Student names: Diala N.

B
Student number: 224735863

DECLARATION BY CANDIDATE

1. I hereby declare that this academic work is submitted in partial

fulfillment of the requirements for the Bachelor of Health Science in
Veterinary Technology at Tshwane University of Technology.
2. This academic work is my own original work and has not been partially
or wholly copied/translated from the academic work of another person.
3. This academic work has not previously been submitted to any other
institution of higher education in South Africa or any other country.
4. I declare that this academic work fully complies with the stipulations of
the TUT Policy on Plagiarism. Each scholarly contribution and all the
sources cited or quoted have been properly attributed and are
indicated and acknowledged by means of a comprehensive list of
references.
5. I understand that the University may impose disciplinary actions
against me should a reasonable suspicion exists that this academic
work is not my own unaided work or that I have failed to properly
acknowledge the source of the information in my academic work”

Name Dr CI Boshoff

Date 18/08/2022
DECLARATION BY COSUPERVISOR

I hereby declare that I have read the proposal submitted for the degree
Bachelor of Health Science in Veterinary, and in my opinion, the
proposed project is sufficient in terms of scope and quality for the degree
Bachelor of Health Science in Veterinary, at Tshwane University of
Technology.”

Name:
Ms A-M Bosman

Date 25/11/2022
1
 © Copyright Tshwane University of Technology
1. IDENTIFICATION

Department : Biomedical Science

Student name : Umbrella project

Degree and course: BHSc IN VETERINARY TECHNOLOGY

Course code : BPVT20

Date: : 18/08/2022

Supervisor : Dr CI Boshoff (PhD)

Co-supervisor : Ms A-M Bosman (MTech, MSc)

2. TITLE

3. BACKGROUND AND JUSTIFICATION

2
Ticks are key vectors of a variety of pathogenic protozoa, bacteria, and viruses that

cause disease and death in humans and animals all over the world (Gondard et al.,

2017). Many tick species parasitize domestic animals and livestock, resulting in

significant economic loss (Rajput et al., 2006; Vesco et al., 2011; Reye et al., 2012). A

tick bite, contact with an infected animal, or consumption of animal products can

transmit tick-borne diseases to humans (Telmadarraiy et al., 2015). There is growing

evidence that tick-borne diseases are spreading geographically, and infection rates

may become a major public health concern in the near future (Estrada-Pena & de la

Fuente, 2014).

A report by Jongejan & Uilenberg (2004) highlighted that the abundance of ticks and

tick-borne pathogens are determined by both biotic and abiotic factors such as the

presence of hosts, rainfall, humidity and temperature. Furthermore, as the

geographical range and incidence of animal reservoir hosts and tick vectors have

expanded, new/novel diseases have emerged in new ecosystems around the world.

According to Ellis and Ramankutty (2008), more than 70% of the world's dry land has

been transformed, primarily through infrastructure or agriculture. A similar study by

Wikel (2018), also emphasised that any compromise on elements that have a direct

or indirect effect on tick populations will result in changes in tick distribution and

prevalence. As a result of the ongoing discovery of new tick-borne pathogens, the

epidemiology and variety of recognized vector-borne illnesses and pathogens are

constantly changing (Paddock et al., 2016).

It is therefore of paramount importance to perform surveys on the distribution of

various tick species in order to estimate the distribution of tick-borne diseases as well

as the ecological conditions to which the ticks are exposed (Sonenshine et al., 2006).

Typically, culture-based, serology and microscopy methods have been used to detect
3
and identify tick pathogens (Clay & Fuqua, 2011)). However, less than 2% of

microorganisms can be isolated in the laboratory because of the low sensitivity of

culturing (Wade, 2002). Nonetheless, pathogen isolation is still the gold standard

since the presence of the isolate enables for additional identification and

characterisation of the pathogen using robust techniques such as polymerase chain

reaction (PCR), next- generation sequencing (NGS), and metagenomics.

Furthermore, studies on tick distribution, population, and disease presence in South

Africa are lacking, and further research is needed to better understand the

distribution and danger of tick-borne diseases occurring in the country.

4. PROBLEM STATEMENT

Ticks are important ectoparasitic arthropods that inflict enormous economic losses

due to their direct or indirect effects on their hosts (Jongejan & Uilenberg, 1994).

They are obligate blood-feeders and vectors of pathogens in both domestic and wild

animals, and they are second only to mosquitoes as zoonotic pathogen disease

spreading agents (de la Fuente et al., 2008; Colwell et al., 2011). In addition, there

are approximately 900 tick species worldwide, and they transmit a wide range of viral,

bacterial, and protozoan infections to their hosts (Jongejan & Uilenberg, 2004).

