Isolation and propagation of an Egyptian Theileria annulata infected cell line and evaluation of its use as a vaccine to protect cattle against field challenge.

Tropical theileriosis is an important protozoan tick-borne disease in cattle. Vaccination using attenuated schizont-infected cell lines is one of the methods used for controlling the disease. This study describes the production of attenuated schizont-infected cell lines from Egypt and an evaluation of its use as a vaccine to protect calves against clinical disease upon field challenge. Two groups of exotic and crossbred male calves were divided into vaccinated and control groups. The vaccinated groups were inoculated with 4 ml (1 × 106 cells/ml) of the attenuated cell line. Three weeks after vaccination, calves of both groups were transported to the New Valley Governorate (Egyptian oasis) where they were kept under field conditions and exposed to the natural Theileria annulata challenge. All animals in the control group showed severe clinical signs and died despite treatment with buparvaquone, which was administered after two days of persistent fever due to a severe drop in packed cell volume (PCV). Animals in the vaccinated group became seropositive without developing severe clinical signs other than transient fever. Post-mortem examinations revealed enlarged and fragile lymph nodes, spleen, and liver with necrosis and hemorrhages. These findings indicate that the Egyptian attenuated cell line was successful in protecting both exotic and crossbred animals against tropical theileriosis under field conditions.

Comparative evaluation of cell-mediated immune response in calves immunized with live-attenuated and killed Theileria annulata vaccines.

Tropical theileriosis is a tick-borne disease caused by the protozoan Theileria annulata and transmitted by numerous species of Ixodid ticks of the genus Hyalomma. The main clinical signs are fever, lymphadenopathy, and anemia responsible for heavy economic losses, including mortality, morbidity, vaccination failure, and treatment cost. Development of poor cell-mediated immunity (CMI) has been observed in the case of many bovine pathogens (bacteria, viruses, and parasites). Quantification of CMI is a prerequisite for evaluating vaccine efficacy against theileriosis caused by T. annulata. The current study evaluated the CMI in calves administered with two types of T. annulata vaccine (live attenuated and killed). We prepared a live attenuated T. annulata vaccine by attenuation in a rabbit model and also prepared killed vaccine from non-attenuated T. annulata. For the evaluation of immune response in experimental groups including control, 20 calves were divided into four different groups (A, B, C, and D). They were either inoculated subcutaneously with live rabbit-propagated-Theileria-infected RBCs (5 × 106) (group A) or with killed T. annulata vaccine (2 × 109 schizonts) with Freund's adjuvant (group B), along with an infected group (group C) and a healthy control group (group D). The protection of vaccinated calves was estimated with challenge infection. Our results showed that with a single shot of live-attenuated and killed vaccine with a booster dose elicited cell-mediated immune responses in immunized calves. We observed a significant elevation in CD4 + and CD8 + T cells in immunized calves. A significant difference in the CD8 + T cell response between the post-challenge stage of killed and live vaccine (p < 0.0001) was observed, whereas no other difference was found at both pre- and post-immunization stages. A similar finding was recorded for the CD4 + T cells at a post-challenge stage, where a significant difference was seen between killed and live vaccine (p < 0.0001). Another significant difference was observed between the CD8 + T cells and CD4 + T cells at the post-challenge stage in the live vaccine group, where there was a significantly higher induction of CD4 + T cell response (p < 0.0001).

Molecular detection of Theileria annulata, Theileria mutans and Theileria velifera but no evidence of Theileria parva infected or vaccinated cattle in Nigeria despite extensive transboundary migrations.

