Parasites of veterinary importance from domestic animals in uMkhanyakude district of KwaZulu-Natal province

This study investigated the occurrence and phylogenetic relationship of protozoan parasites and Ehrlichia infecting domestic animals from three municipalities in uMkhanyakude district of KwaZulu-Natal province, South Africa. A total of 208 blood samples collected from clinically healthy cattle, sheep, goats and dogs from uMkhanyakude district were examined by polymerase chain reaction (PCR) assays, using either genus or species-specific primers to determine the occurrence and phylogenetic relationship of various protozoan parasites and Ehrlichia of veterinary importance. A total of 5/109 (4.6%) cattle were PCR-positive for the presence of Toxoplasma gondii, 33/109 (30.3%) for Babesia bovis, 24/109 (22.02%) for Babesia bigemina and 20/109 (18.3%) for Trypanosoma sp., while 3/10 (30%) of sheep were PCR-positive for Theileria ovis and none of the goats were positive for any of the detected pathogens. The co-infection of 4/109 (3.7%) B. bovis and B. bigemina was detected in cattle. Only Ehrlichia canis was detected in dogs with infection rate of 20/48 (41.7%). Sequences of PCR-positive isolates (B. bovis, B. bigemina, E. canis, T. ovis and T. gondii) showed that they were closely related to their relevant species from various countries. These findings have expanded our knowledge about the prevalence and phylogenetic similarity between protozoan parasites and Ehrlichia isolates of South African origin. To date, this is the first study in South Africa to detect T. gondii infections from cattle blood using PCR.


