Molecular Detection and Phylogenetic Analysis of Anaplasma spp. in Korean Native Goats from Ulsan Metropolitan City,Korea

Introduction

A naplasma spp. are gram-negative tick-borne bacteria belonging to the order of Rickettsiales. They can cause serious infections and damage in humans, cattle, goats, and sheep (Rar and Golovljova 2011, Stuen et al. 2013, Yang et al. 2015). It was reported that Anaplasma capra was infected in 38.9% (7/38) in Korean Jeju goats. (Seong et al. 2015).

The currently recognized species of anaplasmosis are Anaplasma marginale, Anaplasma centrale, Anaplasma bovis, Anaplasma ovis, Anaplasma platys, and Anaplasma phagocytophilum. These pathogens infect various cell types. A. ovis has been most commonly reported in sheep, goats, and wild ruminants in Africa, Asia, and Europe (Inokuma et al. 2001, Battilani et al. 2017, Enkhtaivan et al. 2018). Anaplasma spp. have also been detected in Korean native goats (Seong et al. 2015, Kang et al. 2016b). Seong et al. (2015) detected A. capra in goats and Kang et al. (2016b) detected A. bovis in ticks. A. capra was found also in China and characterized as a new species (Peng et al. 2018, Yang et al. 2018).

The purpose of this study was investigating infection status of goat anaplasmosis in Ulsan Metropolitan City by monitoring the cause of anaplasmosis in goat farms and to provide the basic data on the management and prevention method of goat farms during tick activity.

Materials and Methods

To screen for anaplasmosis, 20 goat farms in Ulsan Metropolitan City were selected for blood sampling from March to November 2016. These farms could be classified based on rearing methods, such as facility-confined (confined), free-grazing (conventional), and half-confined (mixed grazing-confined) farms; 10 farms were confined, 6 were conventional, and 4 were mixed grazing-confined. Goats weighing 40–50 kg were selected for testing. Visiting periods corresponded to spring (March–May), summer (June–August), and fall (September–November). Blood samples were collected once per season (three times in total) from nine farms. The other 11 farms were only visited once or twice due to farm closure or because goats were in the gestation period. Blood samples from 10 goats were taken per farm per season except at one farm, where we took two additional blood samples on the third visit in November. A total of 452 blood samples were collected, accounting for 13% (452/3464) of the goats in Ulsan in 2016. Statistical analysis was performed by chi-squared test.

DNA was extracted from the blood samples using a Maxwell RSC Whole Blood DNA kit (Promega, Madison, WI). DNA were eluted in 50 μL and stored at −70°C until further used. PCR reactions included 2 μL of extracted DNA and 10 pmol of specific primer sets targeting the 16S rRNA gene of Anaplasma spp., GEPs (5′-CTGGCGGCAAGCYTAACACATGCAAGTCGAACGGA-3′) and GEPas (5′-CTTCTTCTRTRGGTACCGTCATTATCTTCCCYAYTG-3′) (Oh et al. 2009), and were run on C1000 Touch TM Thermal Cycler (Bio-Rad, Pleasanton, CA). The size of PCR product was 475 bp. PCR products were sequenced by Macrogen Co. (Seoul, South Korea). The sequences were deposited in GenBank under accession numbers MK409687–MK409735. Searches for sequences homologous to the PCR products were conducted using the National Center for Biotechnology Information (NCBI) BLAST network service and aligned using the MegAlign software package (Windows version 7.1; DNA-STAR, Madison, WI). Phylogenetic trees were generated using neighbor-joining algorithms and the Jukes and Cantor matrix. Support for the topology was calculated using 1000 bootstrap replications.

Results

A total of 452 samples were collected for three seasons and from all farms employing different rearing methods. Forty-nine of the 452 goats (10.8%) had an Anaplasma spp. infection. When analyzed according to seasons, the highest number of infections were observed in summer (13.6%; 19/140), followed by fall (12.3%; 15/122), and spring (7.9%; 15/190), although it was not statistically significant. The infection rate of anaplasmosis was significantly higher in mixed grazing-confined farms (27.1%; 33/122) (χ ² = 60.72, df = 2, p < 0.05) than those in conventional (15.0%; 15/100) and confined farms (0.4%; 1/230) (Table 1).

