RESUMO
The range of the protozoan parasite Theileria parva, which causes East Coast fever in cattle, has been expanding to countries where it has not previously been detected, as a result of cross-border domestic cattle movement. Countries where T. parva has not previously been observed until recently include Cameroon and South Sudan. This raises the issue of the conservation of the p104 antigen gene, on which the nested PCR assay that is widely used for T. parva surveillance in the blood of infected cattle is based. We sampled 40 isolates from six countries widely distributed across the geographical range of the parasite, including eastern, central and southern Africa, for p104 sequence polymorphism. These included parasites from both domestic cattle and the Cape buffalo (Syncerus caffer) wildlife reservoir. The most frequent allelic variants were present in cattle transmissible isolates from multiple widely separated geographical regions in Zambia, Uganda, Kenya, Tanzania, Rwanda and South Africa. These frequent p104 variants were also present in the three component stocks of the Muguga cocktail used for the infection and treatment live immunisation procedure to control T. parva in the field. Other isolates exhibited unique alleles. This includes some of the p104 sequences from Cameroon, which is outside the known range of the Rhipicephalus tick vector and whose origin is therefore unclear. The nested primer oligonucleotides used to generate the amplicons were universally conserved in cattle-derived parasites and a majority of buffalo-derived isolates across the geographical range of the parasite. However, some rare South African buffalo-derived isolates exhibited one or two mismatches with the primer sequences. It therefore remains possible that some p104 alleles may be so divergent that they do not amplify with the current diagnostic primers and are not detectable in surveys, hence the need for increasing knowledge of genetic heterogeneity of diagnostic targets. There was no evidence for positive selection among those p104 mutations that resulted in residue changes. Importantly, the data indicate that the p104-based PCR detection assay should be effective across the majority of the range of T. parva, and if the one or two mismatches are shown in future to result in the primers annealing less efficiently, then the assay can be further improved by introduction of degenerate bases to enable amplification of the less frequent South African buffalo-derived variant p104 genes.
Assuntos
Parasitos , Rhipicephalus , Theileria parva , Theileriose , Animais , Bovinos , Theileria parva/genética , Parasitos/genética , Búfalos/parasitologia , Theileriose/epidemiologia , Theileriose/parasitologia , Rhipicephalus/parasitologia , Reação em Cadeia da Polimerase/veterinária , Variação GenéticaRESUMO
East Coast fever (ECF) is a tick-borne disease of cattle that hinders the development of the livestock industry in eastern, central and southern Africa. The 'Muguga cocktail' live vaccine, delivered by an infection and treatment method (ITM), remains the only immunisation strategy of controlling ECF. However, there are challenges of the live vaccine inducing ECF carrier status in immunised animals and the possibility of lack of protection from parasite strains that are antigenically different from the vaccine strains. In Uganda, there are insufficient data regarding the ECF carrier status and T. parva genetic diversity in vaccinated and associated non-vaccinated cattle to assess the effectiveness of ITM vaccination. Blood was collected from recently ECF vaccinated (98) and non-vaccinated (73) cattle from Iganga district in Eastern Uganda at 120 days post-vaccination. The p104 gene nested PCR was used to screen for T. parva DNA, 11 minisatellite and 3 microsatellite markers (SSR) were used for genotyping. Two minisatellite markers (MS7 and MS19) were used to determine whether ECF carrier status was due to the T. parva vaccine or local strains. The prevalence of T. parva based on p104 nPCR was 61.2% (60/98) (RR 2.234, 95% CI 1.49-3.35, p-value < 0.001) among recently vaccinated cattle and 27.4% (20/73) (RR 1.00) among associated non-vaccinated cattle. The Muguga cocktail vaccine strains were responsible for carrier status in 10 (58.8%) by MS7 and 11 (64.7%) by MS19 in vaccinated cattle. Genotypes of T. parva with different-sized alleles to the vaccine strains that could be potential 'breakthroughs' were detected in 2 (11.8%)) and 4 (23.5%) isolates from vaccinated cattle based on MS7 and MS19 minisatellite markers, respectively. Using 14 SSR markers, T. parva diversity was higher in vaccinated (Na = 2.214, Ne = 1.978, He = 0.465) than associated non-vaccinated (Na = 1.071, Ne = 1.048, He = 0.259) cattle. The principal component analysis (PCA) showed isolates from vaccinated cattle were closely related to those from non-vaccinated cattle. The analysis of molecular variance (AMOVA) revealed high genetic variation (96%) within T. parva isolates from vaccinated and non-vaccinated cattle but low variation (4%) between vaccinated and non-vaccinated cattle. This study reveals the role of ITM in inducing the carrier status and higher T. parva genetic diversity in vaccinated cattle. The low genetic variation between T. parva isolates in both vaccinated and non-vaccinated cattle may be suggestive of the protective role of vaccine strains against genetically related local strains in the study area.
