Presence and Persistence of Amblyomma Hebraeum Augments the Burden of Heartwater in the Southern District of Botswana

Heartwater is a tick-borne, haemoparasitic disease that has the potential to limit the growth of agro-businesses in Botswana. This threat to national food security is attributed to low production and mortality of livestock in subsistence farming of small stock than in commercial farms. This study gives a view of the epidemiological burden of heartwater in Botswana. METHODS Feeding ticks on livestock kept in four Southern sub-districts of Botswana were identied through morphological and molecular analysis. Farmers were interviewed on the management of the heartwater burden within their respective sub-districts. Veterinary clinical case reports were collected to assess the frequency of cases across the Southern District of Botswana. Outcomes of the study revealed; a wide distribution of Amblyomma hebraeum in all four studied sub-districts, gradual annual increase in heartwater cases in Moshupa sub-district and indication of increased impact on indigenous caprine species despite the effort to control vectors by 63% of interviewed farmers.


Background
Amblyomma hebraeum is an invasive tick vector that can contribute to devastating repercussions for the agricultural industry when both the vector and its parasite are present. This tick species acts as a vector of the economically important livestock disease commonly known as heartwater. Heartwater is caused by an intracellular bacterium, Ehrlichia ruminantium, which belongs to the Rickettsiaceae family [1]. Ehrlichia ruminantium can parasitize Amblyomma hebraeum and the vector can drive transmission of the disease in the presence of a suitable ruminant host. Heartwater is a major threat to the national food security in agro-established countries worldwide [2]. In Southern Africa, it has been reported that approximately 90% of stock losses can occur due to untreated heartwater cases alone [3]. Consequently, it is important to manage this economic livestock disease. Heartwater has been managed over the last 50 years through preventative measures such as tick eradication programs, administration of antibiotics and vaccination with a live blood vaccine. The vaccine contains live Ehrlichia ruminantium and is the only commercially available vaccine while the development of the new generations of the vaccine are explored [2], [4].
Botswana has a long history of both arable and pastoral farming. Poverty eradication programmes in Botswana fund the purchase of small stock (goats and sheep) by subsistence farmers to improve their livelihoods [4]. Movement of small stock around the country from heartwater endemic areas can lead to devastating losses of heartwater infection-prone breeds in non-endemic areas. This movement of small stock poses a threat in subsistence farming, as opposed to commercial farming, through the introduction of the heartwater disease in non-endemic areas in the absence of rigorous disease prevention measures as infected or tick-infested ruminants are transported in such areas [5]. This work reports the distribution of ticks that could potentially carry Ehrlichia ruminantium in an established heartwater endemic district

Sample period and sample collection
Sample collection was carried out during the summer seasons of 2017, 2018 and 2019. Three hundred and ninety-one small livestock ruminants (60 cows, 234 goats and 97 sheep) were randomly selected for tick collection from communal farms with no particular regard to a speci c age, sex or breed.

Tick Collection and Identi cation
Ticks were hand-picked from under the tails, around the anus, on belly or groin regions and between hooves of randomly selected livestock. Ticks were carefully transferred into labeled 2 mL tubes and kept in a labeled ziploc bag at room temperature until refrigeration at the end of the sampling day. Ziploc bags were labeled with the sub-district, site coordinates, and date of collection. The ticks collected were morphologically identi ed using a Leica M205 C microscope (Leica Microsystems Ltd, Switzerland) which had a Leica MC190HD camera connected to it. Key features used for identi cation were adopted from guidelines provided by Walker et al (2003) [6].
Tick genomic DNA extraction Ticks were incubated in 1 mL of Quick-DNA Tissue/Insect Miniprep Kit lysis buffer at 56 °C overnight. Tick tissue was dash frozen with liquid nitrogen and ground using a pestle and mortar. Tick tissue was then transferred to bashing tubes from the Quick-DNA Tissue/Insect Miniprep Kit (Zymo Research, USA). DNA extraction was then undertaken according to manufacturer's instructions. Genomic DNA was eluted using 25 µL of nuclease-free water.

