An Overview of the New Vaccine Targeting Cattle Ticks in Uganda

Ticks are among the most important vectors of pathogens that cause some of the most devastating tick-borne diseases (TBDs) in the livestock sector in eastern Africa, resulting in loss of productivity and mortalities. They are among the leading causative agents of diseases in cattle. East Coast Fever for instance causes up to 100% mortalities in untreated exotic European breeds and their crosses. It also contributes 30% losses in calves of local zebu cattle, which depletes the stock for replacement of the national herd. Ticks are vectors of tick-borne pathogens and their control is by acaricides. For livestock farmers, the present methods of tick control using acaricides, have become costly and ineffective due to increasing acaricide tick resistance. This discourages investment in the livestock sector and can lead to food insecurity, thereby impacting on the economy. Alternative methods that reduce the use of acaricides and protect cattle from the risk of tick-borne diseases are urgently needed.

Major ticks of concern in Uganda

In Uganda, the major tick species of concern are Rhipicephalus appendiculatus (also known as the Brown Ear tick), Rhipicephalus (Boophilus) decoloratus (the blue tick) and Amblyomma variegatum.  These transmit Theileria parva, Babebesia bigemina, and Ehrlichia ruminantium respectively which cause East Coast Fever (ECF), Babesiosis, Anaplasmosis, and Heartwater respectively.  

R. appendiculatus, also known as the Brown Ear tick (or Engoha), is a hard tick found predominantly in East, Central and Southern Africa. It is the vector of T. parva the causative agent of ECF in cattle, currently the most fatal and economically important cattle disease in Uganda.
 

Source: Saimo-Kahwa PhD thesis 2017

EastCoastFever kills over 1 million cattle annually in Africa—that's one cow every 30 seconds. It is widespread in Uganda and is the most diagnosed cattle disease and the most economically important tick-borne disease (TBD) in the country.  

Commercial vaccines based on the tick gut protein Bm86 have been successful in controlling the one-host tick R. (Boophilus) microplus in other countries and provide heterologous protection against certain other non-target ixodid tick species. This cross-protection, however, does not extend to the three-host tick R. appendiculatus, the vector of the protozoan parasite Theileria parva, which causes East Coast Fever in Uganda, leaving local cattle vulnerable.

The TicVac-U® vaccine candidate would constitute the most effective and environmentally sound approach for the control of acaricide-resistant R. decoloratus. Based on Ra86 and Bm86 antigen proteins from local ticks, TicVac-U has already shown an efficacy of 86% against East Coast Fever in field trials as well as effective cross-species protection against other tick infestations in Uganda.

Why the need for a new anti-tick solution?

Infection and treatment method (ITM) 

To date, the only practical preventative solution to East Coast Fever, akin to a vaccine, has been the unconventional ITM (Infection and Treatment method) developed in the 1970s, which consists of three stocks of live T. parva parasites (Muguga stock, Kiambu and Serengeti transformed a buffalo strain) referred to as the ‘Muguga cocktail’.  This unconventional vaccine is injected into cattle concurrently with treatment using the long-acting oxytetracycline, in an effort to reduce the strength of T. parva parasites.  

The immunity produced by the ITM is long-lasting from homologous strains, though, there have been breakthroughs due to polymorphism within the parasite stocks.  

Vaccination in Uganda using ITM has for long been hampered due to various constraints, among them regulatory, necessitating registration of the vaccine in Uganda by the National Drug Authority for good reasons. It is a very difficult vaccine to produce, store or distribute.
It possesses logistical and storage problems, requiring cold chain storage (between −80 ◦C and −196 ◦C) to maintain the sporozoite viability. 
A bigger problem of immunisation with live parasites is they persist in immunised cattle, resulting in a carrier state, which causes anxiety and worries about new foreign strains being introduced into areas previously free of them.  

In addition, ITM needs professionals to administer and monitor safely the vaccinated animals since it uses live pathogens. It is also very expensive. It costs at least US$7 per animal, which eliminates its viability for the vast majority of poor pastoral communities across East Africa. Its commercial pack size (packaged between 30 and 40 doses per 0.5 ml straw) further eliminates smallholder farmers (the zero-grazing farmers that keep one or two animals). This has long eliminated its viability in Uganda.   