The extensive livestock management system predominant in Nigeria necessitates active disease surveillance for the early detection and prompt control of transboundary animal diseases. Theileriae are obligate intracellular protozoa which infect both wild and domestic bovidae throughout much of the world causing East Coast Fever (Theileria parva), Tropical or Mediterranean theileriosis (Theileria annulata) or benign theileriosis (Theileria mutans; Theileria velifera). This study aimed to detect and characterize Theileria spp. infecting cattle in Nigeria using conventional PCR and sequencing approach. Five hundred and twenty-two DNA samples obtained from different cattle blood samples were subjected to PCR targeting the 18S rRNA gene of piroplasmida and specifically, the p104 kDa and Tp1 genes for the evidence of infection or vaccination respectively, with T. parva. A total of 269 out of 522 (51.5%) of the cattle tested PCR- positive for DNA of piroplasmida. Nucleotide sequence and phylogenetic analyses showed that the cattle were infected with T. annulata, T. mutans and T. velifera. Piroplasmida DNA was associated with sex (ꭓ2 = 7.2; p = 0.007), breed (ꭓ2 = 115; p = 0.000002) of animals and the state where the samples were collected (ꭓ2 = 78.8; p = 0.000002). None of the samples tested positive for T. parva DNA or showed evidence of vaccination (Tp1 gene). This is the first report on the molecular detection and characterization of T. annulata in the blood of cattle from Nigeria. Continuous surveillance of Nigerian cattle for East Coast Fever (ECF) is encouraged considering the recent report of the disease in cattle in the neighboring country, Cameroon, where unregulated transboundary cattle movement into Nigeria has been observed.

Development of a dual vaccine against East Coast fever and lumpy skin disease.

East Coast fever is an acute bovine disease caused by the apicomplexan parasite Theileria parva and is regarded as one of the most important tick-vectored diseases in Africa. The current vaccination procedure has many drawbacks, as it involves the use of live T. parva sporozoites. As a novel vaccination strategy, we have constructed the recombinant lumpy skin disease virus (LSDV) named LSDV-SODis-p67HA-BLV-Gag, encoding a modified form of the T. parva p67 surface antigen (p67HA), as well as the bovine leukemia virus (BLV) gag gene for the formation of virus-like particles (VLPs) to potentially enhance p67 immunogenicity. In place of the native sequence, the chimeric p67HA antigen has the human tissue plasminogen activator signal sequence and the influenza hemagglutinin A2 transmembrane domain and cytoplasmic tail. p67HA was detected on the surface of infected cells, and VLPs comprising BLV Gag and p67HA were produced. We also show that higher multiple bands observed in western blot analysis are due to glycosylation of p67. The two vaccines, pMExT-p67HA (DNA) and LSDV-SODis-p67HA-BLV-Gag, were tested for immunogenicity in mice. p67-binding antibodies were produced by vaccinated animals, with higher titers detected in mice vaccinated with the recombinant LSDV. This candidate dual vaccine warrants further testing in cattle.

Epidemiology of tick-borne pathogens of cattle and tick control practices in coastal Kenya.

Tick-borne diseases (TBD) are a major constraint to livestock health and productivity in sub-Saharan Africa. Nonetheless, there are relatively few robust epidemiologic studies documenting TBD and its management in different endemic settings in Kenya. Therefore, a cross-sectional study using multi-stage cluster sampling was undertaken to characterize the epidemiology of TBD and management factors among zebu cattle reared under an extensive system in coastal Kenya. Blood samples from 1486 cattle from 160 herds in 14 villages were screened for the presence of tick-borne bacterial and protozoan pathogens using PCR with high-resolution melting analysis and sequencing. Standardized questionnaires were used to collect data on herd structure and herd management practices, and a mixed-effect logistic regression model to identify risk factors for tick-borne pathogens (TBPs). The application of chemical acaricide was the primary method for tick control (96.3%, 154/160), with the amidine group (mainly Triatix®, amitraz) being the most frequently used acaricides. Respondents identified East Coast fever as the most important disease and Butalex® (buparvaquone) was the most commonly administered drug in response to perceived TBD in cattle. The overall animal- and herd-level prevalence for TBPs were 24.2% (95% confidence interval (CI): 22.0-26.4%) and 75.6% (95% CI: 68.2-82.1%), respectively. Cattle were infected with Anaplasma marginale (10.9%, 95% CI: 9.4-12.6), Theileria parva (9.0%, 95% CI: 7.5-10.5), Anaplasma platys (2.6%, 95% CI: 1.9-3.6), Theileria velifera (1.1%, 95% CI: 0.7-1.8), Babesia bigemina (0.5%, 95% CI: 0.2-1.0), and Anaplasma sp. (0.1%, 95% CI: 0.0-0.4). Moreover, 21 cattle (1.4%) were co-infected with two TBPs. None of the assessed potential risk factors for the occurrence of either A. marginale or T. parva in cattle were statistically significant. The intra-herd correlation coefficients (lCCs) computed in this study were 0.29 (A. marginale) and 0.14 (T. parva). This study provides updated molecular-based information on the epidemiological status of TBPs of cattle and herd management practices in coastal Kenya. This information can be used in designing cost-effective control strategies for combating these TBD in the region.