Polymerase Chain Reaction
A fragment of the mitochondrial marker cytochrome c oxidase subunit I (COI) was ampli ed using PCR using universal primers LCO 1490 (5 -GGTCAACAAATCATAAAGATATT GG-3 ) and HCO 2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3) described by Folmer [7] to yield a 710 bp amplicon. Twenty ve µL PCR reaction samples were prepared in PCR vial tubes. Each reaction comprised of 12.5 µL of the Q5® High-Fidelity 2X Master Mix PCR Master Mix (Thermo Fisher Scienti c, USA), 6.5 µL of nuclease free water (VWR International LLC, USA), 1 µL each of forward and reverse primers, and 2 µL of extracted genomic DNA. PCR reaction conditions were as follows; initial denaturation at 98 °C for 30 seconds, followed by 35 cycles of; denaturation at 98 °C for 10 seconds, annealing at 48 °C for 30 seconds and extension at 72 °C for 30 seconds. This was followed by a nal extension at 72 °C for 7 minutes. The reaction was then stopped at 4 °C. The presence of amplicons in PCR products was veri ed through agarose gel electrophoresis.
Agarose gel electrophoresis PCR products were electrophoresed on 1% agarose gel at 120 V for 90 minutes. The agarose gel was stained with ethidium bromide to visualize the amplicons.
Amplicon clean-up PCR products were puri ed using the GeneJET PCR puri cation kit (Thermo Fisher Scienti c, USA), according to manufacturer's instructions.

Sequencing
Bidirectional, Sanger sequencing of the partial COI gene was performed at Inqaba Biotechnical Industries (Pretoria, South Africa).

Molecular identi cation of ticks
Contiguous sequences were analyzed using basic local alignment search tool (BLAST) to con rm matches with submissions made to the NCBI genbank.

Results
Diversity and geographical distribution of tick species A total of 310 feeding ticks collected from 27 different livestock farms during the summer seasons of 2017, 2018 and 2019 were identi ed on basis of morphological features and three tick genera namely, Amblyomma, Hyalomma and Rhipicephalus were found to parasitize livestock (cattle, sheep and goats) in the Southern District of Botswana. Out of the total ticks found, 262 ticks (84.5%) were identi ed as Amblyomma hebraeum and this tick species was found in all four sub-districts of the Southern District of Botswana (Tables 1a & 1b). The other ticks identi ed were Rhipicephalus evertsi mimeticus (11%), Rhipicephalus evertsi evertsi (1.3%) and Hyalomma truncatum (3.2%). These ticks are vectors for theileriosis, anaplasmosis and babesiosis diseases respectively.   Fig. 3, which was identi ed in all four sub-districts of the Southern District of Botswana.
Morphological landmarks that are unique to Amblyomma hebraeum are: 1) the exceptional ornate patterns that embellishes the tick scutum, 2) sensilla distributed through-out the leg, 3) protruding internal and external spur, 4) yellowish bands on the legs, 5) prominent festoons and 6) the anal groove that is present posterior to the tick anus.

Frequency of heartwater cases
The analysis of the heartwater cases collected from the veterinary service centers of four sub-districts showed the highest frequency of con rmed disease in Kanye in 2017 and Moshupa in 2018 and 2019 (Fig. 4).
Moshupa, which had the highest number of heartwater vectors (Table 1b), had the highest number of heartwater cases over the 3 year study period ( Table 2). The trend showed a gradual increase in the number of heartwater cases observed in Moshupa while there was a gradual decrease observed in Goodhope cases (Fig. 4) where the least number of vectors was recorded (see Table 1b and Table 2). Further analysis revealed that small livestock were more affected in in all sub-districts (Table 2) with caprine breeds being the most affected.

Livestock breeds affected by heartwater
The indigenous breeds of caprine and bovine species were found to be more affected than other breeds. On the other hand, crossbreeds of ovine species were cumulatively found to be more affected across subdistricts during the study period (Fig. 5).

Vector control and burden management by subsistence farmers
Having determined the types of vectors, their distribution and the burden of disease that they caused in the southern district of Botswana, we then sought to determine the disease management practices of farmers. The responses of 27 communal farmers show that vector control is practiced across the district. The popular method used by respondents is acaricide dipping (63%) compared to other control methods viz. grease application (30%) and building of a brand-new kraal (7%) (Fig. 6). This observation highlights the effort by some communal farmers in controlling vector infestations and managing disease burden.
The choice of disease management strategies by communal farmers in an effort to handle heartwater disease are presented in Fig. 7. Forty-four percent of farmers reported that they do not take any action in handling the suspected heartwater case while 15% preferred consulting with the veterinary o cer and administering antibiotics themselves.
The survey data in Fig. 8 revealed that 92.6% of farmers do not prevent heartwater through use of an available live vaccine. Furthermore, when a follow-up question was asked, 24 out of the 25 farmers (96%) who do not use the heartwater vaccine explained that they were yet to be educated on the live vaccine as a prevention method or could not afford the live vaccine.