Use of acaricides 

Ugandan farmers, especially in central and western Uganda, have come to rely heavily on the use of chemical acaricides. According to statistics from the Ministry of Agriculture and Animal Industry and Fisheries (MAAIF), Uganda imports approximately 370,000 litres of various acaricides on an annual basis, and another 83,000 litres in imported anti-tickborne disease drugs such as parvaquone, Buparvaquone etc. 

The total annual forex outflow for the country, just to deal with ticks and tickborne diseases (TTBDs) is estimated at (Shs300 billion).  Yet despite this financial effort, the use of acaricides has had a minimal effect and has led to the emergence of acaricide-resistant ticks.   

Though the start of acaricide resistance by ticks in Uganda, first came to light in 2012, the emergence of multiple acaricide-resistant ticks and its implication on tick control was recorded by Vudriko et al, in 2016.  

Today, the country continues to face a worsening emergence of super-resistant R. appendiculatus and R. decoloratus ticks with a very high level of resistance to acaricides.  

In many western regions of Uganda for instance, up to 90% of the most economically significant tick species now show 0% mortality to popular acaricide classes such as amidines, synthetic pyrethroids (SP), organophosphates (OP) and OP-SP co-formulations (COF). 

The major causes of the acaricide failure against ticks could have arisen out of the farmers’ improper and irrational use of the commercial acaricides during application.  This has resulted in a number of farmers increasing the acaricide concentration, over spraying, applying acaricides more frequently and acaricide admixing, including mixing of agrochemicals with acaricides or even use of crop pesticides to spray cattle against ticks as coping methods.   The use of unregistered acaricides as well as fake agrochemicals smuggled into the country has further exacerbated the tick resistance problem.  

In an effort to mitigate the current problem of tick acaricide failure, the Anti-Tick Vaccine Development Initiative (ATVDI) has been at the forefront of research to deliver an innovative alternative immunological vaccine solution that advances the prevention and treatment of ticks and tick-borne diseases, known as TicVac-U.

THE CURRENT ANTI-TICK VACCINE SOLUTION

TicVac-U tick vaccine: Mechanisms of working

The Anti-Tick Vaccine Development Presidential Initiative at the College of Veterinary Medicine, Animal Resources and Bio-security (COVAB), Makerere University, aims to develop a recombinant vaccine using proteins identified from local ticks (Rhipicephalus appendiculatus) that would be used as part of the package in the control of ticks and tick-borne diseases, through vaccination. 

TicVac-U® is the name that has been given to the candidate anti-tick vaccine composed of recombinant Ra86 variant antigens, where Partial DNA sequences of Ra86 variant genes, were synthesized by GeneOne Biotechnologies (Rio de Janeiro, Brazil) optimizing for codon usage of P. pastoris.  

The synthetic sequence was cloned in the pPICZαA plasmid (Invitrogen, USA).  The transformed recombinant P. pastoris cells were selected on YPDS plates containing 100ug/ml Zeocin™ antibiotic (Invitrogen, USA).  The presence of the insert was confirmed by digestion with EcoR1 and Xba1 restriction enzymes (New England Biolabs) and sequencing with 5'AOX1 and 3'AOX1 primers. 

To facilitate the development and commercialization of the TicVac-U® anti-tick vaccine, the proteins must be produced in facilities that follow current Good Manufacturing Practices (cGMP).   

For this purpose, production and in-process tests laboratories for finished products have been set up at the ALFASAN Uganda factory at Namanve and the clinical trials are still being carried out at Ngoma presidential farm.  
However, the team will continue to improve on the vaccine (R&D) continued research and improvement at CoVAB, Makerere University. 

Stall experiments carried out using Ra86-based vaccines have shown a reduction in the number of ticks by 69.7% on hosts., reduced the weight of tick eggs by 21%., stopped attachment of ticks by 60%, and further reduced egg-laying capacity by 50% (Saimo et al 2011; Olds et al., 2012).  
The ability of nymphs to moult into adult ticks was also reduced, in addition to blocking the growth of Theileria parva parasites in the adult ticks (Olds et al,. 2012).  

