Ticks and tick-borne zoonotic pathogens of domestic animals in Lesotho

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Ticks and tick-borne zoonotic pathogens of

domestic animals in Lesotho

SIC Mahlobo

orcid.org 0000-0002-7443-2485

Dissertation submitted in fulfilment of the requirements for the

degree Master of Science in Zoology

at the North-West

University

Supervisor:

Prof MMO Thekisoe

Co-supervisor:

Dr MS Mtshali

Graduation May 2018

28300165

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DECLARATION

I, the undersigned, hereby declare that the work contained in this dissertation is my original work and that I have not previously in its entirety or in part submitted at any university for a degree. I furthermore cede copyright of the dissertation in favour of the North−West University.

Signature: ………..

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DEDICATION

To my family and siblings. I thank you for your continuous support in my studies always praying for me (grandmother, SPP Mahlobo), being proud of all my achievements (my dearest mom, SM Mahlobo) and teaching me the importance of education, humbleness, perseverance and being independent (my late great grandmother, Mrs SA Mahlobo KaMakhathini).

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ACKNOWLEGEMENTS

My genuine and truthful appreciation goes to my supervisors Prof Oriel M.M. Thekisoe and Dr Sibusiso M. Mtshali. In particular, I would like to say to Prof Oriel Thekisoe this project would not have been a success without your continuous support, persistence and inspiration. This reminds me of the lyrics you told us in our first meeting “I came to win, to survive, to conquer and to rise”. To know that to him “Prof.” as a mentor being a hard worker is much more than being intelligent. Those words gave me the courage to press on during challenging days. Reflecting on my journey, I am where I am because of you; continuously pushing me to be the best and to strive for more and now I am better and more refined that before, all the gratitude goes to you Prof.

I would like to show appreciation to all the organization and people that were involved directly or indirectly in this project making it to be successful, Dr Marosi Molomo, Dr Lineo Bohloa and the rest of veterinary officials from Department of Livestock Services, Ministry of Agriculture and Food Security, Maseru, Lesotho, for assistance in tick specimen collection.

I thank my family, my grandmother Philile Mahlobo, my mother Sithethelele Mahlobo, my siblings S’bongimpilo Hlongwane, Sibongumusa Mazibuko and Sibongakonke Mlangeni for your support and always putting a smile on my face during hard days, praying for me and believing in me. To my friends Busisiwe Mazibuko, Sbusiso Shwabede, Philile Mafu, Nobuhle Mbhele, Sydney Gambushe, Malitaba Mlangeni, thank you for your encouraging words, spiritual growth, motivation and advices when I needed, even though most of my work was foreign to some of you, standing by my side and supporting me from the beginning to the end and constantly praying for me and my studies. To my fellow colleagues, Malitaba Mlangeni, Lehlohonolo Mofokeng, Bridget Makhahlela and Clara Van Wyk; who helped me in making this project a success, word of courage, support in mastering my work, brainstorming together, you have become my second family.

To Jani Reeder for helping me in taking picture of the ticks collected and identified in this study. To Ms. Khethiwe Mtshali, Dr Moeti Taioe and Dr Zamantungwa Khumalo, thank you so much for helping me with all the statistical and phylogenetic analysis, result interpretations. Thank you for pushing me to think and work hard. To Mr. Deon Bakkes at ARC-Onderstepoort Veterinary Institution for correcting morphological identification and assigning voucher specimen code to all the ticks identified.

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I thank the North-West University (Potchefstroom Campus), for availing their facilities during this study. I am grateful to the National Research Foundation (NRF) for the Masters Innovation Bursary. The study was financially supported by the NRF Incentive Grant for Rated Researchers and the NWU Institutional Incentive Grant for Rated Researchers. Thank you Lord my God for the plans and thoughts you have for me, keeping me save in the shadow of your winds and guiding me in this journey and giving me hope and a brighter future (Jeremiah 29: 11). Thank you Jesus Christ that in your sufficiency, I am sufficient and that I’m ready for anything and equal to anything through You. Thank you my comforter, Holy Spirit that I can do all things through the strength, confident peace you daily give me and empowering me to do better (Philippians 4: 13).

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ABSTRACT

Ticks are vectors of diseases and transmit infectious pathogens of economic importance, some of these agents have a direct and indirect impact on the livestock industry globally. There is lack of published information on ticks and zoonotic pathogens in Lesotho. This study was therefore formulated to document information of tick species occurring in Lesotho and zoonotic pathogens that they are harbouring, with special focus on

Anaplasma phagocytophilum, Coxiella burnetii and Rickettsia africae pathogens.

A total of 2054 tick specimens were collected from various domestic animals including cattle, sheep, goats and dogs from five Lesotho districts namely, Butha-Buthe, Mafeteng, Maseru, Leribe and Qacha’s Nek. All tick species were identified morphologically under a stereo microscope and submitted to the ARC-Onderstepoort Veterinary Research Tick Museum where species identification was verified and voucher specimen was issued. Seven species were identified falling under four genera, of which three are from family Ixodidae and one from family Argasidae. Ticks collected and identified included

Haemaphysalis elliptica 0.1% (n= 2), Hyalomma rufipes 3.6% (n= 73), H. truncatum 2%

(n= 41), Otobius megnini 18.2% (n= 373), Rhipicephalus decoloratus 17% (n= 349), R.

microplus 10.7% (n= 220), R. evertsi evertsi 46.4% (n= 953) and R. glabroscutatum 2.1%

(n= 43). There was a significant difference at p= 0.005 (ᵪ2_{= 3.072, df= 3) in the overall} species that were identified in five Lesotho districts. However, there was no significant difference at p= 0.06 (ᵪ2_{= 30.072, df= 3) for the abundance of species from each sampled}

district. The CO1 and 18S rRNA genes of the identified ticks were amplified by PCR, sequenced and aligned using MEGA 6 software whereby phylogenetic trees were constructed. The tick genera correctly clustered with their subsequent tick species further indicating that they were identified correctly.

Out of the 247 pooled tick DNA samples screened by PCR for the presence of zoonotic pathogens; overall occurrence of A. phagocytophilum was 7% (18/247) for Butha-Buthe (n= 79), Mafeteng (n= 9), Maseru (n= 30) and Leribe (n= 2). PCR positive samples from

H. truncatum DNA yielded highest number of A. phagocytophilum positive samples with

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with 13%. The R. microplus (n= 3) and O. megnini (n=2) tick specimens were negative for the presence of A. phagocytophilum infections.

The overall occurrence of Coxiella burnetii a gram-negative pleomorphic etiological agent of Query fever (Q fever) was 7% (17/247) for Butha-Buthe (n= 49), Mafeteng (n= 7), Maseru (n= 23) and Leribe (n= 2). Overall infection from cattle, goats and sheep was 22% (17/79), highest infection rate was 33% from H. truncatum followed by, 13.2% from R. e.

evertsi, then 13% from R. decoloratus and the least was 1% from O. megnini. The R. microplus DNA samples were negative for the presence of C. burnetii infections.