Introduction
Protozoan and ehrlichial diseases are significant constraints to the production of livestock in sub-Saharan Africa. In South Africa, around 18% of livestock mortalities are because of protozoan diseases ). These diseases have substantial impact on the country's economic security and poor communities who are dependent on livestock production, as they lead to losses of meat, wool, milk and manure (Perry & Sones 2007;Ringo et al. 2018). In sub-Saharan Africa, little information is available on their presence and distribution. Normally, protozoan parasite infection is thought to result from a complex interaction between pathogens, vectors, vertebrate host and the environment (Weny et al. 2017). Piroplasmosis, trypanosomosis, ehrlichiosis, hepatozoonosis and toxoplasmosis are among parasitic diseases that cause significant threat to the health of domestic animals. Various piroplasm species such as Babesia bovis, Babesia bigemina, Babesia ovis, Babesia motasi, Babesia rossi, Babesia vogelli, Theileria ovis, Theileria lestoquardi, Theileria seperata and Theileria parva have been described in small ruminants. These species are known to be causative agents of babesiosis and theileriosis, respectively (Ijaz et al. 2013;Mohammadi et al. 2017). In southern Africa, B. bovis and B. bigemina are two economically important species infecting cattle and have high prevalence in tropical and subtropical regions , while B. ovis is known to be highly pathogenic in sheep with a mortality ranges of 30% -50% (Ijaz et al. 2013;Ringo et al. 2018). Two species of canine Babesia, B. rossi and B. vogelli, are known to be endemic to South Africa (Matjila et al. 2004). The clinical signs of B. vogelli have not yet been estimated and this led to B. rossi being considered as the most prevalent species in South Africa as it causes severe, often fatal disease (Jacobson 2006).
The most pathogenic member Theileria, particularly in sheep, is known to be T. lestoquardi, while T. ovis is reported to be less pathogenic and usually causes subclinical infection albeit animals subjected to stress may develop significant illness (Durrani et al. 2011). On the contrary, T. seperata is regarded as non-pathogenic but can be fatal to immunocompromised animals or those that are newly introduced to endemic areas (Luo & Yin 1997;Ringo et al. 2018). Following the eradication of This study investigated the occurrence and phylogenetic relationship of protozoan parasites and Ehrlichia infecting domestic animals from three municipalities in uMkhanyakude district of KwaZulu-Natal province, South Africa. A total of 208 blood samples collected from clinically healthy cattle, sheep, goats and dogs from uMkhanyakude district were examined by polymerase chain reaction (PCR) assays, using either genus or species-specific primers to East coast fever, Corridor disease emerged as the most significant form of theileriosis in South African cattle. In areas where common grazing among cattle and infected buffalo occur and where there is an abundance of tick vector species (Rhipicephalus appendiculatus and Rhipicephalus zambeziensis), the disease still poses a serious threat (Uilenberg 1999).
Among the causal agents of chronic, debilitating, emaciating and usually fatal disease in domestic animals, Trypanosoma infections are major causative agents of alopecia, emaciation, lymphadenopathy and anaemia in domesticated animals (World Organization Of animal Health [OIE] 2013). However, the outcome of the infection varies among trypanosome species, livestock species and the virulence of the strains (Connor & Van den Bossche 2004). Trypanosoma vivax, Trypanosoma simiae, Trypanosoma uniforme, Trypanosoma brucei brucei and Trypanosoma congolense are important causative agents of animal African trypanosomosis, also known as nagana in Africa, with tsetse flies acting as biological vectors for the cyclic transmission of the disease in domesticated animals (Steverding 2008). This is attributed to their pathogenicity and effects on productivity (Trail et al. 1994;Wellde et al. 1989).
Toxoplasma gondii is a widespread global zoonotic protozoan parasite that infects a wide range of warm-blooded animals (Howe & Sibley 1995). Humans and animals acquire infection through ingestion of raw and undercooked infected meat that contains viable Toxoplasma tissue cyst or food and drink contaminated with Toxoplasma oocysts excreted from the faeces of infected felids. This makes toxoplasmosis the most important foodborne and waterborne parasitic disease (Bowie et al. 1997;Torgerson et al. 2015). Most animals infected with toxoplasmosis show no clinical manifestation of the disease, but the disease is known to be the leading cause of abortion in sheep.
Ehrlichia canis and Hepatozoon canis are causative agents of canine monocytic ehrlichiosis and canine hepatozoonosis, respectively. The main vector of both pathogens is the brown dog tick, Rhipicephalus sanguineus. Diseases caused by these pathogens occur worldwide and are among the most commonly reported diseases in dogs (Taques et al. 2016;Vieira et al. 2011). Unlike E. canis and other tick-transmitted diseases, ingestion of infected ticks by dogs is the main route of transmission of Hepatozoon rather than through the feeding of the tick on the host. However, alternative routes have been suggested and reported for both pathogens (Aguiar et al. 2007;Ewing & Panciera 2003). Both ehrlichiosis and hepatozoonosis are manifested by a variety of clinical signs that may include, among others, fever, haemophilia, bone marrow failure and death in irreversible cases (Gondim et al. 1998;Mundim et al. 1994).
It is documented that the occurrence of these pathogens hinders the development of livestock sector, which contributes about 49% of agricultural output in South Africa (Terkawi et al. 2011). Furthermore, it is currently unknown whether South African domestic dogs carry zoonotic tick-borne pathogens (TBPs). Therefore, considering dogs as pets and the significance of livestock production in the South African economic landscape, in this study, we determined the occurrence and phylogenetic relationship of parasitic protozoan parasites and Ehrlichia infecting domestic animals in north-eastern KwaZulu-Natal (KZN).

Blood samples
Blood samples were collected from healthy cattle, sheep, goats and dogs in three local municipalities, namely, Mtubatuba, Big 5 Hlabisa and UmHlabuyalingana of the uMkhanyakude district (28°01′25″9 S, 32°17′30″30 E), KZN province, South Africa ( Figure 1). A total of 208 blood samples were obtained from cattle (n = 109), sheep (n = 10), goats (n = 40) and dogs (n = 49). In these municipalities, rural communal farming is predominately practised and forms the main source of income in some households in the area. The owners of the sampled animals did not have any information about the age of the animals nor knowledge on the type of breed for goats and sheep. The cattle breed is Nguni. Sheep are not desired as domestic animals in this province because of cultural beliefs, and hence, only few were available during the sampling period.