East Coast fever, a neglected tropical disease with an outdated vaccine approach?

Since its discovery, bovine theileriosis has caused major socioeconomic losses in sub-Saharan Africa. Acaricide resistance of the intermediate host, paucity of therapeutics, and lack of sufficiently cross-protective vaccines increase the risk of parasite spread due to global warming. Here, we highlight three important areas that require investigation to develop next-generation vaccines.

Theileria annulata SVSP455 interacts with host HSP60.

BACKGROUND: Theileria annulata, a transforming parasite, invades bovine B cells, dendritic cells and macrophages, promoting the uncontrolled proliferation of these cells. This protozoan evolved intricate strategies to subvert host cell signaling pathways related to antiapoptotic signaling to enable survival and proliferation within the host cells. However, the molecular mechanisms of the cell transformation induced by T. annulata remain largely unclear. Although some studies have predicted that the subtelomere-encoded variable secreted protein (SVSP) family plays roles in host-parasite interactions, the evidence for this is limited. METHODS: In the present study, the SVSP455 (TA05545) gene, a member of the SVSP gene family, was used as the target molecule. The expression pattern of SVSP455 in different life-cycle stages of T. annulata infection was explored using a quantitative real-time PCR assay, and the subcellular distribution of SVSP455 was observed using confocal microscopy. The host cell proteins interacting with SVSP455 were screened using the Y2H system, and their interactions were verified in vivo and in vitro using both bimolecular fluorescence complementation and confocal microscopy, and co-immunoprecipitation assays. The role played by SVSP455 in cell transformation was further explored by using overexpression, RNA interference and drug treatment experiments. RESULTS: The highest level of the SVSP455 transcript was detected in the schizont stage of T. annulata, and the protein was located both on the surface of schizonts and in the host cell cytoplasm. In addition, the interaction between SVSP455 and heat shock protein 60 was shown in vitro, and their link may regulate host cell apoptosis in T. annulata-infected cells. CONCLUSION: Our findings are the first to reveal that T. annulata-secreted SVSP455 molecule directly interacts with both exogenous and endogenous bovine HSP60 protein, and that the interaction of SVSP455-HSP60 may manipulate the host cell apoptosis signaling pathway. These results provide insights into cancer-like phenotypes underlying Theilera transformation and therapeutics for protection against other pathogens.

Sequence polymorphisms in a Theileria annulata surface protein (TaSP) known to augment the immunity induced by live attenuated cell line vaccine.

Theileria annulata is a tick-borne protozoan causing tropical theileriosis in cattle. The use of attenuated cell line vaccines in combination with subunit vaccines has been relatively successful as a control method, as exemplified by a recent study in which immunization with a local cell line followed by booster vaccinations with recombinant T. annulata surface protein (TaSP) resulted in 100% protection upon field challenge in Sudan. However, these findings cannot be directly extrapolated to other countries as culture-attenuated live vaccines are generated using local strains and no systematic evaluation of genotype differences between countries has been undertaken. In this study, we sequenced the TaSP gene from T. annulata cell lines and field isolates from Tunisia (n = 28) and compared them to genotypes from Sudan (n = 25) and Morocco (n = 1; AJ316259.1). Our analyses revealed 20 unique TaSP genotypes in the Tunisian samples, which were all novel but similar to genotypes found in Asia. The impact of these polymorphisms on the ability of the TaSP antigen to boost the immunity engendered by live cell line vaccines, especially in Tunisia where studies with TaSP have not been conducted, remains to be examined. Interestingly, phylogenetic analyses of publicly available TaSP sequences resolved the sequences into two clusters with no correlation to the geographical origin of the isolates. The availability of candidate vaccines that were recently attenuated using local strains from Sudan, Tunisia, Egypt and Morocco should be exploited to generate a comprehensive catalogue of genetic variation across this regional collection of attenuated live vaccines.