The overall occurrence of Rickettsia africae which is the most prevalent tick-borne pathogen in sub-Saharan Africa causing African tick-bite fever (ATBF) was 1% (2/247) for Butha-Buthe (n= 7), Maseru (n= 30) and Leribe (n= 2). Overall infection from domestic animals including goats and cattle was 5% (2/37). From all PCR positive samples both R.

e. evertsi and R. decoloratus had 2% infection rate. None of the O. megnini (n= 2), R. microplus (n= 3), H. truncatum (n= 15) tick specimens were PCR positive for the presence

of R. africae.

This current study has provided base line knowledge of the tick species and tick-borne bacterial pathogens (A. phagocytophilum, C. burnetii and R. africae) of economic and zoonotic importance in Lesotho districts. Further studies are needed to determine whether ticks are transmitting these pathogens to livestock and humans.

Key words: Anaplasma phagocytophilum, Coxiella burnetii, Lesotho, PCR, Rickettsia

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LIST OF TABLES

Table 3.1: Morphological identifcation of ticks collected in variuos districts of Lesotho 26

Table 3.2: Primer sequences used to amplify various tick species 29

Table 3.3: Tick species identified from five districts of Lesotho and total number of tick species collected in each district 39

Table 3.4: Lesotho domestic animals where ticks were collected from various districts 40

Table 3.5: BLASTn results for CO1 gene of soft ticks 43

Table 3.6: Soft ticks CO1 gene rates of base substitutions for each nucleotide pair

44

Table 3.7: Soft ticks CO1 gene pairwise distance nucleotide differences found among the taxa 44

Table 3.8: BLASTn results for CO1 gene of hard ticks 48

Table 3.9: Hard ticks CO1 gene rates of base substitutions for each nucleotide pair 48

Table 3.10: Hard ticks CO1 gene pairwise distance nucleotide differences found among the taxa 49

Table 3.11: BLASTn results for 18S rRNA gene of soft ticks 52

Table 3.12: Soft tick 18S gene rates of base substitutions for each nucleotide pair 53

Table 3.13: Soft tick 18S gene pairwise distance nucleotide differences found among the taxa 53

Table 3.14: BLASTn results for 18S rRNA gene of hard ticks 56

Table 3.15: Hard tick 18S gene rates of base substitutions for each nucleotide pair 57

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Table 3.16: Hard tick 18S gene pairwise distance nucleotide differences found among the taxa 57

Table 4.1: Oligonucleotide sequences used to amplify the target pathogens 83

Table 4.2: Overall occurrence of Anaplasma phagocytophilum in ticks from Lesotho districts 85

Table 4.3: Overall occurrence of Coxiella burnetii in ticks from Lesotho districts 87

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LIST OF FIGURES

Figure 1.1: General anatomy of hard ticks showing adult stage of male and female

tick 4

Figure 1.2: Morphological characterization of soft ticks 5

Figure 3.1: One-host tick life cycle, e.g. Rhipicephalus decoloratus 19

Figure 3.2: Two-host life cycle, e.g. Rhipicephalus bursa 19

Figure 3.3: Three-host life cycle, e.g. Rhipicephalus appendiculatus 20

Figure 3.4: Soft tick life cycle, e.g. Ornithodoros moubata; other argasid group may differ considerably 21

Figure 3.5: Map of Lesotho with 10 districts 25

Figure 3.6: The overall prevalence of different tick species from various districts of Lesotho 41

Figure 3.7: Agarose gel electrophoresis of amplified PCR products CO1 gene (upper) 18S gene (lower) of tick samples from various districts in Lesotho 42

Figure 3.8: Nucleotide differences found in the CO1 gene sequences of soft tick species 45

Figure 3.9: Phylogenetic analysis by Maximum Likelihood (ML) method based on the General Time Reversible model 46

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Figure 3.10: Nucleotide differences found in the CO1 gene sequences of hard tick species 50

Figure 3.11: Phylogenetic analysis by Maximum Likelihood (ML) method based on Tamura 3-parameter model 51

Figure 3.12: Nucleotide differences found in the 18S gene sequences of soft tick species 54

Figure 3.13: Phylogenetic analysis by Maximum Likelihood (ML) method based on Kimura 2-parameter model 55

Figure 3.14: Nucleotide differences found in the 18S gene sequences of hard tick species 58

Figure 3.15: Phylogenetic analysis by Maximum Likelihood (ML) method based on the Kimura 2-parameter model 59

Figure 4.1: Gel electrophoresis of A. phagocytophilum amplified PCR product with amplicon size 250bp 84

Figure 4.2: Gel electrophoresis of C. burnetii amplified PCR product with amplicon size of 104 bp 86

Figure 4.3: Gel electrophoresis of R. africae amplified PCR products with amplicon size of 401 bp 88

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LIST OF PLATES

Plate I: Haemaphysalis elliptica 34

Plate II: Hyalomma rufipes 34

Plate III: Hyalomma truncatum 34

Plate IV: Otobius megnini 34

Plate V: Rhipicephalus decoloratus 35

Plate VI: Rhipicephalus microplus 35

Plate VII: Rhipicephalus evertsi evertsi 35

Plate VIII: Rhipicephalus glabroscutatum 35

Plate IX: Eggs 36

Plate X: Larvae 36

Plate XI: Eggs developing into larvae 36

Plate XII: Otobius megnini moulted skin 36

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Plate XIV: Rhipicephalus e. evertsi female 37

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CHAPTER 1 GENERAL INTRODUCTION

1.1 Background

Ticks are ubiquitous blood-feeding ectoparasites infesting terrestrial invertebrates including amphibians, birds, mammals and reptiles globally (Chitimia et al., 2010). These invertebrates form a big clan with insects and spiders, with very small body mostly resembling the spider-shape and they can survive well under desiccation conditions within their habitat of preference (Walker et al., 2003). There are more than 800 tick species that have been identified globally (Walker et al., 2003, Madder et al., 2013). They belong to phylum Arthropoda a group that consists of specimen with external skeleton, which covers internal organs; having a bilateral system with chitinous skeleton articulating appendages and/ or segmentation (Madder et al., 2013). Most of these species occur in various moist places with long grass and dense vegetation, moorland, woodlands, shorelines, heathland, grassland even gardens and urban parks (Parola and Raoult, 2001). The intake amount of the blood meal is not relevant to the body size especially with female ticks, but the effects on the host are severe including blood loss, toxicity, skin damage and paralysis and such effect can be a window for many other infections due to inflamed skin (Walker et al., 2003; Jongejan and Uilenberg, 2004).

