Rickettsiosis

Rickettsiosis

Rickettsias are obligate intracellular gram-negative, small-genome (1.1–1.5 Mb), bacteria that infect several types of cells in the arthropod host or in mammal endothelial cells (1,2). The largest physiopatological effect of a rickettsia infection consists of a small vessel vasculitis as a result of a direct infection of endothelial cells (3). Members of this genus have been classified into four groups: the typhus group (GT), which includes R. prowazekii transmitted by lice and causes epidemic typhus, and Rickettsia typhi, transmitted by fleas and causing endemic typhus; the Spotted fever group (GFM), which includes over 20 species, mostly transmitted by ticks (Rickettsia rickettsii in America, Rickettsia conorii in Europa, and Rickettsia africae in Africa). Other recently identified species such as R. akari, R. felis, and R. australis, sharing GFM and TG characteristics belong to the transitional group (GTR) (2,4). Several arthropods are efficient rickettsia vectors, reservoirs, or amplifiers, and their distribution and ecology affect the epidemiology of rickettsiosis. In Colombia, the presence of several vectors of rickettsia such as ticks of the complex Amblyomma cajennense (5) is renowned. Other vectors are Rhipicephalus sanguineus, or brown dog tick, and Amblyomma ovale or common dog tick (6). Domestic and wild animals are susceptible to infection of some sorts of rickettsia, some others serve as reservoirs or amplifiers in endemic areas. In humans, the severity of clinical symptoms vary depending on the rickettsia involved in the infection. R. rickettsii is the most lethal species for humans (7). Diagnosis of rickettsiosis is generally done by detecting antibodies with indirect immunofluorescence (gold standard). Molecular techniques, such as PCR and sequencing allow a quick and specific diagnosis (7,8).

Biological aspects of the agent. Rickettsias are obliged intracellular bacteria capable of infecting several types of cells in the arthropod host, as well as human endothelial cells. They are coccobacillus ranging from 0.7- 2.0 µm in length and 0.3- 0.5 µm wide; although some rickettsias may have a more elongated filament appearance (1). They have a triple layer cell wall typical of Gram negatives, consisting of an internal and external membrane separated from the peptidoglycan layer and surrounded by a glycocalyx or slime layer (1). Rickettsias have small genomes, between 1.1 and 1.5 Mb as a result of evolution by reduction (loss of genes). The content of G + C ranges between 33 and 29%. Different species have different optimum growth temperatures, but they generally range between 32 and 35°C (2). Rickettsias cannot be visualized with Gram stain, but with Giménez and Giemsa stains.

Taxonomy. During the last decade, the taxonomy of rickettsiae has undergone wide reorganization. The Rickettsial order currently includes the families Rickettsiaceae and Anaplasmataceae, most of whose members are associated with arthropod hosts, and the family Holosporaceae, symbiotic with protists. The traditional bacteriological methods used for identification cannot be applied to the study of rickettsias due to the intracellular nature of this microorganism. Molecular methods have been important for the advances of the taxonomy of these microorganisms (7,9).

Taxonomic molecular studies. Those related with the ribosomal gene 16S are not very useful due to the high homology present between species (≥ 97%). Currently, in order to define genus, group, and species of rickettsia, sequencing five genes is necessary; 16S rRNA (rrs), gltA, ompA, ompB,and sca4 (gene D) (10). According to the aforementioned criteria, in order to recognize and new species of rickettsia it must not show more than one of the following degrees of similarity with other species of validated rickettsia: ≥99.8 and ≥99.9% for genes rrs and gltA, respectively. And ≥98.8, ≥99.2, and ≥99.3% for ompA, ompB and gene D, respectively (10).Other techniques such as PCR-RFLP, MST Multi-Spacer Typing and MLST Multilocus sequence typing, couples are being used to classify a species with good results; nonetheless, they are very elaborate and expensive techniques. Rickettsia experts do not fully agree on classification, and report that its taxonomy is still controversial and continuously evolving (11).

Pathogenic rickettsia groups. Initially, pathogenic members of the genus were divided into two groups by their antigenic characteristics: (i) the typhus group (GT) including species such as R. prowazekii (vectors are lice) and Rickettsia typhi (transmitted by fleas) and (ii) the spotted fever group (GFM) which includes over 20 species mostly transmitted by ticks and associated to infections throughout the world (Rickettsia rickettsii in America, Rickettsia conorii in Europa, and Rickettsia africae in Africa). Other pathogenic species of rickettsia such as R. akari, R. felis, and R. australis comprise the transitional group (GTR). Species of the ancestral group such as R. bellii and R. Canadensis do not have any known pathogenicity (2,4).

Transmission. Molecular studies demonstrate that rickettsias maintained their lifecycle by infecting a wide variety of hosts (9). Rickettsias are among those pathogens transmitted by a wide variety of arthropods such as lice, ticks, fleas, and mites (12) (Table 1).