Due to the high biodiversity of wild and domesticated animals that acts as hosts and

reservoirs for tick-borne microorganisms in South Africa, they are crucial economic

and veterinary health threats in the country. Specifically, to the rural communities of

South Africa, where people practise small-scale or traditional farming, not only as a

source of income, but also for various other reasons (Sansoucy et al., 1995).

Livestock animals are hosts to various insect vectors including ticks, and the

economic losses thereof due to reduced milk yield and cattle mortality caused tick-

4
borne micro-organisms have a negative impact on the communities (Makala et al.,

2003).

The South African Veterinary services, together with the various communities have

implemented various control measures to reduce the tick density within the

communities and on their livestock with the aim of reducing transmission of various

tick-borne microorganisms. These programs employ weekly cattle dips into

acaricides as well as veterinary assistance to ensure healthier livestock in the

communities (Choopa, 2015). However, the overuse of chemical control methods has

previously led to acaricide-resistant tick populations in many communities with a high

rate of transmission of tick-borne organisms from infected to naïve animals, thus

further research is necessary to identify the presence and diversity of tick vectors

circulating on the animals in the area and the tick-borne organisms they transmit, as

well as alternate effective tick control prevention methods.

This project is aimed to train Bachelor of Health Science in Veterinary Technology

students the skill of research. Results obtained will not be communicated to

community and will be reported in a mini-dissertation the partial fulfillment

requirements for the

BHSc in Veterinary Science.

5. AIM
The aim of the study is to train BHSc students in research and laboratory skills by

determining the occurrence and the diversity of the ticks and tick-borne pathogens

circulating in the Hammanskraal area, Gauteng Province, South Africa.

6. OBJECTIVES
Note all objectives will be handled as small projects:

5
 i. Morphological identification of collected ticks ii. Optimization of DNA

extraction: testing the quantity and quality of DNA obtained iii. Optimization

of a PCR reaction: evaluation of three different Taq polymerase master

mixes iv. Genotyping of collected ticks

v. Identification of blood bacteria and protozoa in the collected ticks using

PCR

7. MATERIALS AND METHODS

8. Materials and Methods

8.1. Study design

Ticks will be sourced from farms in the Hammanskraal area. The owners or Animal

Health Technicians (AHT) (State Veterinary Office) will collect the ticks. The

necessary consumables, reagents and equipment will be provided to the collection

owner or AHT. Figure 1 below is the illustration of the summary of the study design.

In brief, live ticks will be collected from cattle manually using sterile forceps at the

different dip tanks in the study area. Different body parts of the animal will be targeted

for tick collection such as the ears, dewlap, axillae, udder, groin, shoulders, dewlap,

belly, and the anus. Information on the tick infestation of the animals and the rest of

the herds at the dip tanks will be captured adequately. Ticks will be morphologically

identified. Furthermore, Genomic DNA will be extracted using different DNA

extraction kits. The DNA will be screened for the presence of various miro-organisms

such as bacteria and parasites using polymerase chain reaction assays (PCR).

6
Figure 1: flow-chart of the study design

7
9. Population / unit of analysis
This study will be conducted in the Hammanskraal area, which is a semi-rural area

situated in the Gauteng Province of South Africa. The veterinarians in the targeted

area will be contacted for assistance in sampling. A workshop will be organized to

raise awareness about the ticks and tick-borne pathogens where the animal owners

will be recruited into the project.

Inclusion criteria:

• For the participants to be included in this study, they must at least have five or

more cattle.

• They must be available during collection of the samples.

Exclusion criteria:

• Individuals which own less than five cattle will be excluded.

10. Data gathering

Some of the data will be gathered in the form of questionnaires will be given to
collaborating owner and or AHT. The questionnaire will be translated into local native
languages to engage all participants fully. The questionnaire will consist of an
information sheet explaining the study to the participants (Appendix A). The
questionnaire will consist of two sections:

• Section 1- Information leaflet (Appendix A) • Section 2-

Questionnaire related to the study (Appendix B).

11. Data analysis

Results and findings will be kept in a laboratory book and the supervisors will sign off
all the experiments. Final results and concluding remarks will be compiled in
minidissertation as partial fulfillment for the BHSc in Veterinary science degree

12. Validity and reliability OR trustworthiness

To ensure validity and reliability, all the instruments will be calibrated by the service
providers. Traceability is important when it comes to the quality of research.

8
Procedures will be followed accordingly, samples will be marked with the dates of

collection, and the name of the collector and anything that is to be stored will be

stored correctly. With the evolution of technology, it might happen that a laboratory

does not have the newest equipment, but alternative methods that can yield good

results will be considered.