1.2 Taxonomy of ticks

According to Walker et al. (2003) and Latif (2013) classification of ticks is as follows Phylum : Arthropoda

Class : Arachnid Subclass : Acari

Order : Parasitiformes Suborder : Ixodida

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Genus : Amblyomma, Anomalohimalaya, Aponomma, Boophilus,

Cosmiomma, Dermacentor, Haemaphysalis, Hyalomma, Ixodes, Nosomma, Margaropus, Rhipicentor, Rhipicephalus, Antricola,

Argas, Ornithodoros, Otobius, Nothoaspis and Nuttalliella

Ticks belong to phylum Arthropoda, class Arachnida which include scorpions, mites, spiders, crustaceans and insects; which are all bilaterally symmetrical with jointed appendages and chitinous skeleton (Mathison and Pritt, 2014). This, class has secondary modification of paired appendages, bear pedipalps and four walking legs. Ticks are distinguishable from insects with a lack of clearly definitive head. Ticks together form their own group under subclass Acari with organisms with segmented fusion bodies and fused opisthosoma (Madder et al., 2013). Order Parasitiformes is a large group consisting of parasitic arthropods represented by respiratory pores with well-developed coxae; ticks can be distinguished from the mites by the presence of toothed hypostome that is exposed; the presence of sensory Haller’s organ on the first tarsus and lastly ticks have relatively larger bodies than microscopic mites (Walker et al., 2003). Ixodida is the suborder of ticks that consist of three families namely the Ixodidae (hard ticks), Argasidae (soft ticks) and Nuttalliellidae (tick of both hard and soft tick features, which has one genus and one species).

Ixodidae family has a total of 14 genera and more than 700 species/subspecies, the Argasidae consist of 5 genera and about 190 species and lastly the Nuttalliellidae only has one species Nuttalliella namaqua (Madder et al., 2013). Absence of the hard plate in body is one characteristic that distinguished soft ticks from hard ticks, with hard ticks having plates for both male and female and referred to as conscutum and scutum respectively (Walker et al., 2003; Chitimia et al., 2010). Mites and ticks are more similar, with an exception of ticks being larger, easily identified without the use of microscope and all of them are significant blood feeding parasites and ticks evoke various reactions to humans (Wall and Shearer, 2008).

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1.3 Tick morphology

Hard ticks are morphological characterized by the presence of tough sclerotized plates on their dorsal surface with scutum covering the entire body of males and in females only the anterior of the dorsal region in unfed nymph, larvae is covered (Figure 1.1). The scutum is an attachment site for dorso-ventral body muscles, cheliceral retractor muscles and other muscles in ixodids, eyes being located on the margins (Walker et al., 2007). The cuticle posterior to the scutum constitutes alloscutum are covered by small setae (Figure. 1.1). Both nymphal and adult stages of soft ticks have a pair of tiny pores, coxal pores (Figure 1.2); that signifies opening of coxal glands between one and two coxae legs (Walker et al., 2007). Fluids are filtered from blood meals and secreted via these pores when in excess.

In soft ticks there is no scutum but, they have a rough integument which is very tough in texture (Parola and Raoult, 2001) (Figure 1.2). Mouthparts have central hypostome (tube used for sucking and piercing) and a pair of palps. The family Nuttalliellidae is characterised by both soft and hard tick morphological characters, having a pseudoscutum that resembles hard ticks and leathery integument resembling soft tick (Sirigireddy, 2008).

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Figure 1.1: General anatomy of hard ticks showing adult stage of male and female tick. Source: Madder et al. (2013)

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Figure 1.2: Morphological characterization of soft ticks. Source: Madder et al. (2013)

1.4 Ticks effect on domestic animals

The impact of ticks in domestic animals depends on several factors including species involved, climatic conditions and infestation on animals by ticks resulting in vulnerability in herds within a region. Based on the study done in 2004 by Jongejan & Uilenburg, short hypostomes in ticks are important for infesting larger hosts. Ticks with long hypostome cause secondary bacterial infections such as loss of teats (depending on the attachment site) leading to calf mortalities. These infestations have a negative effect within livestock industry, limiting productivity of exotic cattle resulting in utilization of expensive and intense chemical tick control that leads to resistance against acaricides applied (Willadsen et al., 1998).

High infestations of ticks cause great damage to animal skin which diminishes the value of hides for the leather production (Jongejan and Uilenberg, 2004). Certain tick species in southern Africa, particularly Ixodes rubicundus a hard tick is known to cause paralysis in sheep, cattle or goats (Madder et al., 2013). In Africa the toxins released by ticks together with saliva result in “Sweating sickness” which is an eczema-like condition that

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affect calves as well as other domestic animals, causing deep, painful wounds in dogs (Jongejan and Uilenburg, 2004).

1.5 Tick relation to human beings

Ticks and tick-borne diseases are of importance in tropical and within subtropical regions. The increase in the zoonotic tick-borne diseases poses a serious risk to public health, within rural areas there is high increase of recreational activities and thus result an increase in human tick bite (Cunha, 2000). It was reported that by the year 1982 there were 15 cases of newly reported pathogens on human tick-borne diseases (Parola and Raoult, 2001; Muruthi, 2015). With regards to the impact caused by these pathogens transmitted by ticks, many studies are being done and there are new documented reports on the emerging diseases that are on the rise (Paddock and Yabsley, 2007; Lwande et

al., 2013).

International human travelling is one of the largest activities that have a great impact on human health; tick-borne diseases associated with travelling are common (Jensenius et

al., 2002). Amblyomma species are vectors of Rickettsia africae that causes African tick

bite fever (ATBF). Distribution of Amblyomma species ranges from Caribbean to sub-African region, this disease is mostly confused with tropical diseases like malaria especially when clinical symptoms are more complicated (Jensenius et al., 2002).

Different tick species are vectors of various pathogens in humans. In USA, California

Argas monolakensis is the most significant tick that feeds on humans and it is well known

to be a vector for Mono Lake virus. In Chile and Peru, the argasid ticks Argas neghmei and Argas moreli respectively are known for their implications on human parasitism within areas that are cohabit of human and chickens (Schwan et al., 1992; Muruthi, 2015).

Crimean-Congo haemorrhagic fever (CCMF) is zoonotic viral disease that is widely distributed in Asia, Africa and Middle East (WHO, 2013). The CCMF virus is transmitted by tick vectors between humans and from human to animal through contact. Hyalomma spp. are said to be the principal vector for transmitting the virus, there has been reports

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on the outbreak of the viral haemorrhagic fever in various countries with 9 - 39% fatality rate by World Health Organization in the year 2013 (Flick and Whitehouse, 2005; WHO, 2013).

1.6 Zoonoses

Diseases and infections that naturally exist in animals but are transmissible to humans are defined as zonooses. Some of the zoonotic pathogens are transmitted by arthropod vectors, which transmit a great range of pathogens to man (Kolo, 2015; Millán et al., 2016). Ticks are significant vectors for medical and public health in several ways (Zivkovic

et al., 2010).

Continuous attachment to the host, feeding on blood for several days to weeks without detachment, is the main characteristic that distinguishes tick vector from other haematophagous specimens (Wikel, 2013). Dating back to the 14th century vector-borne diseases transmitted by tick species have been the cause of problems in vertebrates; places such as Europe have experienced the worst plagues like Black Death, epidemics of yellow fever in America due to pathogens transmitted by ticks (Chitanga et al., 2014).

With the implementations that took place in the 20th century vector-borne disease were controlled and a steady decline was observed; but on the following centuries there was more re-emerging vector-borne diseases compared to prior centuries (Chitanga et al., 2014). According to survey and studies conducted, the cause of the re-emergence is associated with the new global trends including the modern transportation, urbanization, globalization and animal husbandry and amongst other tick-borne diseases is dominating (Gubler, 2009; Chitanga et al., 2014; Estrada-Peña et al., 2012).