Ra86-based anti-tick vaccines can therefore play a part in integrated pest management and control strategies for R. appendiculatus because the Ra86 protein is localized in the tick gut and is known as concealed antigen from the host immune response during normal tick feeding.  
With the uptake of the blood meal by engorging ticks from a vaccinated host, the antibodies and complement damage tick gut wall resulting in death or decreased reproductive capacity.

Immunization with Ra86 gut protein was examined for its impact on nymphs and adult R. appendiculatus ticks after feeding on Ra86-vaccinated cattle.  The moulting of nymphal ticks to the adult stage was significantly reduced in ticks feeding on Ra86 vaccinated animals in comparison to control animals.  This suggests that repeated Ra86 vaccinations would reduce tick populations over successive generations. 
By reducing the nymphal population moulting to adult instars, it had implications on EastCoastFever clinical disease severity T. parva because transmission by adult ticks is commonly associated with more severe disease symptoms when compared to nymph-mediated transmission. Therefore, a reduction of the number of adult ticks before they can transmit T. parva may lead to a reduction of the negative impact of EastCoastFever on animal productivity.   

Dead nymphs (encircled) compared to the moulted young and unfed adult (with legs seen)

Source: Ikwap et al 2021.

Additionally, Ra86 vaccination lowered T. parva infection levels in ticks that fed on vaccinated cattle indicating that targeting the tick gut could affect the uptake of T. parva from infected cattle and/or further development within the vector.  Transmission is reduced by vaccination, resulting in lowering EastCoastFever clinical cases while still advantageously enabling the establishment of immune protection.  Therefore, the use of “concealed antigen” candidates has been successful in effectively reducing tick populations with successive generations.

The health and safety of the TicVac-U® anti-tick solution

The research and development of a protein-based anti-tick vaccine in Uganda that’s based on recombinant DNA technology, has been a concerted and inter-institutional effort and collaboration that has given rise to a superior and cost-effective anti-tick vaccine for tick-borne diseases in Uganda.  TicVac-U® constitutes an effective and environmentally sound approach for the control of ticks and the transmission of the associated tick-borne diseases. 

It particularly represents the first time a conventional anti-tick vaccine has been developed not just in Uganda but the wider East and Central Africa, to mitigate the growing current problem of tick acaricide failure. 

Because it works by stimulating the immune mechanisms against East Coast Fever and other tick-borne diseases, it has no collateral effects on cattle, food safety and public health. This is unlike acaricides which are leading to the accumulation of chemical residues in milk, meat and the environment (from the overly frequent application) with further collateral damage to beneficial arthropod species, consequences of their improper use by farmers.

TicVac-U® being a recombinant protein-based vaccine (PBV) with antigen proteins extracted from the ticks, means that the vaccine proteins are broken down by the immunized cattle and within a few months, there is no tick protein left at all in the animals. This is because antigens are of a biological nature and thus are broken down rapidly in the body, sometimes in a matter of days – meaning an animal can be slaughtered and safely eaten immediately after vaccination. 

With some acaricides, this is not possible, since some require a longer withdrawal period sometimes up to 60days, reflecting the necessary time period for the animal to digest the acaricide and the amount of time needed for acaricide concentration levels in the tissues (muscles) to decrease to a safe, acceptable level for consumption. 

According to the National Drug Authority, farmers are required to withdraw animals from sale or slaughter, during treatment using certain acaricides. Even milk produced during that time period must be disposed of since it won’t be safe for human consumption. Unfortunately, many farmers in Uganda rarely observe these long prescribed withdrawal periods for milk and meat, hence endangering food safety.

SUMMARY

For these reasons, the Anti-Tick Vaccine Development Initiative has remained committed to finding a lasting solution to the problem of ticks and tick resistance to acaricides. 

Vaccination using anti-tick vaccines such as TicVac-U, an East coast fever vaccine, as well as the first vaccine for Babesiosos in Uganda, represents a positive step towards mitigating the growing problem of acaricide failure against the most economically significant tick species affecting the national herd.