Molecular detection of parasitic protozoa and Ehrlichia
Genomic deoxyribonucleic acid (DNA) was extracted using the salting out method adopted from Nasiri et al. (2005) with few modifications. Polymerase chain reaction was used to screen all the samples with genus or species-specific primers obtained from previous studies (Table 1). For each PCR assay, 2 µL of the extracted genomic DNA was added into a 25-µL reaction mixture containing 2.5 µL of 10× standard Taq Reaction Buffer, 0.5 µL of forward and reverse primer (10 µM), 0.5 µL of 10 mM Deoxynucleotide triphosphates (dNTPs), 0.125 µL of Taq DNA polymerase and double distilled water (DDW) to a final volume to 25 µL. The reactions were run on a proFlex thermocycler (Applied Biosystems, California, United States [US]) using the following thermocycling conditions: initial denaturation at 95 ºC for 30 seconds, followed by 35 cycles of denaturation at 95 ºC for 30 s. This was followed by annealing temperature (Table 1) for 1 minute, extension at 68 ºC for 1 min and final extension at 68 ºC for 5 min. Double distilled water was used as a negative control. Synthesised genomic DNA of T. gondii and canine Babesia referred to as g-block (Whitehead scientific-Integrated DNA Technologies, Johannesburg, South Africa) were used as positive controls for Toxoplasma and canine Babesia, respectively. The genomic DNA of T. congolense IL3000, T. b. brucei GuTat1.3 and T. theileri Japan Isolate was used as positive control for Trypanosoma species. The genomic DNA of B. bigemina South African strain, B. bovis SA strain obtained from North-West university, were used as positive control for bovine Babesia. The genomic DNA of T. parva provided by the North-West University was used as positive control for bovine Theileria. Following  the amplification, 5 µL amplicon was analysed by electrophoresis using 1% agarose gel stained with ethidium bromide and visualised under ultraviolet (UV) light. For nested PCR, 1 µL of the primary PCR products was added into a second PCR mixture containing the same reagent composition as described above, except that the nested PCR primers were used instead of the external primers. Reaction mixtures were run as described above.

Sequence alignment and phylogenetic analysis
The PCR-generated fragments were sent to Inqaba Biotechnical Industries (Applied Biosystem, Johannesburg) for purification and direct sequencing in both directions. One to three individually amplified DNA fragments of each selected sample were sequenced. The obtained sequences were compared with similar sequences of the same pathogens from other regions of the world in GenBank. Deoxyribonucleic acid sequences were edited, aligned with Clustal W and visually checked in MEGA 7.0. The genetic distance (p-distance) of the sequences between taxa was also calculated using MEGA version 7.0. Phylogenetic analysis was performed using maximum likelihood method with 1000 bootstrap replicates to estimate the robustness of individual branches ).

Statistical analysis
The proportions for 95% confidence intervals (95% CIs) were computed as CIs for proportions with binomial data using no continuity correction ). This was calculated

Ethical considerations
This study was approved by the Scientific committee of the Integrated Pest Management of North-West University as a no risk study (project number NWU-IPM-2017-003).

Comparative analysis
The BLASTn analysis of the partial sequence of RAP-1 genomic region ( (Figure 3-A1).

Phylogenetic analysis
Retrieved sequences from the amplification of the Rap-1 gene for detection of B. bovis were deposited to GenBank under the accession numbers MN683992 and MN683993. Subsequently, the maximum likelihood tree revealed three major clades with high bootstrap support values as well as divergence estimates (99% identical, p = 0.034) with an average distance of p = 0.053 ( Figure 2 and Table 4). Sequences generated from this study (at 99% bootstrap support) formed a sister clade with the South Africa, China and Brazil sequences at 80% bootstrap. When constructing the phylogenetic tree, the two T. ovis 18S rRNA sequences from this study (MK643269 and MK643268) clustered into a single clade with T. ovis sequences from various countries including South Africa with 100% identity and average divergence between the species was p = 0.022 (Figure 3 and Table 5).

Discussion
Protozoan and ehrlichial diseases are veterinary, medically and economically important contagious diseases affecting the domestic animals in sub-Saharan Africa, and hence, their prevalence and control is very important (Ademola & Onyiche 2013;Ringo et al. 2018). In this study, a low prevalence of protozoan parasites was observed and this could be attributed to the low sample size. It could also be because of the improvement in husbandry systems, better veterinary care and climate change.
There is no epidemiological data on livestock toxoplasmosis in this study area and, to the best of our knowledge, this is the first study that has detected T.   (7), 1870-1874.

