antigen induces schizont-specific CD8+ central memory T cells and confers partial protection against a lethal challenge

The parasite Theileria parva is the causative agent of East Coast fever (ECF), one of the most serious cattle diseases in sub-Saharan Africa, and directly impacts smallholder farmers’ livelihoods. There is an efficient live-parasite vaccine, but issues with transmission of vaccine strains, need of a cold chain, and antibiotics limit its utilization. This has fostered research towards subunit vaccination. Cytotoxic T lymphocytes (CTL) are crucial in combating the infection by lysing T. parva-infected cells. Tp1 is an immunodominant CTL antigen, which induces Tp1-specific responses in 70–80% of cattle of the A18 or A18v haplotype during vaccination with the live vaccine. In this study, human adenovirus serotype 5 (HAd5) and modified vaccinia Ankara (MVA) were assessed for their ability to induce Tp1-specific immunity. Both viral vectors expressing the Tp1 antigen were inoculated in cattle by a heterologous prime-boost vaccination regimen. All 15 animals responded to Tp1 as determined by ELISpot. Of these, 14 reacted to the known Tp1 epitope, assayed by ELISpot and tetramer analyses, with CTL peaking 1-week post-MVA boost. Eleven animals developed CTL with specific cytotoxic activity towards peripheral blood mononuclear cells (PBMC) pulsed with the Tp1 epitope. Moreover, 36% of the animals with a Tp1 epitope-specific response survived a lethal challenge with T. parva 5 weeks post-MVA boost. Reduction of the parasitemia correlated with increased percentages of central memory lymphocytes in the Tp1 epitope-specific CD8+ populations. These results indicate that Tp1 is a promising antigen to include in a subunit vaccine and central memory cells are crucial for clearing the parasite.

An overview of animal health and communication constraints in smallholder farming systems of Machakos County, Kenya

Information sharing is an important pillar for any strategy that aims to control infectious diseases especially in farms and systems that are connected. A study was designed to analyze practices related to sharing of animal health information and identify health constraints affecting livestock keeping in smallholder farming systems of Mwala in Machakos County. Data were collected through focus group discussions (number of groups = 3) and one-on-one questionnaire interviews (n = 172). These were analyzed using both descriptive and thematic approaches. The most (33%; n = 158) feared animal disease was pneumonia which was also the most (62%) frequently reported health problem. Rabies, Newcastle disease, and east coast fever were ranked high by the farmer groups. Current animal management practices, for example, failure to confine dogs, predispose the community to a number of health risks including bites from rabid animals. Smallholder farmers in Mwala utilize a number of options to communicate animal health problems; such sharing is, however, constrained by a number of factors including bad roads, poor coverage of mobile networks, and bad relationships between animal health providers and the farmers themselves. Some of the reasons why farmers delay the reporting of disease problems included the tendency for one to first try local herbal formulations, the bad times when cases are observed, and the unavailability of reliable animal health providers at the time when the farmers need them. This study has provided useful baseline data that can then be used to (1) design a village-based animal disease reporting system which is currently lacking in the surveyed villages and (2) support the county government's efforts to manage animal diseases including zoonoses, through better reporting, for improved health, nutrition, and livelihoods.

Impact of East Coast fever on Grande Comore: assessment taking a participatory epidemiology approach.

East Coast fever (ECF), one of the most serious tick-borne diseases in sub-Saharan and eastern Africa, was introduced to the island of Grande Comore in 2002 through zebu import from Tanzania, resulting in at least a 10% loss of livestock. A participatory epidemiology initiative was launched in 2015 to gain a better understanding of ECF epidemiology. Thirty-six villages were investigated involving 36 focus group sessions and 120 individual questionnaires. Farmers' knowledge of ECF and of priority diseases affecting the country was assessed, and the impacts of ECF and other major diseases were compared by a scoring method. The results showed that 69.4% (95% CI [51.3, 87.5%]) of the farmers had good to very good knowledge of ECF. The most important cattle diseases on Grande Comore were considered to be East Coast fever, heartwater, babesiosis, and cutaneous diseases. About 58% of the farmers (95% CI [49.2, 66.8%]) use curative treatments when cattle were sick. Between January and September 2015, the ECF incidence was estimated at 18.5% (95% CI [15.5, 21.4%]), and 87.5% (95% CI [72.7, 100%]) of the cattle infected by ECF died. The ECF incidence estimated in our study was found to be less when compared to that observed in Tanzania even though the climatic conditions in the Union of the Comoros are suitable for the biological vector of ECF, the tick species Rhipicephalus appendiculatus. Access to chemical treatment and its effectiveness against ECF, as well as controlling borders and organizing quarantine, are discussed.

