As hematophagous ectoparasites, ticks can transmit a variety of zoonoses (1, 2). Babesia, Theileria, Anaplasma, and Ehrlichia are tick-borne pathogens (TBPs) that infect a variety of reservoir animals, including domestic animals (e.g., cattle, sheep, and horses) and wildlife. Babesia and Theileria, belonging to the order of Piroplasmida, can cause babesiosis and theileriosis in animals and occasionally in humans. To date, more than 50 species piroplasmida in domestic and wild species have been reported (3–7). Anaplasma and Ehrlichia belong to the order of Anaplasmataceae, with at least eight validated Anaplasma species and eight identified Ehrlichia species.

Kazakhstan, which covers 2,724,900 km² in Central Asia, is listed as the ninth largest country in the world. TBPs play a vital role in veterinary medicine and public health. Some TBPs, such as Crimean–Congo hemorrhagic fever virus, spotted fever rickettsia, and tick-borne encephalitis virus, have already been reported in Kazakhstan (8–10). However, there have been few reports on the prevalence of piroplasms, Anaplasma, and Ehrlichia in Kazakhstan. In the present study, we aimed to detect Babesia, Theileria, Anaplasma, and Ehrlichia in ticks parasitizing cattle, horses, sheep, pet dogs, and hens in the east, south, and west regions of Kazakhstan.

Five tick species belonging to three genera were identified from 272 representative ticks, namely Hyalomma scupense (n = 126), Hyalomma asiaticum (n = 34), Hyalomma anatolicum (n = 75), Rhipicephalus turanicus (n = 24), and Argas persicus (n = 13). A total of 11 TBPs were detected: Theileria orientalis, Theileria equi, Theileria ovis, Theileria annulata, Babesia occultans, Babesia caballi, Anaplasma phagocytophilum, Anaplasma ovis, and three phylogeny-independent Ehrlichia spp. (shown in Figures 1, 2 and Table 1). The information on the pathogens’ sequence similarities and their geographical distribution in this study is presented in Appendix Table 2.

Furthermore, T. orientalis genotype 1 (Chitose) (PQ056491), T. equi genotype A (PQ056492, PQ056499), and B. caballi genotype B (PQ056500) were confirmed (Figure 3), which were clustered with those from Australia (AB520953), the United States (JX177671), and South Africa (Z15104), respectively.

Kazakhstan is located in Central Asia, bordered by countries such as China, Russia, Kyrgyzstan, and Turkmenistan. International trade of domestic animals and their products is common. In this study, two Babesia species, four Theileria species, two Anaplasma species, and three independent Ehrlichia species were molecularly identified in hard ticks collected in six oblasts of southern and western Kazakhstan. Piroplasms, Anaplasma, and Ehrlichia are tick-borne pathogens of economically and medically important diseases (16–18). Domestic and wild animals play the roles of reservoirs, carriers, and disseminators in the epidemiology of many tick-borne pathogens. When vertebrates become infected, they may develop babesiosis, theileriosis, anaplasmosis, and ehrlichiosis (18–20). These diseases restrict livestock production and even impact public health in developing countries, including Kazakhstan.

In summary, the prevalence rate of both B. occultans and B. caballi in Hy. asiaticum stands at 2.93%. In Hy. scupense, the prevalence rate for T. orientalis and T. annulata is 0.79%, whereas that for T. equi and Ehrlichia spp. is 1.59%. In Rhipicephalus turanicus, the prevalence rate for both A. phagocytophilum and Ehrlichia spp. is 4.17%. In Hy. anatolicum, the prevalence rate for A. phagocytophilum and A. ovis is 1.33%, with T. annulata having a prevalence rate of 8.00%. And in Argas persicus, the prevalence rate for A. phagocytophilum is 7.69%. Furthermore, genotypes 1 (Chitose) of T. orientalis, genotype A of T. equi, and genotype B of B. caballi were confirmed.

Previously, T. annulata and B. caballi were detected in hard ticks in Turkistan oblast (South Kazakhstan) (5). T. annulata, T. orientalis, B. bigemina, B. major, and B. occultans were detected in bovine blood from Turkistan and Jambyl oblasts, and genotypes 1 (Chitose) and 3 (Buffeli) of T. orientalis were further confirmed (21). B. caballi, T. annulata, T. equi, B. occultans, and T. ovis were detected in hard ticks in Almaty and Turkistan oblasts, and T. equi genotype E and B. caballi genotype A were also confirmed (22). In this study, Babesia species (B. caballi genotype B) and two Theileria species (T. orientalis genotype 1 [Chitose] and T. equi genotype A) were found in Kyzylorda and Jetysu oblasts (southern and eastern Kazakhstan) for the first time. These findings indicate more genetic diversity among piroplasms in Kazakhstan.