Theileria equi RAP-1a and RAP-1b proteins contain immunoreactive epitopes and are suitable candidates for vaccine and diagnostics development.

Theileria equi is an obligate intracellular protozoan parasite that causes severe hemolytic anaemia in most equid species. Similar to other apicomplexan parasites, T. equi contains rhoptries whose contents have been implicated in host cell invasion and formation of the parasitophorous vacuole that is crucial for survival of the species within cells. Despite their importance, the composition of T. equi rhoptries and their role(s) in host cell invasion remain unexplored. To gain insight into these issues, we evaluated the expression, immunogenicity, and functional roles of two T. equi rhoptry-associated proteins abbreviated as RAP-1a and RAP-1b. The full-length RAP-1a protein was expressed to perform the analysis but our efforts to express the full-length RAP-1b protein failed due to an unknown reason. We therefore generated synthetic immunogenic peptides that map onto the N- and C-termini of the RAP-1b protein as an alternative approach. Our findings show that both proteins are expressed in the extracellular and intra-erythrocytic merozoite stages of T. equi. Serological analyses show that T. equi-infected horses mount antibody responses that recognise both proteins and correlate with a decrease in T. equi load in both acutely and persistently infected horses. In vitro neutralisation studies show that the T. equi RAP-1a protein contains neutralisation-sensitive epitopes as antibodies developed against the protein significantly inhibited the parasites from invading equine erythrocytes. Conversely, antibodies developed against the RAP-1b synthetic peptides did not neutralise parasite invasion, showing that the protein regions on which the peptides were based are not required for T. equi invasion. Overall, the data shows that T. equi rhoptries and their contents are involved in invasion of host cells and supports T. equi RAP-1 proteins as candidates for developing novel serodiagnosis tools and vaccines.

Spatio-temporal clustering and risk factor analysis of bovine theileriosis (Theileria parva) in Zimbabwe from 1995 to 2018.

Bovine theileriosis (caused by Theileria parva) is the most important tick-borne transboundary animal disease endemic to Zimbabwe, yet its distribution dynamics data in the country remain scant and outdated. A retrospective study was conducted to determine high-risk areas of bovine theileriosis and associated risk factors in Zimbabwe. Records on bovine theileriosis spanning 23 years (January 1995 to December 2018) were obtained from the Epidemiological Unit of the Division of Field Veterinary Services of Zimbabwe (DVSZ). Data were analysed using Studio R® version 11.0 for regression analysis and SatScan® version 9.4.6 for spatio-temporal clustering. Communal farmers (72%), adult cattle (29%), the year 2018 (60%) and the hot wet season (42%) had the highest proportion (p < .050) of bovine theileriosis cases recorded. Seven out of the country's ten provinces and 36 of its 59 districts were affected. Bovine theileriosis was observed to lose seasonality when cases rose exponentially in 2018. Five and four high-risk clusters of bovine theileriosis were detected using one-year and one-month time aggregate, respectively, all within the last eight years of the study (2011-2018). Two potential risk factors (province and farming system) were significantly (p < .050) associated with bovine theileriosis occurrence. Bovine theileriosis was found to be rampant and if left unchecked will spread and adversely affect the whole country. Improved theileriosis surveillance and control is warranted. Recommendations for control and prevention strategies revolve around better farmer awareness about the disease, correct and consistent use of acaricides, cattle movement control and disease surveillance among others.

Development of a Potential Yeast-Based Vaccine Platform for Theileria parva Infection in Cattle.