FIGURE 2:
In phylogenetic tree analysis of Babesia bovis based on RAP-1 gene, the tree was constructed with maximum likelihood method based on the Tamura-3-parameter model, with bootstrap values (expressed as percentages of 1000 replications) superimposed at branching points. The sequences produced in this study are shown with bullet points. The evolutionary distances were computed using the p-distance method. Babesia orientalis was used as an outgroup.
in 45.45% and 1.8% of goats and sheep samples, respectively (Gebremedhin et al. 2014;Gharbi et al. 2013). There are various risk factors such as age, sex, breed and climate conditions which may have contributed to the differences in prevalence in this study and other studies across the world.
Little attention has been given to ovine piroplasmosis compared to bovine piroplasmosis despite its widespread distribution through tropical and subtropical areas. According to Berggoetz et al. (2014), theileriosis in small ruminants can be caused by a number of well-known species such as T. ovis, T. seperata and T. lestoquardi. In this study, T. ovis was the only species detected (6.0%). However, previous studies in South Africa have reported T. ovis with a higher infection rate in small ruminants, whereby Ringo et al. (2018) reported an overall infection of 19.8% and Berggoetz et al. (2014) reported an overall infection of 10.9%. Theileria ovis known to be an agent of benign ovine and caprine theileriosis which has little economic importance (Mtshali et al. 2015) was identified in 30% of sheep and none of the goats in this study. It was suggested that there are two possible reasons for the higher prevalence of TBPs in sheep as compared to goats: firstly, detection of ticks can be hampered by too much hair, which covers the sheep, resulting in persistence and low awareness of TBPs in sheep. Secondly, differences in natural resistance against TBPs among sheep and goats could influence the prevalence of the parasites (Aydin, Aktas & Dumanli 2015, Gebrekidan et al. 2014Rjeibi et al. 2014). Although the pathogen is less pathogenic, it cannot be completely neglected.
The phylogenetic tree of 18S rRNA gene sequences constructed in the present study revealed that T. ovis from this study was placed in the same clade with most of the T. ovis sequences in this tree.
The absence of ovine Babesia sp. in this study is similar to previous reports by Aktas, Altay and Dumanli (2007) and Ringo et al. (2018) who could not detect ovine Babesia sp. in Turkey and South Africa, respectively. Similar results were also reported in Tunisia where B. motasi was not detected (Rjeibi et al. 2016). B. ovis is considered to be one of the most important TBPs in small ruminants and its absence could also be an indication that the pathogen is not common in the study area. From documented literature, B. ovis has only been documented in northern African countries including Algeria and Tunisia (Aouadi et al. 2017;Rjeibi et al. 2014).
Babesia bigemina and B. bovis are the two economically significant species infecting cattle in southern Africa, and they have shown to be present in all provinces of South Africa (Bock et al. 2004;. Generally, the occurrence of both B. bigemina and B. bovis in the study area could be because of the presence and distribution of their tick vectors    Mtshali 2013). Uncontrolled movement of cattle that usually occurs within the province could also be one of the factors for the prevalence of bovine Babesia sp. in all municipalities sampled.
From the phylogenetic tree constructed, it is clear that our isolates showed a close relationship with B. bovis strains from South Africa, Brazil and China. The conservation of nucleotide diversity observed among the RAP-1 sequences has also been observed by  and Ramos et al. (2012) in South Africa and Brazil, respectively. However, the isolates from this study formed a monophyletic grouping that is very distinct from that of other published B. bovis strains. According to , this indicates the presence of micro-heterogeneities between the RAP-1 sequences within B. bovis strains. It is also important to note that the South African sequence KC894394 was obtained from samples collected in Mpumalanga province , hence the lack of 100% identity to those generated in the current study.