As zoonotic pathogens and their vectors, miraculously co-evolve over the years on their molecular mechanism that is responsible for subsidization of growth and survival along with lack of information within Sub-Saharan Africa these factors contribute a lot towards

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problems of evaluating the occurrence of these major effects (Zivkovic et al., 2010; Hotez and Kamath, 2009). There is a wide range of various pathogens transmitted by tick vectors such as viruses, bacteria, protozoa; causing numerous diseases such as Lyme disease, Typhus, Babesiosis, yellow fever, and dengue fever to name few (Mathison and Pritt, 2014).

In southern Africa tick-borne diseases have been reported including the tick-borne relapsing fever (TBRF) caused by Borrelia duttonii, babesiosis caused by Babesia microti, Crimean-Congo hemorrhagic fever (CCHF) caused by Nairovirus virus (Chitanga et al., 2014). Rickettsia aeschlimanii, R. conorii and R. africae causing rickettsioses, anaplasmosis is caused by Anaplasma marginale, A. phagocytophilum, A. centrale, A.

ovis (Chitanga et al., 2014).

Health workers, agriculture and livestock are sectors affected by the CCHF; this tick-borne viral disease has spread in more than 30 countries including Asia, Africa, middle and southeastern Europe. Tick species are both vectors and reservoirs of this virus as the mechanism of transmission is both transstadial and transovarially and the disease has worldwide distribution (Chitanga et al., 2014).

1.6.1 Anaplasmosis

Anaplasma phagocytophilum is an obligate gram-negative intercellular bacterium

colonizing within granulocytic within the host causing anaplasmosis (Chitanga et al., 2014). This disease is known as human granulocytic anaplasmosis in (HGA) in people and referred to as tick-borne fever (TBF) in ruminants (Matei et al., 2015). Over the years

A. phagocytophilum has been detected globally in both domestic and wild animals (Zhang et al., 2016).

According to Kim et al. (2006), recent studies have confirmed the broad existence of anaplasmosis in Asia, America, Europe and Africa; with natural reservoirs namely the

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Odocoileus virginianus and Peromyscus leucopus (white-tailed deer and white-footed

mouse respectively) for Anaplasma species in United States and Korea. In Europe rodents are natural reservoirs for Anaplasma species. Anaplasma phagocytophilum is said to be maintained in the tick-rodent cycle (Ohashi et al., 2005).

In southern Africa there is no scientific report on humans infected or suspected to be infected by A. phagocytophilum other than a report published by Inokuma et al. (2005), for presence of A. phagocytophilum in South African dogs. Over the years many reports have been published on the pathogen in Africa all these were based on serological detection of antibodies used to analyze serum samples (Chitanga et al., 2014).

1.6.2 Query fever

Query fever causative agent is Coxiella burnetii which is an intercellular bacterium that is asymptomatic; domestic animals have been reported as main sources for human infection including sheep, goats and cattle. Compared to R. africae, C. burnetii ranges far less as a zoonotic disease (Mtshali et al., 2015).

Query fever has a worldwide distribution and due to this wide distribution and high infectivity, military and civilian personnel that deploy to other countries are more at risk of being infected (Chen and Ching, 2014). More than 40 vector species of different genera from soft and hard ticks (Argas and Carios, Aponomma, Dermacentor, Haemaphysalis and Rhipicephalus) maintain and harbor Coxiella burnetii pathogen (Sumrandee et al., 2015).

1.6.3 Rickettsiosis

Rickettsia species causes a febrile illness known as rickettsiosis that consist of two groups namely typhus and spotted fever. In sub-Saharan Africa spotted fever group is the most significant tick-borne zoonosis with three species namely Rickettsia africae, R.

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the Middle East (Chitanga et al., 2014). Amblyomma species particularly A. hebraeum in southern Africa are principle vector that transmit R. africae causing ATBF (Parola et al., 2005; Althaus et al., 2010; Chitanga et al., 2014; Inokuma et al., 2005).

Previously Rhipicephalus sanguineus was reported as the main vector of R. conorii, but recently there was a report on this pathogen being isolated from Haemaphysalis spp. in South Africa (Chitanga et al., 2014). According to Chitanga et al. (2014), R.

appendiculatus is the vector responsible for R. aeschlimannii transmission.

Challenges and threats faced globally are real, as the prominence of these emerging zoonotic pathogens are on the increase. Direct proportion of tick-borne diseases influenced by several factors within the environments at large and the constant changes and development contribute a larger percentage. The severity of not knowing the exact geographic distribution of many pathogens infections also affect surveys that have been conducted. New molecular techniques implemented are reliable and this allows expansion towards research scope in diagnostics of vector-borne diseases (Parola et al., 2005; Wang and Crameri, 2014).

In the absence of published data of ticks and tick-borne zoonotic pathogens in Lesotho, this study seeks to establish a base-line data by documenting ticks infesting domestic animals in Lesotho districts. Furthermore, the current study used PCR to detect selected tick-borne zoonotic pathogens of economic, medical and veterinary importance which are harbored by these ticks.

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CHAPTER 2 PROBLEM STATEMENT, AIM AND OBJECTIVES

2.1 Problem statement

Anaplasmosis and rickettsioses are two of many zoonotic diseases that have been widely reported in South Africa (Gummow, 2003). Sixty-four percent of veterinarians suffer from zoonotic diseases particularly those living in rural areas, exposing themselves to zoonotic pathogens being constantly in contact with farm animals (Raoult et al., 2001; Althaus et

al., 2010). A survey conducted by Gummow (2003), suggested Gauteng was leading with

zoonotic diseases followed by KwaZulu-Natal with Northern and Western Cape being the least affected provinces.

Additionally, detailed information in many of these tick-borne zoonoses and their precise distribution and occurrence is lacking. In sub-Saharan Africa there are many cases of underdiagnosed and inadequate awareness within health sectors with most infections and clinical symptoms being confused with those of malaria (Hotez and Kamath, 2009). Furthermore, Zimbabwe and South Africa are two countries known for their endemic occurrence of Rickettsia africae a pathogen known to cause ATBF, which is one of the diseases under spotted fever group. Amblyomma tick species are endemic in these countries and identified as vectors for ATBF; based on serological surveys the infection is reported mostly in travellers returning to North America and Europe from South Africa (Hotez and Kamath, 2009).

Rift Valley Fever, Rabies, Crimean-Congo Haemorrhagic Fever and ATBF are some of the zoonotic diseases that are of interest, nonetheless there is no report or little information reported for illnesses or deaths caused by these pathogens among locals (Althaus et al., 2010). Respiratory, blood-borne ailments and common digestion are alike symptoms displayed by bacteria making it problematic to determine various pathogens depending only on clinical presentation; leading to deterioration risk of condition and delaying treatment (Xia et al., 2015).

