@phdthesis{oai:obihiro.repo.nii.ac.jp:00004888,
 author = {Ringo, Aaron Edmond},
 month = {2023-04-24, 2023-04-24, 2023-04-24},
 note = {2022, application/pdf, Tick-borne diseases (TBDs) pose a major challenge to the livestock industry in many tropical and sub-tropical countries. In East Africa, Tanzania and Kenya are among the countries located just south of the horn of Africa. This region is popularly known for a large population of livestock. However, TBDs in the region present a serious threat to the livestock sector particularly cattle and small ruminants. The most important TBDs in the region are theileriosis, anaplasmosis, babesiosis and ehrlichiosis. Despite the damage incurred by these diseases in Tanzania and Kenya, limited epidemiological data on the occurrence and distribution of TBDs in the two countries is available. Therefore, several molecular studies were carried out in different locations of the two countries to address the problem.
In chapter 1, a total of 245 blood samples from different breeds of cattle were randomly collected on Pemba Island, in Tanzania. Polymerase chain reaction (PCR) and sequencing was used to detect and identify the tick-borne pathogens (TBPs). The assays were performed using primers based on Theileria spp. (18S rRNA), Babesia bovis (SBP-2), B. bigemina (RAP-1a), Anaplasma marginale (MSP-5 and groEL), Ehrlichia ruminantium (pCS20), T. parva (p104), T. mutans (18S rRNA), and T. taurotragi (18S rRNA). PCR screening of cattle samples collected on Pemba Island revealed overall infection rates for Theileria spp. (62.4%), B. bigemina (17.6%), A. marginale (15.9%), E. ruminantium (7.4%) and B. bovis (4.5%). Further analysis using sequences of Theileria spp. (18S rRNA) revealed infection of cattle with T. mutans (68.6%), T. taurotragi (48.4%), T. parva (41.2%), and T. ovis (1.9%). Co-infections of cattle, with up to six TBPs, were revealed in 46.9% of the samples. Sequence analysis indicated that T. parva (p104), E. ruminantium (pCS20) and A. marginale (MSP-5) genes are conserved among cattle blood samples in Pemba, with 99.3% - 100%, 99.6% - 100% and 100% sequence identity values, respectively. In contrast, the B. bigemina (RAP-1a) and B. bovis (SBP-2) gene sequences were relatively diverse with 99.5% - 99.9% and 66.4% - 98.7% sequence identity values, respectively. The phylogenetic analyses revealed that T. parva (p104), E. ruminantium (pCS20) and A. marginale (MSP-5) gene sequences clustered in the same clade with other isolates from other countries. In contrast, the B. bigemina (RAP-1a) and B. bovis (SBP-2) gene sequences showed significant differences in the genotypes, as they appeared in separate clades. The data provided should improve the understanding of the epidemiology of tick-borne diseases, and is expected to improve the approach for diagnosis and control of tick-borne diseases in Tanzania.
In chapter 2, blood samples were collected randomly in 236 cattle of different breeds from Zanzibar Island, Tanzania. The PCR and sequencing were used to screen the samples for detection of TBPs. The assays were performed using primers described in chapter 1. The PCR screening revealed that 64.5% of animals were infected by TBPs, including T. mutans (38.1%), T. parva (34.3%), T. taurotragi (30.9%), A. marginale (10.2%), B. bigemina (5.1%), T. velifera (3.4%) and B. bovis (2.1%). Overall, a total of 86 animals (36.4%) were co-infected with up to five pathogens concomitantly. The pathogens mostly involved in the co-infection were T. parva, T. taurotragi and T. mutans. Sequence analysis indicated that T. parva (p104) and B. bigemina (RAP-1a) genes are diverse among the sampled animals on Zanzibar Island, with 99.64%–100% and 99.51%–100% nucleotide sequence identity value, respectively. In contrast, the A. marginale (MSP-5) and B. bovis (SBP-2) genes are conserved, with 100% and 99.66%-100% nucleotide sequence identity values respectively. The phylogenetic analyses revealed that T. parva (p104) and B. bigemina (RAP-1a) gene sequences showed significant differences of genotypes, as they appear in different clades. Meanwhile, A. marginale (MSP-5) and B. bovis (SBP-2) gene sequences appear in the same clade with other sequences extracted from the NCBI GenBank. The epidemiological findings revealed in this study will provide important information on tick-borne diseases in Tanzania and will be used as scientific basis for planning future control strategies.