RESUMO
Immunisation of livestock with high quality vaccines is considered an essential approach to controlling many animal diseases. The only currently available commercial vaccine to protect cattle from East Coast fever (ECF), a tick-borne disease caused by Theileria parva, is an unconventional "infection and treatment method" (ITM) involving administration of a combination of live T. parva isolates, referred to as the "Muguga cocktail", and simultaneous treatment with long-acting oxytetracycline. Veterinary vaccine research and development typically involves studies designed to demonstrate vaccine quality, safety, and efficacy; however, as there were no such purpose-designed registration studies conducted for the Muguga cocktail, evidence for safety and efficacy is solely based on that which is available in the clinical literature. An extensive systematic review was conducted to analyse the evidence available in the literature in order to establish the safety and efficacy of the Muguga cocktail vaccine. A combination of meta-analyses and narrative summaries was conducted. A total of 61 studies met the criteria to be included in the systematic review. The majority of studies demonstrated or reported in favour of the vaccine with regards to safety and efficacy of the Muguga cocktail vaccine. Proximity to buffalo often resulted in reduced vaccine efficacy, and reports of shed and transmission of vaccine components affected the overall interpretation of safety. Better understanding of control options for this devastating livestock disease is important for policymakers and livestock keepers, enabling them to make informed decisions with regards to the health of their animals and their livelihoods.
RESUMO
East Coast fever (ECF) is an acute fatal tick-borne disease of cattle caused by Theileria parva. It causes major losses in exotic and crossbreed cattle, but this could be prevented by a vaccine of T. parva if the vaccine is selected properly based on information from molecular epidemiology studies. The Muguga cocktail (MC) vaccine (Muguga, Kiambu 5 and Serengeti-transformed strains) has been used on exotic and crossbreed cattle. A total of 254 T. parva samples from vaccinated and unvaccinated cattle were used to understand the genetic diversity of T. parva in Malawi using partial sequences of the Tp1 and Tp2 genes encoding T. parva CD8+ antigens, known to be immunodominant and current candidate antigens for a subunit vaccine. Single nucleotide polymorphisms were observed at 14 positions (3.65%) in Tp1 and 156 positions (33.12%) in Tp2, plus short deletions in Tp1, resulting in 6 and 10 amino acid variants in the Tp1 and Tp2 genes, respectively. Most sequences were either identical or similar to T. parva Muguga and Kiambu 5 strains. This may suggest the possible expansion of vaccine components into unvaccinated cattle, or that a very similar genotype already existed in Malawi. This study provides information that support the use of MC to control ECF in Malawi.
RESUMO
East Coast fever (ECF) is an often fatal, economically important cattle disease that predominantly affects eastern, central, and southern Africa. ECF is controlled through vaccination by means of simultaneous injection of oxytetracycline and cryogenically preserved stabilate containing live, disease-causing parasites. Storage and transportation of the stabilate requires liquid nitrogen, a commodity that is commonly unreliable in low-resource settings. Here we show that storage of conventionally prepared stabilate at -80⯰C for up to 30 days does not significantly affect its ability to infect cultured peripheral blood mononucleated cells or live cattle, suggesting an alternative cold chain that maintains these temperatures could be used to effectively manage ECF.