Molecular epidemiology of Babesia species, Theileria parva, and Anaplasma marginale infecting cattle and the tick control malpractices in Central and Eastern Uganda

East Coast fever, babesiosis, and anaplasmosis are the major tick-borne diseases affecting cattle productivity in Uganda. The emergence of acaricide-resistant ticks is suspected to have caused a rise in hemoparasites. This study sought to detect and characterize hemoparasites among farms in acaricide-failure hotspots of central as compared to the acaricide-failure naïve areas in Eastern Uganda. Nested PCR assays were performed to determine the prevalences of Babesia bovis, Babesia bigemina, Theileria parva, and Anaplasma marginale in cattle blood samples sourced from randomly selected farms. Randomly selected isolates were sequenced to determine the genetic diversity of the parasites using the following marker genes: B. bovis spherical body protein 4, B. bigemina rhoptry-associated protein 1a, T. parva 104 kDa microneme-rhoptry antigen, and A. marginale major surface protein 5. Furthermore, partially and fully engorged adult ticks were collected for taxonomy, and tick-control practices were assessed using a semi-structured questionnaire. The prevalences of B. bigemina, T. parva, and A. marginale in cattle were 17.2, 65.1, and 22.0%, and 10.0, 26.5, and 3% in the central and eastern region, respectively. Whilst, B. bovis was not detected in the farms involved. The sequences for B. bigemina, T. parva, and A. marginale from the central region showed 99% identity with those from the eastern region. Of the 548 ticks collected, 319, 147, 76, and 6 were Rhipicephalus (Boophilus) decoloratus, Rhipicephalus appendiculatus, Amblyomma variegatum, and Rhipicephalus evertsi evertsi, respectively. The Rhipicephalus ticks were more abundant in the central region, whereas A. variegatum ticks were more abundant in the eastern region. Tick control malpractices were found in both Central and Eastern Uganda, and 42 of the 56 surveyed farms lacked appropriate restraining facilities and so they utilized either ropes or a ‘boma’ (enclosure). In summary, B. bigemina, T. parva, A. marginale and their co-infections were more prevalent in the central than eastern region; even though, tick control malpractices were observed in both regions. Therefore, an urgent tick and TBD control strategy is needed.

Increasing the Local Relevance of Epidemiological Research: Situated Knowledge of Cattle Disease Among Basongora Pastoralists in Uganda.

Cattle disease can have severe negative impacts on the livelihoods of the poor, but still, animal disease management and outreach often remain suboptimal in low-income settings. In a study on Basongora pastoralists in Uganda, we examined local priorities, perceptions and practices regarding cattle disease, in order to improve outreach and disease control advisory work in such contexts. We also investigated how participatory epidemiology can be better equipped for gathering situated knowledge. Empirical material obtained in focus group discussions, interviews, participatory mapping, and wealth-ranking was used to perform a thematic, bottom-up analysis. The concepts of situated knowledge and embodied objectivity and insights from participatory research and interdisciplinary dialogue were applied to better embrace local perspectives. Cowdriosis, trypanosomosis, contagious bovine pleuropneumonia, East Coast fever and anthrax were high-priority diseases for participants. Lack of control over the animal health situation and money invested in treatments that did not guarantee recovery were of general importance for disease prioritization. Participants' descriptions of diseases sometimes diverged from textbook definitions. Co-infections, chronic and recurring infections and lack of access to formal knowledge were identified as important factors for differences between formal and situated knowledge. Paying attention to situated knowledge and particular context-specific issues such as proximity to a national park proved to be of special relevance for local understanding and experiences with disease. Another factor was the local importance ascribed to number of cattle, rather than production levels. These factors need to be taken into consideration when formulating disease control advice, as does the complex disease landscape. The results reveal the importance of moving research and advice beyond curing “knowledge-gaps” and creating different ways of understanding disease so that situated knowledge can be considered, and disease control improved.