In chapter 3, a total of 250 blood samples were randomly collected from indigenous cattle of Tanga region, Tanzania. The assays performed were based on primers described in chapter 1. The results show an overall infection rate for T. mutans (48%), A. marginale (32.4%), T. parva (25.6%), T. taurotragi (20.8%) and B. bigemina (13.2%). Co-infections of up to four pathogens were revealed in 44.8% of the cattle samples. Sequence analysis indicated that T. parva (p104) and A. marginale (groEL) genes were conserved among the sampled animals with sequence identity values of 98.92 – 100% and 99.88% – 100%, respectively. On the other hand, the B. bigemina (RAP-1a) gene and the (V4 region of the 18S rRNA of T. mutans genes were diverse among the sampled cattle, indicating the sequence identity values of 99.27% - 100% and 22.45% - 60.77%, respectively. The phylogenetic analyses revealed that T. parva (p104) and A. marginale (groEL) gene sequences of this study were clustered in a clade. In contrast, the B. bigemina (RAP-1a) and the T. mutans (V4 region of the 18S rRNA) gene sequences appeared in the different clades. The findings revealed in this work shows that indigenous breed of cattle reared in free range system in Tanzania can be the source of tick-borne infections in other naïve breeds of cattle in the country. Therefore, this information is useful in the control of tick-borne diseases.
In chapter 4, blood samples were randomly collected from 76 apparently healthy sheep in Machakos and Homa-bay counties in Kenya. The assays were performed using primers based on Theileria spp. (18S rRNA), Anaplasma spp. (16S rRNA), Babesia ovis (18S rRNA), A. ovis (AoMSP-4) and E. ruminantium (pCS20). The overall infection rates of sheep samples collected in Machakos and Homabay counties in Kenya for Theileria spp. (51.3%), Anaplasma spp. (40.8%), A. ovis (34.2%) and E. ruminantium (7.9%). The overall co-infection was (61.8%). All Theileria spp. positive samples were confirmed to be T. ovis on sequencing. A phylogenetic analysis of (18S rRNA) gene sequences revealed that isolates of this study clustered with T. ovis sequences from other regions suggesting this gene is highly conserved. The E. ruminantium (pCS20) sequences were in the same clade on the phylogenetic tree. However, three (AoMSP-4) sequences appeared in the same clade while one sequence formed a separate branch revealing genetic divergence to the other sequences. The 16S rRNA sequencing revealed uncultured Anaplasma spp. and A. ovis. The phylogenetic analyses of uncultured Anaplasma spp. revealed that the two sequences formed a divergent clade signifying genetic differences to other isolates. This study provides important information regarding tick-borne pathogens occurrence and their degree of genetic diversity among sheep in Kenya.