East Coast Fever (ECF), caused by the tick-borne apicomplexan parasite Theileria parva, remains one of the most important livestock diseases in sub-Saharan Africa with more than 1 million cattle dying from infection every year. Disease prevention relies on the so-called "Infection and Treatment Method" (ITM), which is costly, complex, laborious, difficult to standardise on a commercial scale and results in a parasite strain-specific, MHC class I-restricted cytotoxic T cell response. We therefore attempted to develop a safe, affordable, stable, orally applicable and potent subunit vaccine for ECF using five different T. parva schizont antigens (Tp1, Tp2, Tp9, Tp10 and N36) and Saccharomyces cerevisiae as an expression platform. Full-length Tp2 and Tp9 as well as fragments of Tp1 were successfully expressed on the surface of S. cerevisiae. In vitro analyses highlighted that recombinant yeast expressing Tp2 can elicit IFNγ responses using PBMCs from ITM-immunized calves, while Tp2 and Tp9 induced IFNγ responses from enriched bovine CD8+ T cells. A subsequent in vivo study showed that oral administration of heat-inactivated, freeze-dried yeast stably expressing Tp2 increased total murine serum IgG over time, but more importantly, induced Tp2-specific serum IgG antibodies in individual mice compared to the control group. While these results will require subsequent experiments to verify induction of protection in neonatal calves, our data indicates that oral application of yeast expressing Theileria antigens could provide an affordable and easy vaccination platform for sub-Saharan Africa. Evaluation of antigen-specific cellular immune responses, especially cytotoxic CD8+ T cell immunity in cattle will further contribute to the development of a yeast-based vaccine for ECF.

Merozoites of Theileria orientalis buffeli reduce the parasitaemia of T. orientalis ikeda following tick challenge.

Theileria orientalis is a tick-borne protozoal parasite causing anaemia and death in susceptible cattle. This investigation aimed to confirm whether immunisation with the "benign" buffeli genotype of T. orientalis could reduce the parasitaemia of the virulent ikeda genotype. Calves were inoculated intravenously or subcutaneously with bovine blood containing merozoites of T. orientalis buffeli and when recipients became positive by PCR, they and control calves were challenged with unfed nymphs of Haemaphysalis longicornis ticks infected as larvae with T. orientalis ikeda. All calves became positive for the challenge within 12 days after tick application. In the immunised calves, the first wave of parasitaemia with T. orientalis ikeda from 4 to 6 weeks was reduced significantly by >80 % before the parasite burden declined into the carrier state by 9 weeks. The parasitaemias in two calves which exhibited low infections with T. orientalis ikeda shortly after arrival, were also significantly reduced after tick challenge. The results confirm the previous studies on immunity to T. sergenti in Japan, and field experience with theileriosis in endemic zones where the carrier state appears to prevent clinical disease despite repeated, seasonal tick infestations with virulent genotypes of the parasite. This method offers a means to reduce the severity of the first wave of theilerial parasitaemia after tick challenge and possibly recover associated production losses.

Molecular Epidemiology and Characterization of Theileria in Goats.

The study was carried out to determine the prevalence and associated risk factors of theileriosis in goats of Chattogram district, Bangladesh. Molecular characterization of circulating Theileria in this area was also undertaken. A total of 400 samples were collected from goats of different breeds, ages and sex with relevant information of rearing and management. The prevalence of theileriosis was 8.50% (34/400) by polymerase chain reaction though all of those samples were test-negative by microscopic examination. Among different risk factors season, breed and tick infestation were found to be significantly (p ≤ 0.05) associated with the prevalence of theileriosis in goats. Serous nasal discharge and swollen lymph nodes were determined to be significant clinical signs. The Theileria spp. detected in the present study closely resemble isolates which were previously detected in Myanmar and China. Further large scale epidemiological studies are required to identify the circulating species and responsible vectors, which would facilitate control measures for this disease in Bangladesh.

Molecular characterization and population genetics of Theileria parva in Burundi's unvaccinated cattle: Towards the introduction of East Coast fever vaccine.