Mitochondrial phylogeography and population structure of the cattle tick Rhipicephalus appendiculatus in the African Great Lakes region

BackgroundThe ixodid tick Rhipicephalus appendiculatus is the main vector of Theileria parva, wich causes the highly fatal cattle disease East Coast fever (ECF) in sub-Saharan Africa. Rhipicephalus appendiculatus populations differ in their ecology, diapause behaviour and vector competence. Thus, their expansion in new areas may change the genetic structure and consequently affect the vector-pathogen system and disease outcomes. In this study we investigated the genetic distribution of R. appendiculatus across agro-ecological zones (AEZs) in the African Great Lakes region to better understand the epidemiology of ECF and elucidate R. appendiculatus evolutionary history and biogeographical colonization in Africa.MethodsSequencing was performed on two mitochondrial genes (cox1 and 12S rRNA) of 218 ticks collected from cattle across six AEZs along an altitudinal gradient in the Democratic Republic of Congo, Rwanda, Burundi and Tanzania. Phylogenetic relationships between tick populations were determined and evolutionary population dynamics models were assessed by mismach distribution.ResultsPopulation genetic analysis yielded 22 cox1 and 9 12S haplotypes in a total of 209 and 126 nucleotide sequences, respectively. Phylogenetic algorithms grouped these haplotypes for both genes into two major clades (lineages A and B). We observed significant genetic variation segregating the two lineages and low structure among populations with high degree of migration. The observed high gene flow indicates population admixture between AEZs. However, reduced number of migrants was observed between lowlands and highlands. Mismatch analysis detected a signature of rapid demographic and range expansion of lineage A. The star-like pattern of isolated and published haplotypes indicates that the two lineages evolve independently and have been subjected to expansion across Africa.ConclusionsTwo sympatric R. appendiculatus lineages occur in the Great Lakes region. Lineage A, the most diverse and ubiquitous, has experienced rapid population growth and range expansion in all AEZs probably through cattle movement, whereas lineage B, the less abundant, has probably established a founder population from recent colonization events and its occurrence decreases with altitude. These two lineages are sympatric in central and eastern Africa and allopatric in southern Africa. The observed colonization pattern may strongly affect the transmission system and may explain ECF endemic instability in the tick distribution fringes.

Immunization with one Theileria parva strain results in similar level of CTL strain-specificity and protection compared to immunization with the three-component Muguga cocktail in MHC-matched animals

BackgroundThe tick-borne protozoan parasite Theileria parva causes a usually fatal cattle disease known as East Coast fever in sub-Saharan Africa, with devastating consequences for poor small-holder farmers. Immunity to T. parva, believed to be mediated by a cytotoxic T lymphocyte (CTL) response, is induced following natural infection and after vaccination with a live vaccine, known as the Infection and Treatment Method (ITM). The most commonly used version of ITM is a combination of parasites derived from three isolates (Muguga, Kiambu 5 and Serengeti-transformed), known as the “Muguga cocktail”. The use of a vaccine comprising several strains is believed to be required to induce a broad immune response effective against field challenge. In this study we investigated whether immunization with the Muguga cocktail induces a broader CTL response than immunization with a single strain (Muguga).ResultsFour MHC haplotype-matched pairs of cattle were immunized with either the trivalent Muguga cocktail or the single Muguga strain. CTL specificity was assessed on a panel of five different strains, and clonal responses to these strains were also assessed in one of the MHC-matched pairs. We did not find evidence for a broader CTL response in animals immunized with the Muguga cocktail compared to those immunized with the Muguga strain alone, in either the bulk or clonal CTL analyses. This was supported by an in vivo trial in which all vaccinated animals survived challenge with a lethal dose of the Muguga cocktail vaccine stabilate.ConclusionWe did not observe any substantial differences in the immunity generated from animals immunized with either Muguga alone or the Muguga cocktail in the animals tested here, corroborating earlier results showing limited antigenic diversity in the Muguga cocktail. These results may warrant further field studies using single T. parva strains as future vaccine candidates.