The PCR product sequences were compared with known 16S rRNA gene sequences in the GenBank database and were identified as either A. bovis or A. capra. (Fig. 1). They were divided phylogenetically into three groups. Group 1 formed a cluster with the isolates of A. capra and Anaplasma spp., of which GenBank accession numbers were MK490726–MK409735 (n = 10). Group 2 (n = 11) were clustered with A. bovis isolates from ticks, deer, and sheep in Iran, Japan, and Tunisia, of which GenBank accession numbers were MK409688, MK409690, MK409693, MK409702, MK409707, MK409708, MK409710, MK409712, MK409713, MK409715, and MK409725 (n = 11). And Group 3 were clustered with another A. bovis isolate group from giraffe, goats, tick, and canine in China, of which GenBank accession numbers were MK409687, MK409689, MK409691, MK409692, MK409694 to MK409701, MK409703 to MK409706, MK409709, MK409711, MK409714, and MK409716–MK409724 (n = 28) (Fig. 1). Ten samples showed 100% similarity with an A. capra strain identified in China (Fig. 1).

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FIG. 1.

Phylogenetic analysis of PCR-amplified 16S rRNA products of Anaplasma spp. in this study. Phylograms were generated by neighbor-joining analysis with 1000 bootstrapped replicates. Bootstrap support values are shown. Abbreviations of samples: 1, spring; 2, summer; 3, fall. A–I, Korean native goat farms; CV, conventional farm; CF, confined farm; GC, mixed grazing-confined farm. ●: sequences in this study.

Discussion

Goat anaplasmosis in Ulsan City in Korea was highly prevalent in mixed grazing-confined type farms, but the outbreaks were not different between spring, summer, and fall seasons. Anaplasma spp. were detected in blood samples from 49 goats (10.8%) and their 16S rRNA gene sequences were compared with those of previously reported Anaplasma spp. A. bovis (8.6%, n = 39) was the most frequently detected species, followed by A. capra. (2.2%, n = 10). Although A. bovis is the most frequently detected Anaplasma sp. in this study, in the previous studies A. ovis was the most frequently detected species in goats from China and Mongolia (Yang et al. 2015, Enkhtaivan et al. 2018). The main hosts of A. bovis are cattle and buffalo, but the infections have also been detected in goats, dogs, red deer, and water deer (Rar and Golovljova 2011). In recent studies, 5.5% of Haemaphysalis longicornis ticks collected from water deer in 2016 were positive for A. bovis (Kang et al. 2016a), and 26.4% and 4.1% of H. longicornis ticks collected from goats in 2016 were positive for A. bovis and A. phagocytophilum, respectively (Kang et al. 2016b). This confirmed that the tick is easily exposed and infected with A. bovis. Anaplasma spp. infection have been reported in Chinese goats and ticks since 2009, and also reported every year in Republic of Korea (ROK) since the first detection in goats on Jeju island in 2015 (Oh et al. 2009, Seong et al. 2015).

The number of infections was highest in summer and lowest in spring, although it was not statistically significant. This might be related to tick activity in the examined area. The population of H. longicornis, the predominant tick species in ROK, rapidly increases in spring and gradually decreases in fall (Chong et al. 2013). Mixed grazing-confined farms had the highest Anaplasma infection, possibly due to the increased exposure to ticks in mixed grazing-confined farms.

This study found that Anaplasma spp. infection is endemic in goats in Ulsan Metropolitan City. Therefore, more systematic research on Anaplasma spp. detected farms, such as regional and seasonal variation, should be carried out. In addition, establishing a rapid diagnosis system for Anaplasma spp. would allow for the effective prevention and treatment of anaplasmosis.