Assuntos
Doenças dos Bovinos/prevenção & controle , Leucócitos Mononucleares/imunologia , Vacinas Protozoárias/imunologia , Refrigeração/veterinária , Theileria/imunologia , Theileriose/prevenção & controle , Animais , BovinosRESUMO
The live infection and treatment (ITM) vaccination procedure using the trivalent Muguga cocktail is increasingly being used to control East Coast fever, with potential implications for Theileria parva population genetic structure in the field. Transmission of the Kiambu V T. parva component to unvaccinated cattle has previously been described in Uganda. We monitored the T. parva carrier state in vaccinated and control animals on a farm in West Kenya where an ITM stabilate derived from the Kenyan T. parva Marikebuni stock was evaluated for field efficacy. A nested PCR-based Marikebuni-specific marker identified a carrier state in nine of ten vaccinated animals, detectable for a period of two years. We used 22 variable number tandem repeat (VNTR) markers to determine multilocus genotypes (MLGs) of 19 T. parva schizont-infected lymphocyte isolates derived from cattle and field ticks. Two isolates from unimmunized cattle were identical to the Marikebuni vaccination stock. Two cattle isolates were identical to a Muguga cocktail component Kiambu V. Seven isolates from ticks exhibited MLGs that were identical to the Serengeti/Muguga vaccine stocks. Six cattle and two tick-derived stocks exhibited unique MLGs. The data strongly suggest transmission of immunizing genotypes, from Marikebuni vaccine-induced carrier cattle to unimmunized cattle. It is possible that genotypes similar to those in the Muguga cocktail are present in the field in Western Kenya. An alternative hypothesis is that these parasites may have originated from vaccine trial sites in Eastern Uganda. If correct, this suggests that T. parva stocks used for immunization can potentially be disseminated 125 km beyond the immediate vaccination site. Regardless of their origin, the data provide evidence that genotypes similar to those in the Muguga cocktail are circulating in the field in East Africa, alleviating concerns about dissemination of 'alien' T. parva germplasm through live vaccination.
Assuntos
Doenças dos Bovinos/parasitologia , Imunização/veterinária , Theileria parva/genética , Theileriose/parasitologia , Doenças Transmitidas por Carrapatos/parasitologia , Carrapatos/parasitologia , Vacinação/veterinária , Animais , Bovinos , Doenças dos Bovinos/prevenção & controle , Doenças dos Bovinos/transmissão , Genótipo , Quênia/epidemiologia , Tipagem de Sequências Multilocus/veterinária , Reação em Cadeia da Polimerase/veterinária , Theileria parva/imunologia , Theileriose/prevenção & controle , Theileriose/transmissão , Doenças Transmitidas por Carrapatos/prevenção & controle , Doenças Transmitidas por Carrapatos/transmissão , Uganda , Vacinas Atenuadas/imunologiaRESUMO
BACKGROUND: East Coast fever (ECF) caused by Theileria parva is endemic in Rwanda. In this study, the antigenic and genetic diversity of T. parva coupled with immunization and field challenge were undertaken to provide evidence for the introduction of ECF immunization in Rwanda. METHODS: Blood collected from cattle in the field was screened for T. parva using ELISA and PCR targeting the p104 gene. Tp1 and Tp2 gene sequences were generated from field samples and from Gikongoro and Nyakizu isolates. Furthermore, multilocus genotype data was generated using 5 satellite markers and an immunization challenge trial under field conditions using Muguga cocktail vaccine undertaken. RESULTS: Out of 120 samples, 44 and 20 were positive on ELISA and PCR, respectively. Antigenic diversity of the Tp1 and Tp2 gene sequences revealed an abundance of Muguga, Kiambu and Serengeti epitopes in the samples. A further three clusters were observed on both Tp1 and Tp2 phylogenetic trees; two clusters comprising of field samples and vaccine isolates and the third cluster comprising exclusively of Rwanda samples. Both antigens exhibited purifying selection with no positive selection sites. In addition, satellite marker analysis revealed that field samples possessed both shared alleles with Muguga cocktail on all loci and also a higher proportion of unique alleles. The Muguga cocktail (Muguga, Kiambu and Serengeti) genotype compared to other vaccine isolates, was the most represented in the field samples. Further low genetic sub-structuring (FST = 0.037) coupled with linkage disequilibrium between Muguga cocktail and the field samples was observed. Using the above data to guide a field immunization challenge trial comprising 41 immunized and 40 control animals resulted in 85% seroconversion in the immunized animals and an efficacy of vaccination of 81.7%, implying high protection against ECF. CONCLUSIONS: Antigenic and genetic diversity analysis of T. parva facilitated the use of Muguga cocktail vaccine in field conditions. A protection level of 81.7% was achieved, demonstrating the importance of combining molecular tools with field trials to establish the suitability of implementation of immunization campaigns. Based on the information in this study, Muguga cocktail immunization in Rwanda has a potential to produce desirable results.