Genetic engineering of Theileria parva lactate dehydrogenase gene: a new anti-theilerial target

ABSTRACT: Theileria parva is the causative agent of East Coast Fever (ECF), a tick borne disease, which results in major economic losses in cattle. Major problems in dealing with this illness are the high cost of drugs, development of resistance, and absence of effective vaccines. Thus, exploiting new targets for cost effective and higher therapeutic value drugs are imperative. Glycolysis is the main pathway for generation of ATP in T. parva, given its development inside erythrocytes. Thus, the enzymes of this pathway may prove potential targets for designing new-generation anti-theilerials. Lactate dehydrogenase of T. parva (TpLDH) has the highest activity of all glycolytic enzymes and thus we selected this enzyme as the potential therapeutic target. Our study is the first to report the isolation, removal of introns through directed mutagenesis, and cloning of TpLDH and showing that amino acid insertions or deletions most notably corresponded to a 5-amino acid sequence (Asn-91A, Glu-91B, Glu-91C, Trp-91D, Asn-91E) between Ser-91 ve Arg-92 of the enzyme. This region is also present in other apicomplexan such as Babesia bovis, a pathogen of cattle and Plasmodium falciparum, a human pathogen. Providing as the attachment site for the enzyme inhibitors and not being present in LDH of respective hosts, we propose this site as an attractive drug target. The work here is expected to lead new studies on detailed structural and kinetic aspects of apicomplexan LDHs and development of new inhibitors.

In Silico Characterization and Structural Modeling of Dermacentor andersoni p36 Immunosuppressive Protein.

Ticks cause approximately $17–19 billion economic losses to the livestock industry globally. Development of recombinant antitick vaccine is greatly hindered by insufficient knowledge and understanding of proteins expressed by ticks. Ticks secrete immunosuppressant proteins that modulate the host's immune system during blood feeding; these molecules could be a target for antivector vaccine development. Recombinant p36, a 36 kDa immunosuppressor from the saliva of female Dermacentor andersoni, suppresses T-lymphocytes proliferation in vitro. To identify potential unique structural and dynamic properties responsible for the immunosuppressive function of p36 proteins, this study utilized bioinformatic tool to characterize and model structure of D. andersoni p36 protein. Evaluation of p36 protein family as suitable vaccine antigens predicted a p36 homolog in Rhipicephalus appendiculatus, the tick vector of East Coast fever, with an antigenicity score of 0.7701 that compares well with that of Bm86 (0.7681), the protein antigen that constitute commercial tick vaccine Tickgard™. Ab initio modeling of the D. andersoni p36 protein yielded a 3D structure that predicted conserved antigenic region, which has potential of binding immunomodulating ligands including glycerol and lactose, found located within exposed loop, suggesting a likely role in immunosuppressive function of tick p36 proteins. Laboratory confirmation of these preliminary results is necessary in future studies.

Antibodies to in silico selected GPI-anchored Theileria parva proteins neutralize sporozoite infection in vitro

East Coast fever (ECF) caused by Theileria parva kills cattle in East, Central and Southern Africa leading to significant economic losses. Vaccination is used as a control strategy against ECF and is presently dependent on deliberate infection with live sporozoites and simultaneous treatment with a long-acting oxytetracycline. Although effective, this method has serious limitations; the immunity is parasite strain specific and immunized cattle can become life-long asymptomatic carriers of the parasite, posing risk for the spread of the disease. In efforts to develop a subunit vaccine, the role of antibodies in the neutralization of T. parva sporozoites infection of host cells has been investigated and a circumsporozoite protein, p67, is able to induce such neutralizing antibodies. However, the p67 protein only protects a proportion of immunized cattle against T. parva challenge and such protection might be improved by inclusion of additional parasite antigens that neutralize sporozoite infection. In an attempt to identify such antigens, we searched the re-annotated T. parva genome for genes predicted to contain GPI anchor signals, since they are likely to be located on the cell surface, and expressed fragments of six of the selected genes in E. coli. The recombinant proteins were used to raise antisera in mice. Antisera to two proteins, TpMuguga_01g00876 and TpMuguga_01g00939, neutralized sporozoite infectivity to a high degree, while antisera to two additional proteins, TpMuguga_01g00095 and TpMuguga_04g00437, exhibited moderate neutralizing capacity. We conclude that these four antigens are potential vaccine candidates, which should be evaluated further in cattle.