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Bekker et al. (2012), indicates that 41% of the game farmers in South Africa have adapted to mixed farming system that create an uncontrollable co-infection between wild and domestic animals. In Fee State wild species are overstocked into new habitats that they are not adaptable to and for this cause, they are many other diseases that emerge and vectors have also increased.

It has been reported that wild animals are associated more to these emerging zoonotic diseases than domestic animals as stipulated by Bekker et al. (2012). However, Berggoetz et al. (2014) study indicates that there is no significant difference between domestic and wild animal zoonotic diseases, as both these animals are able to work evenly as efficient sources of infection for each other and among themselves. A recent study conducted by Golezardy et al. (2016), indicates that despite the use of acaricides and other intensive control efforts, tick species are able to strive and maintain a great population range and continue in transmitting various pathogens.

There is lack of published scientific data about tick species occurring in Lesotho as well as zoonotic pathogens they are harbouring. This study has strived to pioneer documentation of tick species infesting domestic animals in Lesotho as well as the zoonotic pathogens they are harbouring with particular attention to Anaplasma

phagocytophilum, Coxiella burnetii and Rickettsia africae. In this study, ticks were

collected from cattle, sheep, goats, dogs and horses. Data generated by this study shed light on tick species occurring in Lesotho, their distribution as well as zoonotic pathogens they are harbouring. This data will contribute with to formulation of tick control strategies.

2.2 Aim of the study

The aim of this study was to document information of tick species infesting domestic animals in Lesotho and detecting selected zoonotic tick-borne pathogens they harbour.

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2.2.1 Objectives

 To conduct morphological identification and molecular characterization of tick species infesting domestic animals in various districts of Lesotho.

 To determine phylogenetic position of ticks infesting domestic animals in various districts of Lesotho by maximum likelihood using MEGA6 software.

 To conduct molecular detection of Anaplasma phagocytophilum, Coxiella burnetii and

Rickettsia africae from ticks infesting domestic animals in various districts of Lesotho

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CHAPTER 3 CHARACTERIZATION OF TICKS INFESTING DOMESTIC ANIMALS IN

LESOTHO

3.1 Introduction

Phylum Arthropoda has the largest number of animals, yet they have the smallest number of species that are direct or indirect relative to public health; the group comprises of about 860 species (Horak et al., 2002). Ticks belong to an ancient lineage that dates back to 100 million years; they are a successful group of arthropoda that are found in all habitats of biosphere (Walker, 1999). The success of this phylum is associated with its jointed legs and cuticle. They have adapted to feeding on any terrestrial birds, reptiles and mammals, furthermore there are also reported cases ticks attacking humans and infestation in domesticated animals over the years (Walker et al., 2000; Walker, 1999).

Tick species identified and described are close to ±900; more than 700 of those species belong to the family Ixodidae and the remaining sum belongs to family Argasidae (Walker

et al., 2003). Most tick species are abundant within Afrotropical regions that are richer in

animal fauna and climatic zones are between arid to tropical (Anderson and Magnarelli, 2008). Wild animals are primary source serving as reservoirs for pathogens, which can later be transmissible to domestic animals as well as humans. Many of these diseases from the wildlife cause rabies, avian influenza, salmonellosis, schistosomiasis, rickettsioses, anaplasmosis, babesiosis, African trypanosomiasis, leishmaniasis and most recently is the Ebola virus (Horak et al., 2015; Spickett et al., 2011). Furthermore, contamination with microorganisms via aerosol from respiration plays an important role in transmission of microorganisms between humans and animals (Klous et al., 2016).

In Africa 10 tick genera are well represented in their role of infestation of domestic animals. Seven of the total are ixodids having more than 80 species occurring in South Africa and the remaining three genera are Argasids (Sirigireddy, 2008). Different species have different preference when it comes to geographic distribution and seasonal

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occurrence. All the hard ticks attach to their hosts for much longer periods and they feed slowly, depending on the developmental stage of the tick (larvae, nymph, and adult). Soft ticks of genera Argas and Ornithodoros attach to their host for a short period of time and their common habitat are in the nest or host housing. Ticks of the genus Otobius attaches to host during stages of larvae, nymph and ear canal is their main habitat on the hosts (Horak et al., 2002; Walker et al., 2007; Walker et al., 2003).

Ticks have over many years been regarded as vectors and agents of various diseases that affect livestock and wildlife. These ticks as well as tick-borne diseases occur within specific geographic areas and the range continuously expands and spread intercontinentally (Walker et al., 2003). Regardless of the fact that domestic animals are of preference as common host for ticks to feed on, most tick species occur on wildlife, for completion of the lifecycle, without small wild mammals or birds as host for immature stages most ticks will not be able to moult into adult stage (Horak et al., 2002). During this exchange of feeding on hosts and development, this is where the tick-borne pathogens transmission occurs. As a result, these occurrences pose a high risk for livestock farming and development (Walker et al., 2003).

Ticks have become the most significant concern in medical and veterinary research due to their direct effect on their host including injury at attachment point, blood loss, and paralysis by toxins in tick saliva as well as being efficient vectors of various microorganisms (Walker et al., 2000; Latif, 2013). According to Asokan and Asokan (2016), zoonoses that infect human beings constitute more than 60% of all known infectious diseases; 75% of emerging infectious diseases; 40% is comprised of fungi, 50% bacteria, 80% viruses and 70% Protozoa. Out of 400 known emerging pathogens only 100 occurs in humans (Asokan and Asokan, 2016).

Viruses, bacteria and protozoa are some of the pathogens known to humans transmitted by the tick. With new globalization, change in transportation, animal husbandry and urbanization; these factors are thought to be the cause of re-emerging tick-borne diseases along with other vector-borne diseases (Chitanga et al., 2014). Hotez and

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Kamath (2009) suggested that although tick-borne diseases are known globally but in sub-Saharan Africa there is still insufficient information on these pathogens which is a major factor that contributes towards lack of information on impact assessment on the tick-borne disease.

The inability to control tick-borne pathogens contributes to major challenges such as limitation of livestock production. In return, this burdens the economic stability globally results in loss of billions of dollars annually due to tick infestation and tick-borne pathogens.

For purpose of this dissertation ticks which are commonly referred to as Boophilus

decoloratus or Rhipicephalus (B.) decoloratus and Boophilus microplus of Rhipicephalus (B.) microplus (Beati and Keirans, 2001; Murrell et al., 2000) are referred to as Rhipicephalus decoloratus and R. microplus in the entire document (Horak et al., 2002;

Guglielmone et al., 2010; Spickett et al., 2011).