In conclusion, the studies reveal that TBDs are well distributed in Tanzania and Kenya. Theileria spp. were the most prevalent TBPs in the region led by Theileria mutans, T. parva and T. taurotragi. Moreover, mixed infection in cattle and sheep were the prominent findings in the study areas. These data provide basement epidemiological background which will contribute to the future control strategies of tick-borne diseases in Tanzania and Kenya., マダニ媒介性感染症は、多くの熱帯および亜熱帯地域の畜産業に対し大きな脅威となっている。タンザニアとケニアは、共に東アフリカに位置しており、畜産業が盛んな地域として知られているが、家畜におけるマダニ媒介性寄生虫症の被害は甚大なものとされている。この地域で最も重要な家畜のマダニ媒介性寄生虫症は、一般的にタイレリア症、アナプラズマ症、バベシア症およびエーリキア症とされている。しかしながら、当該地域の家畜におけるマダニ媒介性寄生虫症の発生と分布に関する詳細な疫学的データは限られている。そこで、本研究ではタンザニアとケニヤの主要家畜である牛と羊におけるマダニ媒介性寄生虫症の流行実態の一端を解明するために分子疫学的調査を実施した。
第1章では、タンザニアのペンバ島で採集した245頭の牛の血液サンプル中のマダニ媒介性病原体についてPCR法によるスクリーニングとシークエンス解析を行った。その結果、Theileria spp.（62.4%）、B. bigemina（17.6%）、A. marginale（15.9%）、E. ruminantium（7.4%）およびB. bovis (4.5%)が検出された。Theileria属に属する種の特定を行ったところ、 T. mutans (68.6%)、T. taurotragi (48.4%)、T. parva (41.2%)、およびT. ovis (1.9%) が検出された。PCR産物の塩基配列に基づいて系統解析を行ったところ、T. parva (p104)、 E. ruminantium (pCS20)、および A. marginale (MSP-5) の標的遺伝子塩基配列が、他のアフリカの国と地域の分離株と近縁のクラスターに位置していることが判明した。対照的に、B. bigemina (RAP-1a) と B. bovis (SBP-2) の標的遺伝子配列は、他のアフリカの国と地域で分離された株とは独自のクラスターを形成した。これらのデータは、タンザニアにおけるマダニ媒介性寄生虫症の流行実態の一端を解明し、さらにコントロール対策を構築する上で一助となると考えられる。
第2章では、タンザニアのザンジバル島で採集した236頭の牛の血液サンプル中のマダニ媒介性病原体についてPCR法によるスクリーニングとシークエンス解析を行った。その結果、T. mutans (38.1%)、T. parva (34.3%)、T. taurotragi (30.9%)、A. marginale (10.2%)、B. bigemina (5.1%)、T. velifera (3.4%)およびB. bovis (2.1%)が検出された。系統解析では、T. parva (p104) 、B. bigemina (RAP-1a) およびB. bovis (SBP-2)の標的遺伝子配列は、タンザニア独自のクラスターを形成した。一方、A. marginale (MSP-5)の標的遺伝子配列は、地理的に離れた国及び地域の分離株と近縁のクラスタを形成した。これらの結果は、タンザニアにおけるマダニ媒介性寄生虫症に関する重要な情報を提供し、将来の制御戦略を構築する上で有用な科学的根拠として活用できると考えられる。
第3章では、タンザニアの陸地であるタンガ地域で採集した250頭の牛の血液サンプル中のマダニ媒介性病原体についてPCR法によるスクリーニングとシークエンス解析を行った。その結果、T. mutans (48%)、T. parva (25.6%)、T. taurotragi (20.8%)、B. bigemina (13.2%)およびA. marginale (32.4%)が検出された。系統解析では、T. parva (p104) および A. marginale (groEL) の標的遺伝子配列がタンザニア独自のクラスターを形成した。対照的に、B. bigemina (RAP-1a) および T. mutans (18S rRNA のV4領域) の標的遺伝子配列は、アフリカと世界中で分離された株と近縁関係にあることが判明した。これらの結果は、タンザニアの放し飼いシステムで飼育された土着の牛の品種が、他のナイーブ品種への伝播の感染源になり得ることが示唆された。したがって、これらの情報はタンザニアの内陸地域におけるマダニ媒介性寄生虫症の制御戦略を構築する上有効に活用できると考えられる。
第4章では、ケニアのマチャコスとホマベイ地域の羊76 頭から採集した血液サンプル中のマダニ媒介性病原体についてPCR法によるスクリーニングとシークエンス解析を行った。その結果、T. ovis（51.3％）、A. ovis（34.2％）、およびE. ruminantium（7.9％）が検出された。系統解析では、T. ovis (18S rRNA)の標的遺伝子配列は、ケニア分離株において高度に保存され、アフリカの他の地域やトルコで分離された株と近縁関係にあった。また、E. ruminantium (pCS20)塩基配列は東アフリカで分離された株で独自のクラスターを形成した。A. ovis (AoMSP-4) の標的塩基配列では、３つのケニア分離株が同一クラスターに属し、また、一つの分離株は他の３株と分岐を形成した。これらの結果から、ケニアの羊の間でマダニ媒介性病原体の発生とその遺伝的多様性の関する有用な情報を提供し、小型反芻動物におけるマダニ媒介性寄生虫症の診断と制御戦略の構築に有用であると考えられる。
以上のように、この研究ではタンザニアとケニアの主要家畜である牛と羊におけるマダニ媒介性寄生虫症の流行実態の一端を明らかにした。そのなかでもT. mutans、 T. parvaおよび T. taurotragi 最も広く分布しているマダニ媒介性病原体であることが明らかとなった。さらに、多くの牛と羊が複数のマダニ媒介病原体に同時感染していることが示唆された。これらのデータは、タンザニアとケニアにおける家畜のマダニ媒介性寄生虫症の制御戦略構築に役立つ基礎データを提供するものと考えられる。, 博士学位論文
大学院畜産学研究科 獣医学専攻
Doctoral Program of Veterinary Science},
 school = {帯広畜産大学},
 title = {Epidemiological study on tick-borne parasitic diseases in livestock from Tanzania and Kenya},
 year = {}
}