Assuntos
Antígenos de Protozoários/imunologia , DNA Satélite/genética , Imunização/veterinária , Theileria parva , Theileriose , Animais , Variação Antigênica , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/parasitologia , Doenças dos Bovinos/prevenção & controle , Ensaio de Imunoadsorção Enzimática/veterinária , Genes de Protozoários , Marcadores Genéticos , Variação Genética , Filogenia , Reação em Cadeia da Polimerase/veterinária , Polimorfismo Genético , Vacinas Protozoárias/imunologia , Ruanda , Linfócitos T/imunologia , Theileria parva/genética , Theileria parva/imunologia , Theileriose/imunologia , Theileriose/prevenção & controle , Vacinação/veterináriaRESUMO
BACKGROUND: Theileria parva causes East Coast fever (ECF), one of the most economically important tick-borne diseases of cattle in sub-Saharan Africa. A live immunisation approach using the infection and treatment method (ITM) provides a strong long-term strain-restricted immunity. However, it typically induces a tick-transmissible carrier state in cattle and may lead to spread of antigenically distinct parasites. Thus, understanding the genetic composition of T. parva is needed prior to the use of the ITM vaccine in new areas. This study examined the sequence diversity and the evolutionary and biogeographical dynamics of T. parva within the African Great Lakes region to better understand the epidemiology of ECF and to assure vaccine safety. Genetic analyses were performed using sequences of two antigen-coding genes, Tp1 and Tp2, generated among 119 T. parva samples collected from cattle in four agro-ecological zones of DRC and Burundi. RESULTS: The results provided evidence of nucleotide and amino acid polymorphisms in both antigens, resulting in 11 and 10 distinct nucleotide alleles, that predicted 6 and 9 protein variants in Tp1 and Tp2, respectively. Theileria parva samples showed high variation within populations and a moderate biogeographical sub-structuring due to the widespread major genotypes. The diversity was greater in samples from lowlands and midlands areas compared to those from highlands and other African countries. The evolutionary dynamics modelling revealed a signal of selective evolution which was not preferentially detected within the epitope-coding regions, suggesting that the observed polymorphism could be more related to gene flow rather than recent host immune-based selection. Most alleles isolated in the Great Lakes region were closely related to the components of the trivalent Muguga vaccine. CONCLUSIONS: Our findings suggest that the extensive sequence diversity of T. parva and its biogeographical distribution mainly depend on host migration and agro-ecological conditions driving tick population dynamics. Such patterns are likely to contribute to the epidemic and unstable endemic situations of ECF in the region. However, the fact that ubiquitous alleles are genetically similar to the components of the Muguga vaccine together with the limited geographical clustering may justify testing the existing trivalent vaccine for cross-immunity in the region.
Assuntos
Variação Antigênica , Antígenos de Protozoários/genética , Theileria parva/genética , África Central , Antígenos de Protozoários/imunologia , Genótipo , Polimorfismo Genético , Análise de Sequência de DNA , Theileria parva/imunologiaRESUMO
Integrative management of wildlife and livestock requires a clear understanding of the diseases transmitted between the two populations. The tick-borne protozoan parasite Theileria parva causes two distinct diseases in cattle, East Coast fever and Corridor disease, following infection with parasites derived from cattle or buffalo, respectively. In this study, cattle were immunized with a live sporozoite vaccine containing three T. parva isolates (the Muguga cocktail), which has been used extensively and successfully in the field to protect against cattle-derived T. parva infection. The cattle were exposed in a natural field challenge site containing buffalo but no other cattle. The vaccine had no effect on the survival outcome in vaccinated animals compared to unvaccinated controls: nine out of the 12 cattle in each group succumbed to T. parva infection. The vaccine also had no effect on the clinical course of the disease. A combination of clinical and post mortem observations and laboratory analyses confirmed that the animals died of Corridor disease. The results clearly indicate that the Muguga cocktail vaccine does not provide protection against buffalo-derived T. parva at this site and highlight the need to evaluate the impact of the composition of challenge T. parva populations on vaccine success in areas where buffalo and cattle are present.