Discussion
This study reports on the trends for con rmed heartwater cases, distribution of heartwater vectors, identi cation of heartwater susceptible breeds kept by Batswana farmers, as well as tick control measures and burden management as practiced in Southern District farms in Botswana. This study submits that the heartwater vector is present across the Southern District of Botswana as a basis of current and potential increased disease burden that could occur in the future. The approach to determining this burden was to study the association of two disease determinants, that is, the frequency of disease and its vector distribution. These determinants were selected as they rank among the most investigated drivers of vector-borne disease. Previous studies in Africa, including Botswana, have reported on the distribution of Amblyomma tick vectors [8]- [12]. However, this is the rst study to investigate the association of heartwater vector (Amblyomma hebraeum) with heartwater clinical cases in Botswana.
The current investigation demonstrated that certainly the heartwater endemicity of the area relates with geographic extension of vector species. Additionally, it is important to note that the true burden of heartwater is likely grossly underestimated by the presented clinical heartwater cases as the current diagnostic method relies on post-mortem, microscopic visualization of Ehrlichia ruminantium on Giemsa stained brain smears. All affected and at-risk animals in a single kraal or farm are therefore not included in the presented work.
The heartwater vector as well as clinical heartwater cases were predominantly associated with hardveld Districts (Good Hope, Kanye and Moshupa) than the transition sandveld-hardveld district (Jwaneng). This indicates that Amblyomma hebraeum ticks prefer humid, rocky and denser vegetation of hardvelds contrary to the dry, sandy and less dense vegetation of sandvelds [13]. The survival and the distribution of various tick species depend on these humid environments to avoid desiccation and predators. The predominantly collected heartwater vector shares similar ecological preferences with babesios, anaplasmosis and theileriosis vectors. These aforementioned vectors are reported to cause the most important tick-borne diseases in Botswana [14].
Eradication of Amblyomma hebraeum by acaricide dipping was the most practiced tick control measure used by respondents in this study across the district compared to other control tick methods. In most countries acaricide use is a government initiative [5]. This tick control method in some countries was reported to have been successful, while in some countries like South Africa, acaricide use has been unsuccessful in eradicating some tick species [15]. The exact attribution of the persistence of ticks and resultant heartwater cases across the district in the background of concerted vector control measures is yet to be investigated. The ndings however highlight the need for effective tick control measures.
The choice of livestock breeds kept by farmers within the district are shown to vary in host susceptibility to heartwater infections. In this study, heartwater disease was reported to be more frequent in both indigenous and cross breeds than in exotic breeds. This reported data on susceptibility may be attributed either to livestock trade practices that occur from non-endemic to heartwater endemic farms, pathogen transmission to ticks by carrier animals that survived heartwater infections or low immune resistance due to heat-stress.
Livestock trade of various breed types is adopted by farmers with the intention of upgrading local phenotypes that satisfy the demand by local and international consumers. For this reason, exotic livestock are moved to endemic areas in the effort of adapting indigenous breeds through the existing breed development programs in Botswana [16]. Kabi et al (2016) reviewed the effects of phenotypic modi cation through inter-breeding and noted that disease tolerant traits may be lost due to limitations to skill techniques in matching genotypes during cross breeding practices [17]. As a result, cross breed livestock may lack desired traits such as endemic disease tolerance and adaptation to high temperature in districts. This evaluation may be the reason for heartwater susceptibility of cross breed ovine species in this study. Stringent tick/disease control measures in an attempt to protect naïve exotic breeds traded into endemic areas may also put indigenous breeds at risk. Exotic breeds due to their prized status can be protected with the live vaccine and treated to recovery. In the process of this treatment, carrier exotic breeds may transmit heartwater infection to tick vectors within the endemic areas. Stringent tick control may also inadvertently select for acaricide resistant tick populations. Consequently, indigenous and cross-breeds are at a higher risk of disease transmission. Moreover, asymptomatic carriers in farms may be affected by the disease once the immune resistance is lowered due to the sensitivity to climatic changes or impacted iby environmental factors such as drought. The presented ndings on heartwater susceptibility of Botswana breeds however are acutely limited and require con rmatory work to elucidate the reasons for higher heartwater disease rates in indigenous breeds of caprine and bovine species.

Conclusion
This study nds validation in endorsing interventional approaches in averting conceivable endemic risks that can occur with growing distribution of Amblyomma hebraeum across Botswana. Education of farmers on heartwater disease prevention and management is pivotal. Furthermore, the determinants of the persistence of the vector need to be investigated. These efforts will assist in reducing the burden of heartwater and improving livestock productivity.  Study area map indicating sample sites within the heartwater-endemic Southern District, Botswana. Note:

Declarations
The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.