13. ETHICS
This study will be conducted taking into consideration the requirement of the ethics

clearance approved by the Faculty of Science Research Ethics Committee (FCRE) at

the Tshwane University of Technology (TUT), where the study will be registered.

Access to the Hammanskraal community will be obtained through the municipal

committee upon explaining the purpose of the project. The study expectations and

respective obligations by both the participants and investigators will be explained and

any question will be answered.

No animal will be handled by TUT staff or student, collection of ticks will be done in

collaborating with the owner and / or the AHT.

14. OCCUPATIONAL HEALTH AND SAFETY

Any occupational health and safety risks will be minimised, for example:

Where applicable:

• Researchers will be provided with the appropriate protective clothing and

equipment for collection of the samples.

• Safe and reliable transport will be used to transport the researchers and the

samples

• Ticks will be collected in 70% ethanol and will be transport to the laboratory in

doubled bag containers as prescribed by State Veterinary Office.

9
 • Safe and reputable accommodation will be used during sample collection.

• The support of the community leaders and members during the awareness and

subsequent to the awareness will be acknowledged.

10
15. TIME SCHEDULE

Table 1: Time schedule.

YEAR 2022 2023

MONTH March to November February March to July August September October

ACTION
Proposal
Sample collection
Laboratory work
Write mini dissertation
Oral presentation of
results
Submission of
minidissertation

16. BUDGET
Provide a clear, complete, realistic and affordable budget on the costs of the research

project and indicate where the funds needed to conduct the project will be obtained

(the researchers responsibility/ funding from grant etc.). Please note this includes

printing, petrol, transport costs etc.

11
Table 2: Budget.
Expenses Cost
Printing R 200.00
Fuel costs for meeting with the state R1000.00
Veterinarians
Fuel costs for meeting with community R1500.00
Leaders
Telephone and Internet R800.00
Arrangement of the awareness R 25 000.00
Sample collection and analysis R 25 000.00
TOTAL R53 500.00

17. RECORDS

All study information will be stored at the Department of Biomedical Sciences,

Tshwane University of Technology for a minimum of 3 years.

18. INTELLECTUAL PROPERTY

Not applicable.

19. DEVIATIONS

Any deviations will be recorded and submitted to the Department of Biomedical

Sciences Committee for Post Graduate Studies and the Faculty of Science

Committee for Research Ethics for reconsideration.

20. DISSEMINATION OF RESEARCH OUTCOMES

The results from this research will provide educational feedback to the staff and

community members of Hammanskraal area where the research will be conducted.

21. PUBLICATIONS

12
The results extracted from this project will not be published in any journal as it is for

the training purpose of the fourth level students in the Department of Biomedical

Sciences. A mini-dissertation will be compiled in partial fulfillment of the BHSc in

Veterinary Technology degree.

22. REFERENCES

CANGI, N., PINARELLO, V., BOURNEZ, L., LEFRANÇOIS, T., ALBINA, E., NEVES,

L. & VACHIÉRY, N. 2017. Efficient high-throughput molecular method to detect

Ehrlichia ruminantium in ticks. Parasites & vectors, 10(1),1-12.

CHAISI, M.E., BAXTER, J.R., HOVE, P., CHOOPA, C.N., OOSTHUIZEN, M.C.,

BRAYTON, K.A., KHUMALO, Z.T., MUTSHEMBELE, A.M., MTSHALI, M.S. &

COLLINS, N.E. 2017. Comparison of three nucleic acid-based tests for detecting

Anaplasma marginale and Anaplasma centrale in cattle. Onderstepoort Journal of

Veterinary Research, 84(1), 1-9.

CHOOPA, C.N. 2015. Diagnosis of tick-borne diseases in cattle in Bushbuckridge

Mpumalanga South Africa and identification of Theileria parva carriers (Doctoral

dissertation, University of Pretoria).

CLAY, K. & FUQUA, C. 2010. The tick microbiome: diversity, distribution and

influence of the internal microbial community for a blood-feeding disease vector. In

Critical needs and gaps in understanding prevention, amelioration, and resolution of

Lyme and other tick-borne diseases: the short-term and long-term outcomes.

Workshop report.

National Academies Press, Washington, DC.

13
COLWELL, D.D., DANTAS-TORRES, F. & OTRANTO, D. 2011. Vector-borne

parasitic zoonoses: emerging scenarios and new perspectives. Veterinary

parasitology, 182(1),14-21.