As scientists continue to improve on the vaccine (R&D) through continued research and improvement at CoVAB, the most effective way forward is to use integrated methods of tick and tick-borne disease control that should include the use of vaccination and some tick control using acaricides, especially during the first 3 months after vaccination with TicVac-U tick vaccine. 

REFERENCES

Allan, F. K., & Peters, A. R. (2021). Safety and Efficacy of the East Coast Fever Muguga Cocktail Vaccine: A Systematic Review. Vaccines, 9(11), 1318. https://doi.org/10.3390/vaccines9111318

Byaruhanga, J., Tayebwa, D. S., Eneku, W., Afayoa, M., Mutebi, F., Ndyanabo, S., Kakooza, S., Okwee-Acai, J., Tweyongyere, R., Wampande, E. M., & Vudriko, P. (2017). Retrospective study on cattle and poultry diseases in Uganda. International Journal of Veterinary Science and Medicine, 5(2), pp.168–174. https://doi.org/10.1016/j.ijvsm.2017.07.001

De la Fuente, J., Kopáček, P., Lew‐Tabor, A. and Maritz‐Olivier, C., 2016. Strategies for new and improved vaccines against ticks and tick‐borne diseases. Parasite Immunology, 38(12), pp.754-769.

Githaka,N.W., Kanduma, E.G., Wieland, B., Daghourth, M.A., Bishop, R.P (2022) Acaricide resistance in livestock ticks infesting cattle in Africa: Current status and potential mitigation strategies. Current Research in Parasitology & Vector-Borne Diseases, 2: 100090. https://doi.org/10.1016/j.crpvbd.2022.100090 

Nahamya Joshua (2021) NDA, cattle farmers blame each other on tick resistance in Western Uganda. The Cooperator [Online] at https://thecooperator.news/nda-cattle-farmers-blame-each-other-on-tick-resistance-in-western-uganda/

Ndawula Jr, C. and Tabor, A.E., 2020. Cocktail anti-tick vaccines: The unforeseen constraints and approaches toward enhanced efficacies. Vaccines, 8(3), p.457.

NDA (2021) National Drug Authority (NDA) operation on falsified/counterfeited veterinary drugs; tick burn spray [Online] at https://www.nda.or.ug/national-drug-authority-nda-operation-on-falsified-counterfeited-veterinary-drugs-tick-burn-spray/

Obara, I., Githaka, N., Nijhof, A. et al. (2020). The Rhipicephalus appendiculatus tick vector of Theileria parva is absent from Cape buffalo (Syncerus caffer) populations and associated ecosystems in northern Uganda. Parasitol Res 119:2363–2367 https://doi.org/10.1007/s00436-020-06728-x 

Patel, E., Mwaura, S., Di Giulio, G. et al. (2019) 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. BMC Vet Res 15, 46. https://doi.org/10.1186/s12917-019-1787-y 

Saimo-Kahwa, Margaret. (2014). ‘Characterization and Production of recombinant protein Ra92A from Rhipicephalus appendiculatus aiming tick vaccine antigen in Uganda’. Conference Paper presented at the meeting of Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Brazil. 

Vudriko, P., Okwee-Acai, J., Tayebwa, D.S. et al. (2016) Emergence of multi-acaricide resistant Rhipicephalus ticks and its implication on chemical tick control in Uganda. Parasites Vectors 9(4) pp.1-13. https://doi.org/10.1186/s13071-015-1278-3

Vudriko, P., Okwee-Acai, J., Tayebwa, D.S. et al. (2017) Evidence-based tick acaricide resistance intervention strategy in Uganda. Ticks

Vudriko, P., Okwee-Acai, J., Byaruhanga, J., Tayebwa, D. S., Omara, R., Muhindo, J. B., Lagu, C., Umemiya-Shirafuji, R., Xuan, X., & Suzuki, H. (2018). Evidence-based tick acaricide resistance intervention strategy in Uganda: Concept and feedback of farmers and stakeholders. Ticks and tick-borne diseases, 9(2), pp.254–265. https://doi.org/10.1016/j.ttbdis.2017.09.011 

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