3.1.1 Seasonal occurrence

Most arthropods exhibit photoperiodically controlled life cycle in synchronizing their developmental stages with climatic conditions that are favorable (Madder et al., 2013). Certain ticks display semivoltine (require more than a year to complete their life cycle) examples include Ixodes rubicundus, Otobius megnini (Madder et al., 2013); univoltine (one cycle per year) example include Hyalomma rufipes, H. truncatum (Walker et al., 2003) and multivoltine (more than one cycle per year) example include Rhipicephalus

evertsi evertsi, R. decoloratus (Walker et al., 2007). Seasonality is displayed in tick life

cycles, in which most species (adult ticks) are more active and feed at the initiation of the rain. Due to the significance of economic growth, tick-borne pathogens and vector tick need further studies in order to understand their (ticks) seasonal occurrence; in return, this can be of major significance in controlling the ticks and tick-borne diseases (Walker

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3.1.2 Life cycle

All ticks need to go through four stages of development before completion of the life cycle. Tick species have four stages namely; embroyonated egg, larva, one or more nymphal stages and then adult stage (Figure 3.1. - 3.4.) (Madder et al., 2013). Each post-embryonic stage requires a blood meal. In Argasidae, the development is gradual with multiple nymphal stages before molting to adult stage and this requires multiple hosts for each stage for example Otobius megnini (Walker et al., 2003). Ixodidae cycle accelerate by having only one nymphal stage, each stage seeks a host, feeds and then drops of to develop to the next stage within a natural environment for example Amblyomma

hebraeum. For hard ticks they require three host life cycle prior to completion; in some

cases, the life cycle can be shorted when the nymph remain on the host after feeding resulting in one/two host before a life cycle is completed for example R. microplus (Walker

et al., 2007).

3.1.2.1 Hard ticks

Engorged adult female ticks detach from a host after feeding and after few days they start oviposition, whereby they lay single large batch of eggs in a sheltered spot and then they die (Madder et al., 2013). Certain period passes by (weeks/months) six-legged larvae hatch from the eggs. “Seed” or “peppers” are the common names used for larvae, as they resemble the small seed or crunched peppercorns. Some larvae of certain species climb up the stems of vegetation waiting for passing hosts that they can attached to, whereas some larvae of other species only wait on the ground and climb and attach on a host passing by. This makes it possible for a host (human or animal) to be bitten by several larvae simultaneously (Horak et al., 2002; Madder et al., 2013).

After attachment, they feed and then a period of quiescence follows while partial metamorphosis occurs within the larva skin. The tick molts into nymphal stage, few days are required for integument to harden before they feed again; they attach, feed and go through quiescence period then molt to final stage (adult), which also requires couple of days for integument to harden before they attach (Madder et al., 2013). Mating of ticks occurs on the host. Pheromones play a significant role in the behavior of ticks and in ticks

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finding a host and for mating (Parola and Raoult, 2001; Walker et al., 2003). Partially engorged adult males when attracted to the engorged adult females, migrates to where the female is and then they mate. The female detaches and drops off to the ground to lay eggs, while the male remains in the host for several months before dying (Walker et al., 2003; Madder et al., 2013).

Ticks are adapted to feed on various hosts it can be one/two/three hosts in order to complete their lifecycle. Ticks that feed only on one host for all entire developmental stages are one-host ticks for example Rhipicephalus microplus which they remain on the host during all stages and only engorged female adults, detach to lay eggs (Figure 3.1.) (Walker et al., 2003; Neto et al., 2011). Certain ticks feed on two hosts during their developmental stages and are referred to as two-host ticks (Figure 3.2.) for example R.

evertsi evertsi. These ticks feed on the first host during larval and nymphal stage only.

They drop off afterwards and attach to a different host (second host) for their final blood meal; when they are adults, the female will drop off to lay eggs after feeding and completion of mating (Madder et al., 2013). For a three-host tick (Figure 3.3), feeding takes place on different host on for each developmental stage for example Amblyomma

hebraeum. After feeding, they drop off and attach onto a new host (larva to adult). The

adult females lay batch of eggs (400 - 120 000) depending on the species (Jongejan and Uilenberg, 2004; Walker et al., 2003; Madder et al., 2013).

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Figure 3.1: One-host tick life cycle, e.g. Rhipicephalus decoloratus. Source: Walker et

al. (2003)

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3.1.2.2 Soft ticks

Tick species of Argasidae have a uniform pattern and more diverse life cycle when compared to Ixodidae species, some soft ticks seek host by questing on low-lying vegetation, whereas most are nest parasites residing in sheltered environment (caves, nest, and burrows) (Madder et al., 2013). Biochemical substrates such as carbon dioxide, heat and movement serves as a stimulant that guides host-seeking behaviour (Madder

et al., 2013). When the soft ticks feed the cuticle expands, but does not grow to

accommodate the large volume of blood meal ingested. Because of this soft ticks feed rapidly as a result females oviposit frequently depositing small egg masses <500 eggs per cycle. There are 2 - 7 nymphal stages in their life cycle (Madder et al., 2013).

Most soft tick species larvae seek host and attaches to feed for 15 – 30 minutes before dropping off to moult in sand, duff/cracks and crevices of natural habitat. The

Ornithodoros larvae do not feed and moult immediately to the next stage (nymphal). First

Figure 3.3: Three-host life cycle, e.g. Rhipicephalus appendiculatus. Source: Walker

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nymphal stages resembles those of adult with absence of dimorphism evidence and genital pore, in return they attack the host, feed rapidly and moult again to another nymphal stage as shown in Figure 3.4 (Madder et al., 2013). This cycle can be repetitive up to seven times before the last moult into adult stage. When the soft tick becomes sexually active, no blood meal is required for initiating gametogenesis. Mating occurs both times (before and after) feeding but off the host. Otobius species adult do not feed at all (Walker et al., 2003; Madder et al., 2013).

After feeding oviposition commences and completion of soft tick life cycle remains vigorous, seek new host, feed and oviposit again. The pattern of repetitive gonotropic cycles permits soft ticks to spread their progeny over time across a span of countless years. Diapause can be a factor in regulation of the time for growth of several soft tick species that survive in empty nest and borrows upon waiting for host return or new arrival (Madder et al., 2013).

Figure 3.4: Soft tick life cycle, e.g. Ornithodoros moubata; other argasid group may

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3.1.3 Economic importance of ticks

Ticks rank second to mosquitos as life threatening vectors in domestic animals and human diseases as they serve as important vectors of bacteria, viruses, some helminthes and protozoan parasites amongst others for humans and animals (Andreotti et al., 2011).

Ticks have a specialized specific diet as a meal they only feed on blood from birds, reptiles, mammals, amphibians; the feeding technique of ticks can result in a detrimental effect on the host as certain ticks secrete a cementing material to fasten themselves to the host (Jongejan and Uilenburg, 2004). According to Soneshine (1991), during the process of feeding, ticks concentrate blood nutrients in their gut allowing them clear water excess from their bodies; making use of salivary glands in pumping by-product of the blood meal into the host. This course enables them to transfer and transmit pathogens to hosts (human or animal). Infected tick needs to feed for more than two consecutive days for the transmission to occur as there is no automatic or immediate effects as transmission occur through a tick-bite only (Soneshine, 1991).