DANTAS-TORRES, F., CHOMEL, B.B. & OTRANTO, D. 2012. Ticks and tick-borne

diseases: a One Health perspective. Trends in parasitology, 28(10), 437-446.

de la FUENTE, J., ESTRADA-PENA, A., VENZAL, J.M., KOCAN, K.M. &

SONENSHINE, D.E. 2008. Overview: ticks as vectors of pathogens that cause

disease in humans and animals. Front Biosci, 13(13), 6938-6946.

ELLIS, E.C. & RAMANKUTTY, N. 2008. Putting people in the map: anthropogenic

biomes of the world. Frontiers in Ecology and the Environment, 6(8),439-447.

Estrada-Peña, A. and de la Fuente, J., 2014. The ecology of ticks and epidemiology
of tick-borne viral diseases. Antiviral research, 108:04-128.

GONDARD, M., CABEZAS-CRUZ, A., CHARLES, R.A., VAYSSIER-TAUSSAT, M.,

ALBINA, E. & MOUTAILLER, S. 2017. Ticks and tick-borne pathogens of the
Caribbean: Current understanding and future directions for more comprehensive
surveillance. Frontiers in Cellular and Infection Microbiology, 7:490.

JONGEJAN, F.R.A.N.S. & UILENBERG, G. 1994. Ticks and control methods. Revue

scientifique et technique (International Office of Epizootics), 13(4),1201-1226.

KUMAR, S., STECHER, G. & TAMURA, K. 2016. MEGA7: molecular evolutionary

genetics analysis version 7.0 for bigger datasets. Molecular biology and evolution,

33(7), 1870-1874.

MAKALA, L.H., MANGANI, P., FUJISAKI, K. & NAGASAWA, H. 2003. The current

status of major tick borne diseases in Zambia. Veterinary Research, 34(1), 27-45.
PADDOCK, C.D., LANE, R.S., STAPLES, J.E. & LABRUNA, M.B. 2016. Changing

14
paradigms for tick-borne diseases in the Americas. In Global health impacts of

vector- borne diseases: workshop summary (pp. 221-258). Washington, DC, USA:

National

Academies Press.

Rajput, Z.I., Hu, S.H., Chen, W.J., Arijo, A.G. and Xiao, C.W., 2006. Importance of
ticks and their chemical and immunological control in livestock. Journal of Zhejiang
University Science B, 7(11), pp.912-921.

REYE, A.L., ARINOLA, O.G., HÜBSCHEN, J.M. & MULLER, C.P. 2012. Pathogen
prevalence in ticks collected from the vegetation and livestock in Nigeria. Applied and
Environmental Microbiology, 78(8),562-2568.

SANSOUCY, R., JABBAR, M.A., EHUI, S.K. & FITZHUGH, H. 1995. The contribution

of livestock to food security and sustainable development: Keynote paper. Livestock

Development Strategies for Low Income Countries, pp.9-26.

SONENSHINE, D.E., KOCAN, K.M. & DE LA FUENTE, J. 2006. Tick control: further

thoughts on a research agenda. Trends in parasitology, 22(12),550-551.

STOLTSZ, H., BYARUHANGA, C., TROSKIE, M., MAKGABO, M., OOSTHUIZEN,

M.C., COLLINS, N.E. & NEVES, L. 2020. Improved detection of Babesia bigemina

from various geographical areas in Africa using quantitative PCR and reverse line

blot hybridisation. Ticks and Tick-borne Diseases, 11(4), 101415.

TELMADARRAIY, Z., CHINIKAR, S., VATANDOOST, H., FAGHIHI, F. & HOSSEINI-

CHEGENI, A. 2015. Vectors of Crimean Congo hemorrhagic fever virus in Iran.
Journal of arthropod-borne diseases, 9(2), 137.

VESCO, U., KNAP, N., LABRUNA, M.B., AVŠIČ-ŽUPANC, T., ESTRADA-PEÑA, A.,
GUGLIELMONE, A.A., BECHARA, G.H., GUEYE, A., LAKOS, A., GRINDATTO, A. &

15
CONTE, V. 2011. An integrated database on ticks and tick-borne zoonoses in the
tropics and subtropics with special reference to developing and emerging countries.
Experimental and applied acarology, 54(1), 65-83.

WADE, W. 2002. Unculturable bacteria—the uncharacterized organisms that cause

oral infections. Journal of the Royal Society of Medicine, 95(2), 81-83.

WIKEL, S.K. 2018. Ticks and tick-borne infections: complex ecology, agents, and

host interactions. Veterinary sciences, 5(2),60.

YOUNG, A.S., GROOCOCK, C.M. & KARIUKI, D.P. 1988. Integrated control of ticks
and tick-borne diseases of cattle in Africa. Parasitology, 96(2), 403-432.

16