Following from the recent demonstrations of R. microplus in southern-eastern Kenya, this study confirmed the presence of B. bovis DNA in cattle blood from the same region. This was necessary to instigate monitoring and control measures because the economic burden of ticks and tick-borne diseases in Africa  and also globally is high . Climate change has contributed to expanded range of tick vectors increasing the risk of tick-borne diseases in Africa . Bovine babesiosis is one of the four major livestock diseases that is prevalent in Kenya [21, 26–30]. Until recently, bovine babesiosis in Kenya was presumed to be caused by B. bigemina, transmitted by R. decoloratus which is endemic in many regions in Kenya . Recent recordings of R. microplus in Kwale , indicated an urgent need for surveillance of B. bovis to update epidemiological data on bovine babesiosis.
Screening of the cattle samples with two B. bovis and one B. bigemina specific probes, we observed that B. bovis was present in 17.2% (87/506) of the animals while 13.8% (70/506) of the samples were positive for B. bigemina. Co-infections with the two Babesia spp were observed in 26 (5.1%) of the animals screened (Table 3). This is the first study to report significant occurrence of the pathogenic B. bovis in Kenya and East African region. The findings indicate that B. bovis infections do occur in significant numbers in this region but are probably disregarded in most studies screening for tick pathogens because of the belief that only the endemic B. bigemina is present.
In this study, the highest prevalence of B. bovis was observed in Vanga located within the Lunga Lunga sub-County which borders Tanzania. There is a livestock market in Tanga, a port city in Northeast Tanzania and at the Kenya border post at Lunga Lunga where cattle from Tanzania are purchased by Kenyans from neighbouring counties. There is a holding area at Lunga Lunga and another in Msambweni in Kwale along the major highway from the border for keeping recently acquired animals that are in transit to neighbouring counties. The Lunga Lunga market is frequented by Kenyan traders from as far as Tana River County while some are from Somalia. The extensive movement and transborder trade of infected and tick-infested animals is regarded as the major driving factor that is responsible for introduction of tick vectors and Babesia spp into new areas . A similar pattern of transboundary cattle trade contributing to the spread of R. microplus and possibly its associated pathogens has been reported in Central and West Africa where animal movement across national boundaries is common . The finding of high prevalence of the pathogenic B. bovis reported in this study indicates a possible threat of fatal bovine babesiosis across eastern Africa countries where most cattle would be lacking endemic stability to the disease. These observations together with the recent confirmation of occurrence of R. microplus in Kenya provide a rationale to implement urgent surveillance to determine and monitor the spread of B. bovis. Effective measures should also be instituted to control the spread of R. microplus to limit establishment of B. bovis babesiosis.
The molecular tests used have added both reliability and sensitivity to the detection of B. bovis infections in Kenya. Previously, for an active B. bovis infection, a diagnosis was confirmed by the presence of two pairs of small pear-shaped bi-lobed parasites in giemsa-stained blood smears. However, the presence of B. bigemina in a blood smear may not necessarily indicate clinical babesiosis, as symptoms can be due to resurgence of a chronic infection. In Kenya, diagnosis of redwater is confirmed by observation of the characteristic red urine and therefore microscopic examination of blood smears is seldom performed. In national diagnostic and research laboratories, detection of circulating B. bigemina antibodies is used for disease surveillance [26, 27, 31]. Molecular PCR techniques have been used to detect and differentiate Babesia parasites with high sensitivity and specificity [32–34]. Based on PCR amplification and sequencing of the B. bovis spherical body protein-4 (SBP-4) gene, Moumouni et al.  previously reported a B. bovis prevalence of 12.3% and 23.7% in Kajiado and Machakos Counties, respectively. Babesia bigemina was found to be more prevalent in Kajiado. Using the reverse line blot (RLB) assay targeting the V4 hypervariable region of Babesia rRNA, Njiiri et al.,  reported a B. bovis prevalence of 2.2 % in Busia County, Western Kenya. In this study we used a well validated Taqman probe multiplex assay based on B. bovis 18S , B. bovis cytochrome b  and B. bigemina cytochrome b  that can detect, differentiate B. bovis from B. bigemina and has the potential to quantify levels of infection. High specificities and efficiencies of above 96% were observed with these primer and probe sets (Figure 2) confirming the efficiency and usefulness of these assays in detecting, quantifying, and discriminating B. bovis infections (Figure 2, Table 2). In our study, a total of 61 animals (12.1%) were found to have B. bovis parasites only using both the 18S and cytochrome b primers while 44 animals (8.7%) had B. bigemina only. There was a weak agreement between the two B. bovis detection assays (Kappa = 0.36). In 33 blood samples where B. bovis was detected by both probes used, the 18S gene was detected in significantly lower copies compared cytochrome b (Wilcoxon matched pairs signed rank test (p <0.0001). The difference in sensitivities between the B. bovis 18S and cytochrome b primers may be due to differences in abundance of the two gene products. The B. bovis genome has been shown to contain three rRNA operons . Although information on the number of mitochondria in Babesia parasites is lacking, some apicomplexan parasites, such as Toxoplasma gondii and Plasmodium falciparum have been reported to have only a single mitochondrion per parasite . The mitochondrial DNA of Plasmodium falciparum comprises approximately 20 copies of a 6 kbp linear genome per cell [37, 38] encoding three protein coding genes including cytochrome b . Therefore, the quantities detected by different genes could reflect the gene copies per individual organisms as well as level of parasitaemia which was not determined in this study. More B. bovis positive samples were detected with the cytochrome b primers compared to the 18S indicating the higher sensitivity obtained by using this gene as compared to the nuclear-encoded 18S rRNA gene. Therefore, based on the data obtained in this study and theoretical predictions, the B. bovis cytochrome b primers would be the best target for a routine field diagnostic assay. However, it would appear from all these studies that B. bovis is well established in Kenya, while its biological vector, R. microplus, has also been confirmed to occur .
Animals that recover from babesiosis become carriers of babesia parasites for life and can develop the disease again if they undergo physiological stresses such as nutritional restriction or co-infection with another infection. Babesiosis is therefore a costly chronic animal disease in endemic areas, because of frequent resurgence, especially if the animals are experiencing stress . Factors such as animal breed, type of agro-ecological zone (AEZ) and livestock production systems are important risk factors associated with babesiosis infections . Majority of farmers in Kwale County which has an estimated 190,988 zebu cattle and 5,475 dairy crosses  practice open grazing system which has previously been shown to be significantly associated with high prevalence of B. bigemina infections in Murang’a County in Kenya . Kwale County, which is a gateway to mainland Kenya for livestock purchased from the Tanzanian border market, could be acting as focal source of B. bovis infections to the rest of the country.
Ticks and tick-borne diseases are the biggest threat to sustainable cattle production in Kenya which has close to 18 million cattle . Although this study reports high prevalence of B. bovis in three sub-Counties in Kwale, the situation in the rest of the country is unknown. The spread and clinical consequences of B. bovis infections in Kenya could add severe burdens on survival and productivity of cattle enterprises, especially because of occurrence of multiple co-infections is common, thereby intensifying the impacts of other serious infections such as East Coast fever. Therefore, the demonstration of the parasite at the prevalence revealed indicate its establishment and local transmission. It is recommended that molecular diagnostics including the qPCR used in this study be added to routine surveillance to detect and differentiate the two Babesia parasites with the aim of obtaining a complete picture of the prevailing status of bovine babesiosis in the country. Such dataset is critical in designing disease and vector control programs both locally and for neighbouring countries.