In South Africa Ambyomma hebraeum, A. marmoreum, Haemaphysalis elliptica, H.

silacea, Hyalomma marginatum rufipes, H. truncatum, Ixodes pilosus group, I. rubicundus, Rhipicephalus decoloratus, R. appendiculatus, R. evertsi evertsi, R. follies, R. gertrudae, R. glabroscutatum, R. maculates, R. muehlensi, R. sanguineus, R. simus, R. warburton, R. zambeziensis, and R. pulchellus are reported to feed on humans (Horak et al., 2002). According to Ginsberg (2008), an individual tick can be infected with more

than one pathogen; these multiple infections can be of medical significance due to the fact that co-infection can escalate several symptoms in both animals and humans. Zoonosis contributes to socio-economic problems in agro-exporting nations and can be a serious factor on exportation of animal products (Rojas, 2011).

According to Ndhlovu et al. (2009), damage caused by ticks is seen on hide, teats and scrotum, udders, blood loss, injection of toxins, tick worry and myiasis due to sites that are damaged by maggots and other secondary microbial infection. Some tick infestation

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can cause flaccid, ascending and fatal paralysis; humans bitten by the tick can have an allergic reaction or anaphylactic reaction (Sparagano et al., 1999; Hlatshwayo, 2000; Ndhlovu et al., 2009).

Several ticks are known to be associated with productivity loss, depending on the pathogen that they harbor and transmit, such effects are more apparent in commercial farming than communal farms where they are minimal (Ndhlovu et al., 2009).

According to Ndhlovu et al. (2009), economic damage threshold and economic threshold concept are essential when comparing tick and tick-borne pathogens. In Zimbabwe, there was a survey done to estimate the cost due to tick-borne disease; the cost was at US$ 5.6 million per annum (Mukhebi et al., 1999). For Angola, Botswana, Malawi, Mozambique, South Africa, Swaziland, Tanzania, and Zambia the estimate was at US$ 44.7 million due to other diseases caused by Ehrlichia ruminantium specifically transmitted by A. hebraeum and A. variegatum in southern Africa (Allsopp and McBride, 2009; Ndhlovu et al., 2009).

Certain measures need to be taken into consideration in controlling ticks such as vegetation management (burning, herbicides treatment, cutting and drainage of wet areas) to modify habitats (Stuen et al., 2013). These are most common strategies but they are short-lived and resulting in ecological damage. Acaricides can be used directly onto domestic and wild animals in eliminating all ticks attached to the host. Chemical compounds such as organophosphates can result in toxicity towards humans and animals when used to control the ticks within an environment. According to Parola and Raoult (2001), biological methods used to control ticks; includes introduction of a natural predator (spider, ants or beetles), parasites (nematodes, mites and insects) and bacterial pathogens of tick immunization of hosts against ticks and mass release sterilization in males. So far, the most effective solution is integrated pest management which includes several control measures in tandem with environmental management (Parola and Raoult, 2001; Stuen et al., 2013).

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3.2 Aim of the study

The Mountain Kingdom of Lesotho (here in referred to as Lesotho) is a landlocked mountainous country which is completely surrounded by South Africa, with borders stretching 909 km in length covering an area of 30.355 square km of land

(http://www.golesotho.co.za). Lesotho has coolest climate than other regions with same

latitude, because of its elevation. There is no scientific data published on tick species occurring in Lesotho as well as zoonotic pathogens they are harboring. In this chapter, this study has seeked to pioneer documentation of tick species infesting domestic animals in Lesotho.

This chapter was aimed at recording tick species that infest domestic animals (cattle, goats, sheep, horses and dogs) in various districts of Lesotho.

3.3 Materials and Methods 3.3.1 Study area

Lesotho is a country known for its clear crystal water which nourishes green pastures for domestic animals and the only country globally that is entirely more than 1000 m above sea level (www.golesotho.co.za). Lesotho has a total of 10 districts (Figure 3.5). Ticks were collected from domestic animals i.e. horses, goats, cattle, sheep and dogs using sterile forceps with a fine tip. Among collection sites is Butha-Buthe district known as and the only town within this district; Mafeteng district (known as the place of the passers-by) is located 76 km south and the town is named after Emile Roland, nicknamed as “Lefeta” meaning a traveller; Maseru district which includes the capital city which is situated near the Caledon River; Leribe district, which is known for the gun tower, crafts and dinosaur prints and Qacha’s Nek district which is situated beneath the Letloepe hill/rock formation, the only home town for snake park.

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3.3.2 Tick collection

Ticks identified in this study were collected from sheep, goats, horses and cattle from communal and commercial farmers in Butha-Buthe, Mafeteng, Maseru, Leribe and Qacha’s Nek. These sampled districts are represented by star in Figure 3.5. Ticks were picked up from different body parts depending on the area of abundance and where visible such as ears, head, neck, abdominal areas and perineum.

3.4 Morphological identification of ticks

Ticks were identified using published identification guides, namely, “Ticks of Domestic Animals in Africa: A Guide to Identification of Species” by Walker et al. (2007); “Ticks: Tick identification” by Madder et al. (2013) and “Illustration guide to identification of African tick species” by Latif (2013). The main features of distinguishing tick species are

Figure 3.5: Map of Lesotho with 10 districts. Source:

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shown in Table 3.1. In addition, reliable internet sources were used for further confirmation of identified species (http://www.afrivip.org/education/arthropod-vectors). Ticks were submitted to the tick museum of Agricultural Research Council (ARC) - Onderstepoort Veterinary Research Institute where identification was confirmed and voucher specimen were issued (Appendix I).

Table 3.1: Morphological features used in identification of ticks collected in various

districts of Lesotho

Tick species Ticks features

Haemaphysalis elliptica Eyes are absent and festoon present. Small in size and yellow-brown in color; the mouthparts form a distinctive conical shape; lateral extension is large. Coxae 1-3 spurs are medium.

Hyalomma rufipes Males are large with, dark-brown bodies and large mouthparts. The scapular groove is steep, scutum is evenly rounded, dark in color and punctation size is small, distribution is dense. Eyes are oval. The legs are red and white banded. Body shape is more elongated. Females have wide shield of genital aperture with broad posterior margin.

Hyalomma truncatum Punctation size is small on the scutum and the distribution localized. The scutum is dark in color and the eyes are round in shape. In female the genital aperture is distinctive transversely elongated and oval, with a U- shape on the posteriorly side. Males have distinct groove extending to the eyes.

Otobius megnini There is no distinctive margin between ventral and dorsal sides of the body. The conscutum and scutum are absent. The nymph integuments have short rigid spines covering the body. Eyes are absent and mouthparts are on the ventral view (anterior on the first nymphal stage).

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3.5 Molecular identification of ticks 3.5.1 Isolation of ticks DNA

All the ticks were morphologically identified and grouped according to species and host they were collected from. Genomic DNA was extracted from 2054 ticks resulting in a total of 247 pooled DNA samples from five Lesotho districts. Prior to DNA extraction, tick specimens were removed from 70% ethanol containers and were transferred into new empty 1.5 ml tubes and left to dry-out on the bench. A pool of five individual specimens (adults, nymphae and larvae) for each species were dissected and crushed into sterile 1.5 ml tubes. DNA was isolated from the ticks (adult, nymphae, larva and eggs) using salting-out method described by Nasiri et al. (2005) which was conducted as follows.