Genetic diversity and population structure of Theileria parva in South Sudan

Theileria parva is a parasitic protozoan that causes East Coast fever (ECF), an economically important disease of cattle in eastern, central and southern Africa. In South Sudan, ECF is considered a major constraint for livestock development in regions where the disease is endemic. To obtain insights into the dynamics of T. parva in South Sudan, population genetic analysis was performed. Out of the 751 samples included in this study, 178 blood samples were positive for T. parva by species-specific PCR, were collected from cattle from four regions in South Sudan (Bor = 62; Juba = 45; Kajo keji = 41 and Yei = 30) were genotyped using 14 microsatellite markers spanning the four chromosomes. The T. parva Muguga strain was included in the study as a reference. Linkage disequilibrium was evident when populations from the four regions were treated as a single entity, but, when populations were analyzed separately, linkage disequilibrium was observed in Bor, Juba and Kajo keji. Juba region had a higher multiplicity of infection than the other three regions. Principal components analysis revealed a degree of sub-structure between isolates from each region, suggesting that populations are partially distinct, with genetic exchange and gene flow being limited between parasites in the four geographically separated populations studied. Panmixia was observed within individual populations. Overall T. parva population genetic analyses of four populations in South Sudan exhibited a low level of genetic exchange between the populations, but a high level of genetic diversity within each population.

Occurrence of tick-borne haemoparasites in cattle in the Mungwi District, Northern Province, Zambia

Little is known about the occurrence of haemoparasites in cattle in communal grazing areas of Mungwi District of Northern Province, Zambia. Clinical signs and post mortem lesions are pathognomonic of mixed tick-borne infections especially babesiosis, anaplasmosis and East Coast fever. The main objective of this study was to screen selected communal herds of cattle for tick-borne haemoparasites, and identify the tick vectors associated with the high cattle mortalities due to suspected tick-borne diseases in the local breeds of cattle grazing along the banks of the Chambeshi River in Mungwi District, Northern Province, Zambia. A total of 299 cattle blood samples were collected from July to September 2010 from Kapamba (n = 50), Chifulo (n = 102), Chisanga (n = 38), Kowa (n = 95) and Mungwi central (n = 14) in the Mungwi District. A total of 5288 ticks were also collected from the sampled cattle from April to July 2011. DNA was extracted from the cattle blood and the hypervariable region of the parasite small subunit rRNA gene was amplified and subjected to the reverse line blot (RLB) hybridization assay. The results of the RLB assay revealed the presence of tick-borne haemoparasites in 259 (86.6%) cattle blood samples occurring either as single (11.0%) or mixed (75.6%) infections. The most prevalent species present were the benign Theileria mutans (54.5%) and T. velifera (51.5%). Anaplasma marginale (25.7%), Babesia bovis (7.7%) and B. bigemina (3.3%) DNA were also detected in the samples. Only one sample (from Kapamba) tested positive for the presence of T. parva. This was an unexpected finding; also because the tick vector, Rhipicephalus appendiculatus, was identified on animals from Kowa (14.0%), Chisanga (8.5%), Chifulo (6.0%) and Kapamba (1.4%). One sample (from Kapamba) tested positive for the presence of Ehrlichia ruminantium even though Amblyomma variegatum ticks were identified from 52.9% of the sampled animals from all study areas. There was significant positive association between T. mutans and T. velifera (p < 0.001) infections, and between A. marginale and B. bovis (p = 0.005). The presence of R. microplus tick vectors on cattle was significantly associated with B. bovis (odds ratio, OR = 28.4, p < 0.001) and A. marginale (OR = 42.0, p < 0.001) infections, while A. variegatum presence was significantly associated with T. mutans (OR = 213.0, p < 0.001) and T. velifera (OR = 459.0, p < 0.001) infections. Rhipicephalus decoloratus was significantly associated with B. bigemina (OR = 21.6, p = 0.004) and A. marginale (OR = 28.5, p < 0.001). Multivariable analysis showed a significant association between location and tick-borne pathogen status for A. marginale (p < 0.001), T. mutans (p = 0.004), T. velifera (p = 0.003) and T. taurotragi (p = 0.005). The results of our study suggest that the cause of cattle mortalities in Mungwi during the winter outbreaks is mainly due to A. marginale, B. bovis and B. bigemina infections. This was confirmed by the clinical manifestation of the disease in the affected cattle and the tick species identified on the animals. The relatively low prevalence of T. parva, B. bigemina, B. bovis and E. ruminantium could indicate the existence of endemic instability with a pool of susceptible cattle and the occurrence of disease outbreaks.