2.3 Tick collection and tick PCR for detection of T. equiand B. caballi
Ticks were collected from the areas surrounding the farms that were confirmed to be positive for T. equi. BioQuip’s tick drag was used to monitor ticks. Ticks were removed from traps, and transported to the laboratory where they were stored at -70°C until species identification by microscopic morphology on a cold table using standard keys (Yamaguti N, 1971). Up to 30 specimens were analyzed by species.
RESULTS AND DISCUSSION
During 2016-2017 in ROK, one of 1,650 horses (0.06%) was confirmedT. equi antigen and antibody (Table 2 ). In addition,Theileria spp. was found in 2 of the 1,650 horses (0.12%), which showed seronegative for T. equi antibody test. In the previous study in ROK, 83 samples in Jeju, 37 in Gyeongbuk and 104 in Gyeonggi were analyzed for EP pathogens (Seo et al., 2013), and the antigen positive rate of T. equi was 0.9%. B. caballi was not detected both studies. In this study, the samples were collected at national level in Korea, which were Jeju (n = 661), Gyeongbuk (n = 114), Gyeonggi (n = 378), and the other 12 regions (n = 497) (Table 2 ). In this study, T. equi was detected in Ulsan, while T. equi was found in Gyeonggi during 2007-2010 in the previous study (Table 2 ). The positive regions between two studies was not related epidemiologically each other. Ticks are an important vector to transmit EP. It has been known that there are 15 tick species that transmit B. caballi - 7 Dermacentorspp., 6 Hyalomma spp., and 2 Rhipicephalus spp. (Wise et al., 2013). And there are 14 T. equi- transmissible tick species, which are 4 Dermacentor spp., 4 Hyalomma spp., 5Rhipicephalus spp., and Amblyomma cajennense (Wise et al., 2013). In Korea, the predominant horse tick species isHaemaphysalis longicornis (Seo et al., 2016). To date, EP-transmissible tick species have not been found in ROK (Jiang et al., 2019; Yun et al., 2014). We collected the 62 ticks in T. equi -positive horse ranch, which were 52 ticks (2 nymphs and 50 larvae) in 2016 and 10 (larvae only) in 2017. All of them were H. longicornis , in which EP pathogens were not detected. In 2010, the positive rate of T. equi was 0.9%, while in 2017, that was 0.06%. It might be implied that EP in the ROK has not spread from Gyeonggi after 2010. This result might be due to absence of the transmissible tick species of EP pathogens in ROK and no transport ofT. equi -positive horses. However, the artificial infection experiments have reported that the propagation of T. equi byH. longicornis is possible (Ikadai et al., 2007). Therefore, it is needed consecutively to examine EP pathogens in horse ticks with identification of collected horse ticks.
The antibody titer of T. equi -positive horse at the first test was 54.63 %I using T. equi antibody test by cELISA kit. The second and third test of it were performed at 4 months and 1 year later, in which T. equi -antibody titers were showed 56.79 %I and 67.33 %I, respectively. At that time, we have also tested for 5 co-breeding horses with T. equi -positive horse in Ulsan at the same time to bleed T. equi -positive horse. No antibodies of T. equi orB. caballi were found in the other 5 co-breeding horses. In other countries, seroprevalence of T. equi in horses have been various from 78.8% to 0.0% depending on its infection and sanitation status. Sudan has the highest seroprevalence at 78.8%, followed by Mongolia (72.8%), Brazil (34.8%), and China (3.8%) (Boldbaatar et al., 2005; Elata et al., 2020; Li et al., 2019; Souza et al., 2019; Xuan et al., 2002). In Japan, no T. equi antibody-positive cases were found (Huang et al., 2006). Meanwhile, the seroprevalence (0.06%) of T. equi in ROK was close to “EP-free” status. B. caballi -positive horse was not found until now, but the seroprevalence of B. caballi was also different from each country. The seroprevalence ofB. caballi was the highest in Mongolia at 40.1%, followed by Brazil (27.2%), China (20.1%), Japan (7%), and Sudan (5.1%) (Boldbaatar et al., 2005; Elata et al., 2020; Li et al., 2019; Souza et al., 2019; Xuan et al., 2002). To sustain “EP-free” status in ROK, a thorough border quarantine inspection should be conducted continuously and the monitoring of domesticated horses of EP should be performed consecutively. In addition, a preemptive monitoring system for horse ticks should be established, implementing the examination of EP pathogens.
Three of 1,650 horses (0.18%) were Babesia spp. andTheileria spp. dulex PCR-positive, which were collected from Ulsan, Gyeonggi-do, and Gyeongbuk-do (Table 1 and 2). All 3 positive samples were negative for B. caballi PCR. T. equi PCR revealed that one sample (0.06%) from Ulsan was positive (Table 2 ), in which T. equi antibody was also detected (Table 2 ). The 17H107 only identified as T. equi in this study was from a horse ranch located in Ulsan regions. This T. equi strain exhibited the highest genetic homology with two strains in ROK (HM229407, HM229408) through phylogenetic analysis of 18S rRNA gene (Fig. 1A ). The T. equi found in this study was confirmed to be the same genotype as the previously detected Korean strain (Fig. 1A ). Compared with those of other countries, 17H107 showed highly homologous with the strains found in Chinese (KF559357) and Spanish (AY534882) horse blood and the strain identified in Mongolian horse ticks (JQ657703) (Fig. 1A ). It was genetically similar to strains prevalent in China, Mongolia, and Spain (Fig. 1A ). In 2008, EP-positivity was confirmed in horses imported illegally from Mexico into USA (Short et al., 2012), indicating the importance of EP prevention through border quarantine. It is important to quarantine animals at international border for national-free infectious disease control. In addition, the phylogenetic analysis will give the molecular epidemiologic data between each country.
In this study, Theileria spp. was confirmed in 2 of 1,650 horses (Fig. 1B ). The Theileria spp. identified in this study, 1st _427 and 2nd _128, was found to be similar to the T. buffeli derived from cattle and T. luwenshuni derived from deer in the ROK. In addition, the genetic homology was high with T. luwenshuni , T. orientalis , andT. sinensis from China and T. annulata from India (Fig. 1B ). T. luwenshuni , T. orientalis ,T. sinensis , and T. annulata have never been observed to infect horses in any previous report. Furthermore, there are no reports of T. sinensis and T. annulata among 4 Theileriaspp. in Korea. Meanwhile, T. luwenshuni and T. orientalishave been reported in infected water deer and cattle in the ROK, respectively (Han et al., 2017; Park et al., 2019). Two horses confirmed with the infection of Theileria spp. showed no clinical signs. Thus, Theileria spp. identified in 2 of 1,650 horses might be non-to-mildly pathogenic Theileria spp., not T. annulata . It is needed to further identification of Theileria spp. at species level.
In conclusion, EP should be regularly monitored in horses reared within the ROK to maintain the EP-free status of the country. Furthermore, strengthening the surveillance system for this disease by examining the status of the distribution of horse ticks and pathogens in the ticks is important for proactively monitoring horse tick-borne infectious diseases, such as EP.