Rhipicephalus decoloratus Males have distinctive cornua; in adanal, spurs on ventral plates are distinct and visible in dorsal view. Appendages are narrow in fed specimens. Females have hypostome teeth 3+3 (column), coxae 1 spur is distinct and present in 2 and 3 coxae.

Rhipicephalus microplus Hypostome teeth in female are 4+4 column, with coxae 1 spur distinct. The general aperture lips shape is U. In males the ventral plates spur is distinct and not visible in dorsal view. appendages present.

Rhipicephalus evertsi evertsi Medium in size, with pale orange legs uniform over each segment. Scutum margin are sinuous. The scutum is dark in color and eyes are convex. The scapular groove is shallow with medium punctations. Caudal appendage is absent in fed specimens.

Rhipicephalus glabroscutatum Cervical field is large and straight, the scutum color is brown and slightly convex. Palps are blunt and round. Genital appearance is smooth and shiny. Marginal grooves and posterior grooves are distinct and shallow. Eyes are beady, orbited. Coxae 1 are large.

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Eppendorf tubes (1.5 ml) containing the crushed ticks were filled with 410 µl of DNA extraction buffer [(10 mM Tris-HCl, pH8.0), 10 mM EDTA and 1% sodium dodecyl sulphate (SDS)]. Eighty microliters of 10% SDS was added along with 5 µl of proteinase K (Pro-K). Samples were incubated for an hour at 55°C for DNA lysis and digestion after finger vortex of the contents. After an hour, incubation period 5 µl of Pro-K was added and the samples were incubated overnight at the same temperature of 55°C to complete digestion. On the next day, the samples were centrifuged at 12000 rpm for 5 minutes; the supernatant was transferred to the second batch of sterile 1.5 ml Eppendorf, adding 500 µl of the supernatant to each tube. Hundred and eighty microliters of 5M NaCl was added to each tube containing the supernatant content and the mixture was vortexed vigorously for 30 seconds followed by centrifugation at a full speed (15 000 rpm) for 5 minutes. The supernatant was transferred to the third final 1.5 ml Eppendorf tubes and ice-cold 420 µl isopropanol (Propan-2-ol) was added to each tube and the content was mixed by gently inverting the tubes 5 times. The content was centrifuged at full speed for 15 minutes at 4°C to precipitate the extracted DNA. The supernatant was discarded and the isopropanol was removed by swirling the tube slowly. Finally, the pellet was washed with 70% ethanol and finger vortex and then spun for 5 minutes; this final step was repeated twice. Samples were left open to air dry on the bench for an hour at room temperature, covered with lint-free paper towel and the DNA pellet was dissolved in 200 µl of double distilled water (DDW). A Nano drop spectrophotometer (Thermo Fischer, USA) was used to confirm the presence of the DNA before storage at -20°C until used.

3.5.2 PCR amplification of tick DNA

PCR was conducted using a total volume of 25 µl, containing 12.5 µl of Amplitaq Gold® 360 Master Mix (Applied Biosystem, USA), 1.5 µl of each primer (10 µM of each primer), using the primers (10 µM each) for amplification of 710 bp CO1 and 780 bp of 18S rRNA genes (Table 3.2), 2 µl of template DNA and 7.5 µl of double distilled water (DDW) to adjust the volume. Haemaphysalis longicornis DNA obtained from tick colony of the National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine Japan was used as a positive control and DDW was used as a negative control.

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PCR conditions for CO1 primers were: activation at 94ºC for 5 minutes, followed by 36 cycles at 94ºC for 30 seconds, annealing at 47ºC for 50 seconds, extension at 72ºC for 2 minutes and final extension at 72ºC for 10 minutes.

For 18S rRNA the PCR conditions were: activation at 95ºC for 10 minutes, followed by 35 cycles at 95ºC for 30 seconds, annealing at 52ºC for 30 seconds, extension at 72ºC for 1 minutes and final extension at 72ºC for 7 minutes.

It is important to note that the DNA used for molecular analysis was from Butha-Buthe, Mafeteng and few samples from Maseru and Leribe, for all PCR experiments. Most tick samples from Maseru, Leribe and few for Qacha’s Nek were kept in glyceral during collection by the veterinarians, which resulted in poor DNA quality which could not be used for PCR.

Table 3.2: Primer sequences used to amplify tick species DNA from Lesotho districts

Pathogen Primers Nucleotide sequence Reference

Ticks CO1F CO1R LCO1490: 5'-GGTCAACAAATCATAAAGATATTGG-3' HCO2198: 5'-TAAACTTCAGGGTGAC CAAAAAATCA-3' Folmer et al. (1994) 18S rRNA F 18S rRNA R 18S-F: 5'-CATTAAATCAGTTATGGTTCC-3' 18S-R: 5'-CGCCGCAATACGAATGC-3' Lv et al. (2014)

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3.5.3 Agarose gel electrophoresis

The PCR amplification for all the samples were confirmed by gel electrophoresis, using 1.5% agarose prepared with 1x TAE buffer (40 mM Tris, 20 mM Acetic acid, 1mM EDTA, at pH 8.0). Five microliter of PCR product and 1 µl of 6x Purple loading dye (BioLabs, New England) were mixed and loaded into the wells. A volume of 5 µl of 100 bp molecular weight marker (GeneRuler Thermo Scientific, South Africa) was used for conforming the amplified product size. The gel was stained with ethidium bromide for visualization under UV light. Gel electrophoresis was performed at 100V for 30 minutes using a mini-sub cell GT electrophoresis system (Bio-Rad, UK). Gel images were captured using ENDURO™ GDS image system (Labnet International, Inc., US).

3.5.4 Purification of PCR products and sequencing

PCR amplicons of tick DNA were purified using QIAquick Gel Purification Kit following manufactures protocol (Qiagen, USA). The positive PCR amplicons were cut out from 1.5% agarose gel and placed into 1.5 ml eppendorf tubes. Following the manufacturer’s protocol, 600 µl of QG buffer was added on the 1.5 ml tubes and incubated at 50ºC for 10 minutes, vortexing the tubes in-between 2 - 3 minutes during incubation period. The gel was dissolved completely with a yellow colour mixture. A total volume of 300 µl of isopropanol was added on the mixture to increase DNA yield fragments. The mixture was then transferred by pouring into QIAquick spin column with 2 ml collection tubes and centrifuged at maximum speed (15 000 rpm) for 1 minute. The supernatant was discarded. To remove all traces of agarose gel, 500 µl of QG buffer was added and the column were spun at maximum speed for 1 minute. A volume of 750 µl PE buffer was added and the column was left to stand on the bench for 5 minutes, and then centrifuged for a minute. The supernatant was discarded and spun for additional 1 minute at 13 000 rpm to make sure there were no traces of ethanol from the PE buffer. QIAquick column were placed into clean 1.5 ml eppendorf tubes and 50 µl of EB buffer was added to eluted the DNA and centrifuge at 13 000 rpm for 1 minute. The presence of elute DNA was confirmed by gel electrophoresis. Purified DNA samples were sent to Inqaba Biotechnological Industries Pty Ltd (R.S.A) for sequencing.

Ticks and tick-borne zoonotic pathogens of domestic animals in Lesotho