We have comprehensively described piroplasm parasite community in wildlife and cattle population of the greater Kafue ecosystem by a meta-barcoding strategy using illumina MiSeq sequencing platform and ASV based bioinformatics pipeline. This approach has an advantage of generating high resolution data which enables discriminating sequences down to a single nucleotide difference(41,42). This is ideal to reveal cryptic variation and diversity in parasite community in a population which is ordinarily masked when looked at genus or species level(30). The robustness of RLB-NGS (illumina MiSeq) approach in piroplasm investigations is well demonstrated in previous studies done on laboratory isolates, trial livestock and captive wild/domestic buffalos(30,43). Here we present the use of this method in different species of wild populations in the national park and in the wildlife-livestock interface area. A revealed parasite community of 45 ASVs and 23 piroplasm species consisting 4 genera (Babesia, Theileria, Hepatozoon and Colpodella) were identified in wildlife and cattle population from the study area.
The study identified 16 sequences of Theileria species, subspecies and variants (Table 4). As an important natural reservoir host, buffalo had the highest diversity of ten theileria species or sub-species infection compared to other wildlife species. Importantly, three genotypes of T. parva (OTU23 comprising ASV15, 86 and 101) were obtained from buffalo, providing important epidemiological data for cattle in the area. This finding is consistent with a previous report from a serological study involving buffalos (15). Indeed, T. parva ASV15 was detected in cattle (Na032), suggesting possible spillover of T. parva from wildlife to domestic animals.
The presence of non-pathogenic T. sp. (buffalo) (26 of 53; 49.1%) and T. sp. (bougasvlei) (10 of 53; 18.9%) in buffalo (Table 4) is of diagnostic importance as it affects the accurate detection of T. parva in mixed infections when performing hybridization PCR assay diagnosis(44). In addition to buffalo, this study found T. sp (buffalo) circulating in cattle population (1 of 230; 0.4%). This finding deviates from previous studies in southern Africa which classified T. sp (buffalo) as a buffalo specific parasite(22,45). In fact, other studies conducted in Kenya also identified T. sp (buffalo) from cattle, suggesting that T. sp (buffalo) infection in cattle occurs in the field where buffalo and cattle share pasture(46,47). T. sp (buffalo) is considered to transiently infect cattle and no carrier state is proved, same as Corridor disease of buffalo-derived T. parva infection. Nevertheless, more knowledge on the infection epidemiology and pathogenicity to cattle will be required. Further, the presence of T. taurotragi circulating in cattle population is consistent with findings in other similar studies(25) .The characterization of parasite community and molecular ecology raises awareness on the consequences and limitations of specific diagnostic tests and further cautions the interpretation of the results used for diagnostics or surveillance in a specified area.
Babesia was predominantly observed in cattle but also detected in Wild dogs. Babesia bigemina (31 of 230; 13.5%) and B. occultans (4 of 230; 1.7%) were the only species detected in cattle (Table 4), of which B. bigemina is a pathogenic parasite to cattle causing a clinical disorder of babesiosis, also known as Redwater. These findings are similar to other comparable studies in southern Africa where the presence of Babesia in cattle and wild animals particularly buffalo was assessed(45). To the best of our knowledge, this is the first report of the non-pathogenic B. ocultans in Zambia. However, its specific vectors, impact on cattle, diagnostic consequence in Babesia mixed infection or implication of infection to wildlife are not evaluated.
Despite not being classified in the order of piroplasm but Euccidiidae, Apicomplexan species of Hepatozoon canis and Hepatozoon sp. were detected in African lion and wild dog samples, respectively. Divergent to other arthropod-borne parasites transmitted through the vector`s salivary glands at the time of feeding, Hepatozoon are transmitted to the canid host exclusively by ingestion of infected vectors (ticks) during grooming(48,49). They cause subclinical infection in wild canids while domestic dogs yield to clinical infection(50). Previous studies on free ranging wild canids in Zambia have indicated the widespread presence of Hepatozoon sp. in lions(51). This finding highlights the considered epidemiologic role of wild canids as sylvatic reservoir of canine Hepatozoon and presents the risks of likely spillover of Hepatozoon infections to domestic canids in the interface area.
Genera of Colpodella are part of apicomplexan organisms that are originally known to be free-living. However, recent studies have revealed the parasitic nature of Colpodella sp. as a Human Erythrocyte Parasite (HEP) that has lately been reported to cause relapsing fever and neurological symptoms in human(52,53). Furthermore, the detection of Colpodella sp. in ticks suggests that this parasite may potentially be transmitted by a tick vector(52). We detected a Colpodella sequence from one of the cattle samples, with the sequence similarity of 79.6% with the reported human cases (GQ411073; Colpodella sp. HEP). The sequence detected from our cattle sample showed perfect match (100% identity) to GenBank MN103986 (Colpodellidae clone PL31), reported in raccoon dog in Poland(54). Thus, the detection of Colpodella sp. from cattle sample implies to support those findings that some of the Colpodella species are associated with vertebrates, and possibly cause disease. It is largely undetermined what vector is involved, how the parasite is maintained, and the risk that the cattle may pose for human infection. It would be important to determine the zoonotic scale of Colpodella infection to rule out incidental infections.
The study has revealed the piroplasms parasite community circulating in wildlife and cattle population but also highlighted the parasites that are present in both wildlife and cattle which may signify the possible overspill of wildlife parasites into cattle population in common instances of sharing common resource niche of pasture and water at the wildlife-livestock interface. This is particularly important since Zambia`s cattle population stronghold is in the Itezhi Tezhi district which is adjacent to the KNP. This is cardinal as accurate diagnosis and effective control (vaccinations) of piroplasms need to take the parasite community data into account.
The growth of the game ranching in Zambia has steered an increased large scale movement of seed stock of wildlife from national parks and GMAs to game ranches dispersed throughout the country. Conversely, restocking programs of depleted conservation areas with wildlife from national parks with profuse numbers to restore ecological functions of these areas in line with commitment of the global theme of decade of ecosystem restoration(55). Despite the obvious conservation benefit of these programs there is a potential risk of spread of endemic piroplasms and its vectors into expanded new areas to the detriment of livestock production in the absence of deliberate and specific strategy of accurate diagnosis and wildlife movement controls(56)
The fine scale results of metagenomic analysis of parasite communities has applications in the assessment and development of area specific vaccines candidates. This implies the vaccination programs conforming to local strains and not vaccinations based on cross reacting strains. This prevents introduction of foreign strains to the area from vaccination with exotic cross reacting strains.
Molecular epidemiology based on the strength of knowledge of cryptic parasite community and diversity is essential in identifying and tracing source of infection or outbreaks. Mapping of these parasites in all major livestock landscapes beyond the Kafue ecosystem using metagenomic approach may benefit the piroplasm control in Zambia through high resolution data to precisely guide diagnosis, vaccination and movement controls