Molecular Detection and Genotyping of Theileria spp. in Deer (Cervidae) : 사슴에서 범안열원충(Theileria)의 분자생물학적 검출과 유전형 분석

국문 초록 (Abstract)

Theileria는 가축과 야생 반추동물을 감염시키는 진드기 매개 병원체이다. 숙주동물에서 림프구에서 증식한 다음 적혈구를 침입하여 병원성을 나타내며 참진드기가 Theileria의 감염을 매개한다. 주요 증상은 발열, 빈혈, 식욕부진, 황달 및 생산성 감소 등이다. 이러한 이유로 Theileria는 축산농가에 위협적인 존재로 인식된다. 국내에서는 Theileria를 비롯한 진드기 매개 병원체에 대한 다양한 연구가 진행되고 있지만, 고라니를 비롯한 사슴의 Theileria 감염에 대한 연구는 거의 없다. 이 연구에서, 총 160마리의 사슴 혈액을 채취했으며 DNA를 추출하였다. 유전자 증폭을 위해 추출된 DNA 샘플은 PCR을 진행하였다. 우선, 시료에서 주혈원충 감염을 확인하기 위해 주혈원충 공통 프라이머를 이용하였으며 다음 단계로 범안열원충 특이 유전자 부위를 증폭하였다. PCR 양성 시료는 염기서열을 분석하여 계통수를 작성하였다. 전체 감염률은 8.1%(13/160)로 확인되었다. 지역별로는 북부와 남부가 모두 100%로 가장 높은 감염률을 보였지만 시료수가 각각 5와 1로 적었다는 한계가 존재하였다. 중부 지역은 2.9%(4/140)로 가장 낮은 감염률을 나타내었다. 계절별로는 봄철 감염률이 18.4%(9/49)로 가장 높았다. 그 다음으로 여름이 8.9% (4/45)의 감염률을 보였다. 가을과 겨울의 시료는 확보되지 않았다. 지역과 계절, 두 가지 요인이 통계학적 유의성을 나타내었다. 계통수를 이용한 계통발생학적 분석은 염소나 양과 같은 반추동물을 감염시키는 종인 Theileria luwensuni로 동정되었다. 기후변화로 인한 진드기 분포가 확대되고 있는 상황에서 범안열원충의 중요성은 과거보다 증가하고 있다. 향후 더 많은 시료를 확보하여 범안열원충의 전국적인 분포 현황을 조사할 필요가 있어 보인다.

핵심어: 범안열원충, 진드기, 유전형, 고라니, 계통분석, 감염률

다국어 초록 (Multilingual Abstract)

Theileria is an opportunistic pathogen that infects livestock and wild ruminants. It proliferates in lymphocytes and penetrates red blood cells. The hard tick mediates Theileria infection. Major signs and symptoms of Theileria infection include fever, anemia, anorexia, jaundice, and decreased milk production. Due to these symptoms, Theileria is considered a major threat to livestock farming. Various studies have been conducted on tick-borne pathogens including Theileria in Korea, but only few studies have focused on Theileria infection in deer, including the Korean water deer. In this study, we collected 160 deer blood samples. These samples were then processed to extract DNA, and polymerase chain reaction (PCR) was performed for DNA amplification. The first PCR was performed to locate piroplasms in samples. The second PCR was performed to detect Theileria. Next, PCR positive samples were analyzed through the construction of a phylogenetic tree. Data regarding species, region, and season were recorded as possible. Our results showed that overall infection rate of Theileria was 8.1% (13/160). Infection rates of 100% were observed in the northern and southern regions. However, a limitation of this analysis was the small sample size, where we analyzed 5 and 1 sample from the northern and southern regions, respectively. The central region exhibited the lowest infection rate of 2.9% (4/140). Infection rates also differed based on seasons. The infection rate was the highest, (18.4%, 9/49) in spring, followed by that in summer (8.9%, 4/45). However, 0% infection rates were seen during autumn and winter seasons. Phylogenetic analysis indicated that PCR-positive samples were identical to Theileria luwenshuni which usually infects small ruminants such as goat and sheep. Owing to the enormous economic losses caused by Theileria in livestock farming, it is becoming increasingly important. Consequently, additional investigations should be conducted on various species in a wider area.

Keywords: Theileria, tick, genotyping, Korean water deer, phylogeny, incidence

목차 (Table of Contents)

• Chapter1 Literature Review 1
• 1. Introduction 1
• 2. Biology 3
• 2.1 Taxonomy 3
• 2.2 Life cycle 3
• 2.3 Transmission 4
• 3. Regional distribution of ticks 7
• 3.1 North America and Caribbean 7
• 3.2 South Africa, Spain and Sweden 8
• 3.2 China, Japan and Korea 9
• 4. Prevalence of tick-borne pathogens in Korea 11
• 4.1 Wild Korean water deer (KWD, Hydropotes inermis argyropus) 11
• 4.2 Dogs 11
• 4.3 Horses 12
• 5. Tick-borne Diseases 13
• 5.1 Babesiosis 13
• 5.2 Theileriosis 15
• 5.3 Anaplasmosis and ehrichiosis 16
• 5.4 Lyme borreliosis 18
• 5.5 Cremean-Congo Hemorrhagic Fever virus (CCHFV) infection 20
• 5.6 Tick-borne Bunyavirus infection (SFTS) 21
• 5.7 Alphavirus (Togaviridae) and Flaviviurs (Flaviviridae) infection 22
• 5.8 Q fever 23
• 5.9 ASF 24
• 6. Prevention of tick-borne diseases in human 26
• 7. Conclusion 28
• References 29
• Chapter2 Molecular Detection and Genotyping of Theileria spp. in Deer (Cervidae) 46
• 1. Introduction 46
• 2. Materials and Methods 50
• 2.1. Study area and blood sample collection 50
• 2.2. DNA extraction and PCR assay 52
• 2.3. Statistical and phylogenetic analysis 57
• 3. Results 58
• 3.1. Infection rates calculated using PCR 58
• 3.2 Sequencing and phylogenetic analysis 62
• 4. Discussion 64
• 5. Conclusion 69
• References 70
• (Abstract) 80
• (초 록) 82