Theileria parva (T. parva) is a protozoan parasite that causes East Coast fever (ECF). The disease is endemic in Burundi and is a major constraint to livestock development. In this study, the parasite prevalence in cattle in six regions namely; Northern, Southern, Eastern, Western, Central and North Eastern was estimated. Furthermore, the sequence diversity of p67, Tp1 and Tp2 genes was assessed coupled with the population genetic structure of T. parva using five satellite markers. The prevalence of ECF was 30% (332/1109) on microscopy, 60% (860/1431) on ELISA and 79% (158/200) on p104 gene PCR. Phylogenetic analysis of p67 gene revealed that only allele 1 was present in the field samples. Furthermore, phylogenetic analysis of Tp1 and Tp2 showed that the majority of samples clustered with Muguga, Kiambu and Serengeti and shared similar epitopes. On the other hand, genetic analysis revealed that field samples shared only two alleles with Muguga Cocktail. The populations from the different regions indicated low genetic differentiation (FST = 0.047) coupled with linkage disequilibrium and non-panmixia. A low to moderate genetic differentiation (FST = 0.065) was also observed between samples and Muguga cocktail. In conclusion, the data presented revealed the presence of a parasite population that shared similar epitopes with Muguga Cocktail and was moderately genetically differentiated from it. Thus, use of Muguga Cocktail vaccine in Burundi is likely to confer protection against T. parva in field challenge trials.

An alternative cold chain for storing and transporting East Coast fever vaccine.

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.

Sequence diversity of cytotoxic T cell antigens and satellite marker analysis of Theileria parva informs the immunization against East Coast fever in Rwanda.

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.

The efficacy of toltrazuril treatment for reducing the infection intensity of Theileria orientalis Ikeda type in dairy calves.

The aim of this study was to test the hypothesis that toltrazuril administered at 4 weeks post-turnout reduces the infection intensity of Theileria orientalis Ikeda type in dairy calves and so prevents serious clinical disease in these animals at 2-3 months of age. Two groups of 40 dairy calves on two separate dairy farms in the Waikato were followed for 16 weeks post-turnout onto pasture. On each farm, 20 calves were randomly selected and orally treated with toltrazuril (15 mg/kg) at 4 weeks post-turnout, whilst the remaining 20 calves were left untreated. All 40 calves were blood sampled and weighed at 2, 4, 6, 8, 12, and 16 weeks post-turnout i.e. 6 samplings per calf. A random subset of 10 calves from each treatment group on each farm were faecal sampled at each visit. The blood samples were used to estimate the T. orientalis Ikeda type infection intensity and haematocrit for each calf and the faecal samples were used to estimate the number of coccidia oocysts per gram of faeces. Three linear mixed effects models, to evaluate the effect of toltrazuril treatment on infection intensity, haematocrit (HCT) and weight respectively were fitted to the data. No calves on either farm developed clinical theileriosis or coccidiosis and the three mixed effects linear models, controlling for the effect of farm and days from turnout, showed that there was no effect of treatment on infection intensity (p = 0.81), on HCT (p = 0.99) and on weight gain (p = 0.79). In conclusion, this study showed no evidence supporting the use of toltrazuril to control T. orientalis Ikeda type infection levels and prevent disease.

Effect of chemical tick control practices on tick infestation and Theileria parva infection in an intensive dairy production region of Uganda.

Chemical tick control is a major means of preventing East Coast fever (ECF), especially in sub-Saharan Africa. However, in southwestern Uganda, improper tick control practices have led to severe acaricide resistance. The objectives of this study were to determine the risk factors associated with tick infestation in dairy cattle and Theileria parva infection, and to generate evidence for the prioritization of effective countermeasures for tick control. A cross-sectional study was conducted in 30 farms in Mbarara District, and information on tick control practices and tick infestation were collected. Tick samples were collected from 13 farms to test tick acaricide efficacy. A total of 420 blood samples from calves to adults of exotic- and cross-breed dairy cattle were collected, and T. parva diagnosis via polymerase chain reaction was performed. All the 13 tick populations tested were resistant to deltamethrin (synthetic pyrethroid). Resistance to single-formulation organophosphate-chlorfenvinphos was 39 % (5/13); co-formulations (chlorpyrifos + cypermethrin), 69 % (9/13); and amitraz (amidine), 85 % (11/13). The overall prevalence of T. parva infection at the individual-level was 45.2 % (190/420, 95 % confidence interval (CI): 40.4-50.1), and that at the farm-level was 83 % (25/30, 95 %CI: 65-94). A good quality cattle crush was a preventive factor for tick infestation (odds ratio (OR): 0.32, 95 %CI: 0.15-0.63, p =  0.001). Well-managed acaricide storage (OR: 0.36, 95 %CI: 0.17-0.76, p =  0.008), and a good quality measuring cylinder for acaricide were preventive factors (OR: 0.32, 95 %CI: 0.11-0.93, p = 0.036) for T. parva infection. The risk factors for T. parva infection were a longer period of acaricide use of the same brand (OR: 1.06, 95 %CI: 1.01-1.10, p = 0.012), and a higher frequency (twice a week) of acaricide use rather than once a week (OR: 11.70, 95 %CI: 1.95-70.13, p = 0.007). These risk factors should be given high intervention priority in order to effectively control ticks and prevent T. parva infections in dairy farms. Teaching proper practices to dairy farmers and to technical staff should be used to overcome the severe challenge of acaricide resistance in Mbarara District.

