Orientia tsutsugamushi (from Japanese tsutsuga meaning "illness", and mushi meaning "insect") is a mite-borne bacterium belonging to the family Rickettsiaceae and is responsible for a disease called scrub typhus in humans. It is a natural and an obligate intracellular parasite of mites belonging to the family Trombiculidae. With a genome of only 2.0–2.7 Mb, it has the most repeated DNA sequences among bacterial genomes sequenced so far. The disease, scrub typhus, occurs when infected mite larvae bite humans. This infection can prove fatal if prompt doxycycline therapy is not started.
Orientia tsutsugamushi infection was first reported in Japan by Hakuju Hashimoto in 1810, and to the Western world by Theobald Adrian Palm in 1878. Naosuke Hayashi first described it in 1920, giving the name Theileria tsutsugamushi. Owing to its unique properties, it was renamed Orientia tsutsugamushi in 1995. Unlike other Gram-negative bacteria, it is not easily stained with Gram stain, as its cell wall is devoid of lipophosphoglycan and peptidoglycan. With highly variable membrane protein, a 56-kDa protein, the bacterium can be antigenically classified into many strains (sub-types). The classic strains are Karp (which accounts for about 50% of all infections), Gilliam (25%), Kato (less than 10%), Shimokoshi, Kuroki and Kawasaki. Within each strain, enormous variability further exists.
Orientia tsutsugamushi is naturally maintained in the mite population by transmission from female to its eggs (transovarial transmission), and from the eggs to larvae and then to adults (transstadial transmission). The mite larvae, called chiggers, are natural ectoparasites of rodents. Humans get infected upon accidental contact with infected chiggers. A scar-like scab called eschar is a good indicator of infection, but is not ubiquitous. The bacterium is endemic to the so-called Tsutsugamushi Triangle, a region covering the Russian Far East in the north, Japan in the east, northern Australia in the south, and Afghanistan in the west. One million infections are estimated to occur annually. Antibiotics such as azithromycin and doxycycline are the main prescription drugs; chloramphenicol and tetracyclin are also effective. Diagnosis of the infection requires techniques such as Weil–Felix test, rapid immunochromatographic test, immunofluorescence assays, and polymerase chain reaction. There is no vaccine for the infection.
History The earliest record of O. tsutsugamushi infection was in the 3rd century (313 C.E.) in China. Japanese were also familiar with the link between the infection and mites for centuries. They gave several names such as shima-mushi, akamushi (red mite) or kedani (hairy mite) disease of northern Japan, and most popularly as tsutsugamushi (from tsutsuga meaning fever or harm or illness, and mushi meaning bug or insect). Japanese physician Hakuju Hashimoto gave the first medical account from Niigata Prefecture in 1810. He recorded the prevalence of infection along the banks of the upper tributaries of Shinano River. The first report to the Western world was made by Theobald Adrian Palm, a physician of the Edinburgh Medical Missionary Society at Niigata in 1878. Describing his first-hand experience, Palm wrote:
The aetiology of the disease was unknown until the early 20th century. In 1908, a mite theory of the transmission of tsutsugamushi disease was postulated by Taichi Kitashima and Mikinosuke Miyajima. In 1915, a British zoologist, Stanley Hirst, suggested that the larvae of mite Microtrombidium akamushi (later renamed Leptotrombidium akamushi) which he found on the ears of field mice could carry and transmit the infection. In 1917, Mataro Nagayo and colleagues gave the first complete description of the developmental stages such as egg, nymph, larva, and adult of the mite. They also asserted that only the larvae bites mammals, and are thus the only carriers of the parasites. But then, the actual infectious agent was not known, and it was generally attributed to either a virus or a protozoan.
The causative pathogen was first identified by Naosuke Hayashi in 1920. Confident that the organism was a protozoan, Hayashi concluded, stating, "I have reached the conclusion that the virus of the disease is the species of Piroplasma [protozoan] in question... I consider the organism in Tsutsugamushi disease as a hitherto undescribed species, and at the suggestion of Dr. Henry B. Ward designate it as Theileria tsutsugamushi." Discovering the similarities with the bacterium R. prowazekii, Mataro Nagayo and colleagues gave a new classification with the name Rickettsia orientalis in 1930. (R. prowazekii is a causative bacterium of epidemic typhus first discovered by American physicians Howard Taylor Ricketts and Russell M. Wilder in 1910, and described by a Brazilian physician Henrique da Rocha Lima in 1916.)
The taxonomic confusion worsened. In 1931, Norio Ogata gave the name Rickettsia tsutsugamushi, while Rinya Kawamüra and Yoso Imagawa independently introduced the name Rickettsia akamushi. Kawamüra and Imagawa discovered that the bacteria are stored in the salivary glands of mites, and that mites feed on body (lymph) fluid, thereby establishing the fact that mites transmit the parasites during feeding.
For more than 60 years there was no consensus on the choice of name – both R. orientalis and R. tsutsugamushi were equally used. Akira Tamura and colleagues reported in 1991 the structural differences of the bacterium from Rickettsia species that warranted a separate genus, and proposed the name Orientia tsutsugamushi. Finally, in 1995, they made a new classification based on the morphological and biochemical properties, formally creating the new name O. tsutsugamushi.