Only three Anaplasma species were previously reported in bovine blood samples in Kazakhstan, namely A. ovis in Turkistan oblast, A. marginale in Kyzylorda oblast, and A. centrale in North Kazakhstan oblast (GenBank accession nos.: PQ133423, PQ038050, and PQ038051). In 2015, our team detected A. phagocytophilum in Hy. asiaticum ticks in Almaty oblast (KU723458). Here, A. phagocytophilum was first screened out in Turkistan and Kyzylorda oblasts. In Kazakhstan’s neighboring countries, A. ovis strains were detected in Hy. marginatum, Rh. turanicus, and Dermacentor spp. ticks in Kyrgyzstan and clustered with those in China (MG869525) (23). A. ovis strains in Rh. turanicus and Hy. anatolicum ticks were detected in China and clustered with those in Tunisia (KY659323), Pakistan (MT311202), Italy (GQ130291), and Turkey (OQ167969) (24). In this study, A. ovis was detected in Hy. anatolicum in Jambyl oblast, and it showed an independent clade, although it is comparatively close to those found in sheep blood in Mongolia (LC194134) and China (JN400673). A. phagocytophilum was commonly detected in hard ticks. Meanwhile, it was rarely found in soft ticks, including A. lahorensis, A. japonicus, and A. persicus in China (GenBank accession nos.: MG668811, MN795629, and ON807566). Here, A. phagocytophilum strains were detected in A. persicus and hard ticks (e.g., Hy. anatolicum, Hy. scupense, and Rh. turanicus), and they showed high genetic diversity, especially in the 74–84 bp fragment with U02528 as the original sequence for comparison (Appendix Table 3). To date, 14 genotypes have been reported in A. phagocytophilum. Given the lack of data on Anaplasma in Central Asia, more investigation on Anaplasma should be done in the future.

To date, Ehrlichia includes eight validated species, such as E. chaffeensis, E. ewingii, and E. canis, along with numerous indeterminate species reported. Previously, multiple indeterminate Ehrlichia strains were detected in Amblyomma longirostre, Am. cajennense, Am. romitii, Rh. microplus, and Rh. pusillus ticks (25, 26). In the present study, three phylogeny-independent Ehrlichia strains were detected. One strain originated from pet dog ticks (Rh. turanicus) from a veterinary clinical hospital in Almaty oblast, the second one was from cattle ticks (Hy. scupense) in Turkistan oblast, and the third one was from horse ticks (Hy. scupense) in Aktobe oblast. The discovery and distribution of Ehrlichia species are closely related to their natural hosts and geographical locations. Expanding the sampling to include more tick species, domestic animals, wildlife, and additional sites will be important for future investigations of Ehrlichia species in Central Asia.

Interestingly, we detected A. ovis, B. caballi, and T. equi in ticks collected from cattle, despite the fact that A. ovis is generally considered to be primarily detected in sheep (27, 28), while B. caballi and T. equi are typically found in horses or equines (29, 30). Domestic animals such as cattle, horses, sheep, and camels are natural hosts for species such as Hy. asiaticum, Hy. scupense, Hy. anatolicum, and Rh. turanicus (31, 32). Occasionally, these animals may also host Argas persicus (23). According to reports, ticks infected with T. equi and A. ovis are unable to directly transmit the pathogens to the offspring, while ticks infected with B. caballi can directly pass it on to the next tick generation (6, 33, 34). Moreover, A. ovis does not have strict host specificity and has been detected in cattle in addition to sheep and goats (35–37), which is consistent with our findings. In this study, T. equi and A. ovis were detected in ticks collected from cattle. This could be due to the ticks migrating to cattle after feeding on infected animals. As for B. caballi, it may be carried by the ticks or the cattle themselves. Regarding these findings, future research may delve deeper into aspects such as expanding the range of hosts for tick – borne diseases, the interaction between hosts and pathogens, and the migration of tick vectors and hosts. Therefore, we speculate that cross-species transmission may have occurred as ticks fed on the blood of different hosts. Such cross-species transmission has the potential to cause unknown diseases or symptoms in new hosts, posing a potential threat to public health and animal welfare. Furthermore, these finding underscores the need for further research into the host range and transmission patterns of these pathogens to better understand their distribution and epidemiology in nature.

In this study, although we could not determine whether these TBPs originated from the engorged ticks or their hosts, we still believe that multiple piroplasms, Anaplasmas, and Ehrlichia exist in Kazakhstan. Due to the lack of more data in Kazakhstan and its neighboring countries (especially in Central Asian countries), the taxonomy of TBPs at the level of species and genotype needs further research.

Two species of Babesia (B. occultans and B. caballi), four species of Theileria (T. annulata, T. ovis, T. equi, and T. orientalis), two species of Anaplasma (A. phagocytophilum and A. ovis), and three phylogeny-independent Ehrlichia species were detected in 259 hard ticks and 13 soft ticks in six oblasts in Kazakhstan. The genotype 1 (Chitose) of T. orientalis, genotype B of B. caballi, and genotype A of T. equi were further confirmed. These findings expand the geographical distribution and knowledge of TBPs in Central Asia, especially in Kazakhstan.