With regard to the analysis of SpeI-AvaI restriction fragment sequence of B. bigemina isolates, the highest nucleotide identity was 89.0%. It was recently discovered that the SpeI-AvaI nested PCR assay specific for the detection of B. bigemina DNA also amplified a homologous fragment derived from Babesia ovata (Sivakimar et al. 2012). Nevertheless, this may not be the case in the present study because the presence of B. ovata has not yet been reported in the country's cattle Yoshinari et al. 2013). To date, only five countries (Japan, Korea, China, Mongolia and Thailand) have reported the occurrence of B. ovata in cattle (Suh 1987;Sivakumar et al. 2012;Yoshinari et al. 2013).
Theileria parva is considered as the most significant theilerial species in sub-Saharan Africa and known to cause widespread morbidity and mortality in endemic areas. The absence of T. parva in the present study is comparable to results from a recent study in some parts of Nigeria where a 0,0% prevalence of the pathogen was reported (Okorafor & Nzeako 2014). However, results of this study were not comparable to a study by Yusufmia et al. (2010) who reported a T. parva prevalence of 6.7% in cattle from South Africa. However, observations of the current study were not really surprising as it is known that Corridor disease is mainly restricted to buffaloes in South Africa because of strict preventative measures of the government that aim to ensure that the parasite is not introduced to cattle (Yusufmia et al. 2010). The specimens from this study were also obtained from apparently healthy cattle. In addition, South Africa is considered free of T. parva, except in designated Corridor disease-infected areas such as the Kruger National Park and Hluhluwe-iMfolozi Park that contain various wildlife species.
Animal trypanosomiasis acts as a serious impediment to animal husbandry in all tsetse fly infested regions of sub-Saharan Africa (Nimpaye et al. 2011). In the present study, 18.3% of cattle showed the presence of Trypanosoma DNA in their blood. Uilenberg (1998), Van den Bossche et al. (2006) and Mamabolo et al. (2009) documented that tsetse fly vectors prefer cattle as their hosts as compared to other animals. The presence of Trypanosoma sp. in cattle is an indication that the cattle from uMkhanyakude district have encountered tsetse flies and its low prevalence may be attributed to the low sample size. These findings agree with a previous study by Mamabolo et al. (2009) who reported a Trypanosoma sp. prevalence of 18.4% in cattle in KZN. No Trypanosoma DNA was detected from goats, sheep and dogs, respectively. The absence of Trypanosoma in sheep and goats may be because of several factors such as the low tsetse feeding activity related to their small size and anti-feeding behaviour such as leg kicks and stamping, tail and ear flicks, head movement and skin rippling. According to Kniepert (1981), in communal grazing area, they attack cattle and leave most of the small ruminants uninfected. Canine African Trypanosomiasis (CAT) is seldomly reported (Gow, Simpson & Picozzi 2007;Keck et al. 2009 Ehrlichiosis is considered one of the most economically significant infectious diseases affecting small ruminants in tropical and subtropical regions (Ringo et al. 2018 (Penzhorn et al. 2017), or vectors, or the prevalence was too low to detect with our sample size. At present, in South Africa, B. vogelli has only been detected from the Free State and Onderstepoort Veterinary Academic Hospital, which is an indication that Babesia vogeli infection is not as widely spread as B. rossi in South Africa (Matjila et al. 2004(Matjila et al. , 2008. The absence of H. canis in this study is in agreement with studies by Criado-Fornelio et al. (2003) and Matjila et al. (2008) who could not detect this pathogen in domestic dogs from Europe and South Africa, respectively. H. canis has been reported from South African domestic dogs but only in wildlife. As H. canis is transmitted by ingestion of ticks, its absence in domestic dogs may be attributed to the fact that domestic dogs do not feed on live prey which reduce the probability of ingesting infected ticks with their prey (Baneth, Samish & Shkap 2007). The parasite also seems to be present in high numbers in reticuloendothelial cells, so blood samples are unlikely to harbour-infected cells (Conceição-Silva et al. 1988).
In conclusion, the findings of this study have expanded our knowledge on the prevalence and phylogenetic similarity between protozoan parasites and Ehrlichia isolates of South African origin. To date, this is the first study to detect T. gondii infections in cattle using conventional PCR in South Africa.