Biology
Orientia tsutsugamushi is a Gram-negative bacterium and is a permanent (obligate) parasite in mites. Within a single host cell, O. tsutsugamushi rapidly divides into many individuals. A unicellular organism, it is oval shaped and measures 0.5 to 0.8 μm wide and 1.2 to 3.0 μm long. Due to similarity, it was previously classified in the genus Rickettsia among other bacteria, but later assigned a separate genus, Orientia, which it shares (as of 2010) only with Candidatus Orientia chuto. It is broader but shorter than other rickettsial bacteria, which are rod shaped and on average measure 0.25 to 0.3 μm wide and 0.8 to 1 μm long. During reproduction, it divides (by binary fission) into two daughter cells by the process of budding. While undergoing budding, it accumulates on the host cell surface, unlike other bacteria. One complete budding cycle takes 9 to 18 hours.
The bacterium is enclosed by a cell wall on the outside and cell membrane on the inside. The cell covering takes up stains such as Giemsa and Gimenez stains. Although its cell wall has a classic bacterial double layer, its outer leaflet is much thicker than the inner one, which is just the opposite in Rickettsia species. A capsule layer that forms a spherical halo in other bacteria is missing. The cell wall is less rigid due to the absence of peptidoglycan, which is otherwise characteristic of the rigid cell walls of other bacteria. Classic bacterial lipophosphoglycans such as muramic acid, glucosamine, hydroxy fatty acids, heptose, and 2-keto-3-deoxyoctonic acid are also absent in the cell wall. Due to the absence of peptidoglycan, the bacterium is naturally resistant to all β-lactam antibiotics (such as penicillin), to which Rickettsia species are normally sensitive to. Its genome totally lacks the genes for lipophosphoglycan synthesis, but does contain some for those of peptidoglycan. Important genes essential for peptidoglycan synthesis such as alr, dapF and PBP1 are missing: alr encodes an enzyme L-alanine racemase, which converts L-alanine to D-alanine in the first step of peptidoglycan synthesis pathway; dapF encodes diaminopimelate epimerase, which convert LL-2,6-diaminoheptanedioate (L,L-DAP) to meso-diaminoheptanedioate (meso-DAP); and PBP1 encodes penicillin-binding protein-1 (PBP1), which converts periplasmic lipid II to peptidoglycan. Thus, the bacterium cannot synthesise a typical peptidoglycan cell wall, and instead makes a peptidoglycan-like structure on its surface. The cell membrane is also chemically different in its protein composition, and this difference gives rise to strain variations within the species itself.
The bacterium is highly virulent, such that its isolation and cell culture are done only in a laboratory facility with biosafety level 3. Unlike other bacteria which can easily grow on different culture media, rickettsiales can be cultured only in living cells. O. tsutsugamushi specifically can be grown only in the yolk sacs of developing chicken embryos and in cultured cell lines such as HeLa, BHK, Vero, and L929. In contrast to Rickettsia species which reside in the nucleus of the host cell, O. tsutsugamushi mostly grows within the cytoplasm of the host cell. Genetically, it differs from other Rickettsia by only 9%. Even though adaptation to obligate intracellular parasitism among bacteria generally results in a reduced genome, it has a genome size of about 2.0–2.7 Mb depending on the strains, which is comparatively larger than those of other rickettsiales – two times larger than that of Rickettsia prowazekii, the most well-known member. The entire genome is distributed in a single circular chromosome. Whole genome sequences are available only for Ikeda and Boryong strains, both from the Republic of Korea. The genome of the Ikeda strain is 2,008,987 base pairs (bp) long, and contains 1,967 protein-coding genes. The Boryong strain is larger with 2,127,051 bp and 2,179 protein-coding genes.
Genome comparison shows only 657 core genes among the different strains. With about 42-47% of repetitive sequences, O. tsutsugamushi has the most highly repeated bacterial genome sequenced as of 2013. The repeated DNA sequence includes short repetitive sequences, transposable elements (including insertion sequence elements, miniature inverted-repeat transposable elements, a Group II intron), and a greatly amplified Integrative and Conjugative Element (ICE) called the rickettsial amplified genetic element (RAGE). RAGE is also found in other rickettsial bacteria. In O. tsutsugamushi, however, RAGE contains a number of genes including tra genes typical of type IV secretion systems and gene for ankyrin repeat–containing protein. Ankyrin repeat–containing proteins are secreted through a type I secretion system into the host cell. The precise role of type IV secretion system in O. tsutsugamushi is not known. It may be involved in horizontal gene transfer between the different strains.
Life cycle and transmission
Orientia tsutsugamushi is naturally transmitted in the mite population belonging to the genus Leptotrombidium. It can be transmitted by a female to its eggs through the process called transovarial transmission, and from the eggs to larvae and adults through the process of transstadial transmission. Thus, the bacterial life cycle is maintained entirely in mites. Infection to rodents and humans is an accidental transmission from the bite of mite larvae, and not required for reproduction or survival of the bacterium. In fact, in humans the transmission is stopped, and the bacterium meets a dead end. However, uninfected mites can acquire the infection from infected rodents.