Monitoring vaccinated cattle for induction and longevity of persistent tick-transmissible infection: Implications for wider deployment of live vaccination against East Coast fever in Tanzania.

The infection and treatment (ITM) procedure remains the only available method of immunization against Theileria parva infection. One constraint to deployment is the perception that the carrier state induced by ITM could result in enhanced disease problems. More than one million cattle have been ITM vaccinated in pastoralist systems in Tanzania over the last 2 decades. We present the results of a longitudinal study of six groups of cattle in Maasai villages in northern Tanzania exposed to natural tick challenge for between 2 weeks and 14 years post-vaccination. The p104 nested PCR revealed a higher frequency of T. parva carriers among vaccinates (30%) compared with controls (8%) (OR = 4.89, p = .000), with the highest frequency of carriers found in calves vaccinated 6 months previously, although carrier state was also detected in cattle vaccinated >10 years prior to the study. Variable number tandem repeat genotype analysis revealed 6 MS7 alleles with sizes ranging from 150 bp to 500 bp, but only two alleles were detected in cattle vaccinated >4 years earlier, relative to five alleles detected in recently vaccinated cattle and controls. In terms of heterozygosity, diversity was maximal in calves vaccinated within the last 2 weeks (h = 0.776) but lowest in cattle vaccinated 4 years earlier (h = 0.375). The analysis suggested close genetic relatedness of parasites in vaccinated and unvaccinated groups and up to 96% of variation was within rather than between the groups. These results confirm that ITM leads to a long-term T. parva carrier state in cattle and the detection of vaccine component VNTR in co-grazing unvaccinated cattle suggests potential vaccine transmission by ticks. However, vaccination stocks did not totally replace local genotypes, at least in cattle populations. These findings should mitigate concerns that ITM modifies T. parva field populations in a way that enhances disease in the medium term.

Current status of tropical theileriosis in Northern Africa: A review of recent epidemiological investigations and implications for control.

Tropical theileriosis caused by the apicomplexan hemoparasite Theileria annulata is a tick-borne disease that constraints livestock production in parts of Europe, Asia and Africa. Four Hyalomma tick species transmit T. annulata in at least eight Africa countries (Mauritania, Morocco, Algeria, Tunisia, Egypt, Sudan, South Sudan and Ethiopia). The two dominant T. annulata vector ticks present in Africa, H. scupense and H. anatolicum, underlie two different patterns of transmission, which in turn greatly influence the epidemiology of tropical theileriosis. H. dromedarii and H. lusitanicum are also capable of transmitting T. annulata in North Africa, but their roles are associated with specific production systems and agro-ecological contexts. The emergence of resistance to the most widely used theilericidal compound, buparvaquone, continues to limit the effectiveness of chemotherapy. In addition, acaricide use is increasingly becoming unsustainable. Deployable T. annulata attenuated live vaccines established from local strains in Tunisia, Sudan and Egypt are available, and recent work has indicated that these vaccines can be protective under conditions of natural transmission. However, vaccination programmes may vary over space and time due to differences in the prevalence of disease amongst cattle populations, as well seasonal variation in vector activity. We review recent descriptive and analytical surveys on the epidemiology of T. annulata infection with reference to (a) demographic aspects such as breeds and ages of cattle herds previously exposed to distinct T. annulata infection pressures and (b) seasonal dynamics of tick activity and disease transmission. We then discuss how the wider endemic patterns that we delineate can underpin the development and execution of future vaccination programmes. We also outline options for integrated control measures targeting tick vectors and husbandry practices.