Competent vectors of rickettsiosis transmission. Several arthropods are efficient rickettsia vectors, reservoirs, or amplifiers, and their distribution and ecology affect the epidemiology of rickettsiosis. RMSF (Rocky Mountain Spotted Fever) cases in the United States (transmitted by Dermacentor variabilis and D. andersoni), as well as the Mediterranean Boutonneuse fever (transmitted by Rhipicephalus sanguineus) are more prevalent in late spring and summer, since these ticks are more active during these seasons (7). In contrast, tick activity in Colombia is constant and increases during the dry season between December and February. The most known vectors are summarized in table 1.

Main vectors of SFG rickettsiosis in Colombia. Despite the large amount of information available in the country in regards to ticks, little is known about the vectors of rickettsiosis have been involved in the outbreaks.

In Colombia, we know about several species of the complex Amblyomma cajennense such as: Amblyomma mixtum and Amblyomma patinoi (Figure 1) In a large part of the Colombian territory (5,13).

Another vector involved in the transmission of rickettsia commonly found in Colombia is Rhipicephalus sanguineus, or brown dog tick (Figure 2).

Amblyomma ovale (Figure 3) is a tick species hosted in dogs, recently involved in Brazil as a vector of rickettsias. It is commonly found in Colombia, therefore posing a significant risk for Rickettsia transmission (6).

A new species of SFG rickettsia was reported in the Department of Córdoba; this new species was named Candidatus Rickettsia sp., Colombianensi strain, and it was detected in Amblyomma dissimile (14) (Figure 4), a species commonly found in reptiles (iguanas, snakes, and turtles).

Although more studies are needed to better characterize this new species of rickettsia, so far it is considered as a species with pathogenic potential since it belongs to the SFG group, characterized by having most of is species as pathogenic for humans. Another reason to consider Candidatus Rickettsia sp., Colombianensi strain as potentially pathogenic, is that it has a 99% genetic identity with Rickettsia tamurae, a species of the GFM group, and associated to human disease. This species is reported mainly in Japan in Amblyomma testudinarium (15, 16). However, vigilance of human rickettsiosis cases in the areas where this new species was described would be the best way to establish its pathogenicity.

Pathogenesis. Pathogenesis for all rickettsias is similar; molecular characteristics and gene expression contribute to the pathogenicity among different species of rickettsia. Rickettsiae expresses two major surface proteins called external membrane protein A (rOmpA) and B (rOmpB), both present in SFG rickettsias but rOmpB is found only in rickettsias of the GT group. They play an important role in cell adhesion and therefore are considered in the potential development of vaccines (17, 18). Studies performed on R. conorii demonstrated that the adhesion of rOmpB to Ku70 (receptor) and RickA protein result in a recruitment of the protein complex Arp2/3 (Arp: actin-related protein) which controls actin polymerization, resulting in rickettsia phagocytosis (19, 20, 21). Once inside the cytoplasm, rickettsia escapes from the phagocytic vacuole and enters the cytoplasm. The proteins involved in the liberation are phospholipases and hemolysins, phospholipase D (PLD) and others such as the B1 patatine precursor coded by the pat1 gene, and two hemolysins coded by genes tlyA and tlyC (22, 23, 24).

The largest pathophysiological effect of rickettsia infections consists of a small vessel vasculitis due to a direct infection of endothelial cells. This vasculitis is due to an increase in vascular permeability (as a result of a rupture of the endothelial cell unions), generalized vascular swelling, edema, increase of the interaction between leukocytes and endothelium, and the release of vasoactive mediators that promote coagulation and the production of pro-inflammatory cytokines (3).

The important role of dendritic cells in innate and acquired immunity against rickettsia has been recently demonstrated. Early resistance to the infection is attributed to IFN production and natural killer cells, and the resulting activation of endothelial cells, dendritic cells, and macrophages. IFN and FNT are essential as a primary defense against Rickettsia infections (25).

Clinical manifestations in animals. As in humans, canines are susceptible to infection with some species of Rickettsia. Infected canines present fever, lethargy, vomit, depression, anorexia, petechia, ecchymosis, epistaxis, conjunctivitis, diarrhea, weight loss, and dehydration. As the infection progresses, additional signs may appear, including ocular lesions, blood disorders (anemia, thrombocytopenia, and mild leukopenia), joint pain, and neurologic abnormalities (paraparesis, or tetraparesis, ataxia, vestibular syndrome). They may also have a mild infection depending on the species of rickettsia (26).

Dogs play an important role as biological host of ticks and increase the population of infected ticks, which at some point could make close contact with human population and cause the disease (27). Dogs in endemic rickettsiosis areas act as sentinel animals in epidemiological studies (28). In 2008, Pinter et al. reported that dogs are important sentinels to detect the presence of R. rickettsii in areas where A. aureolatum ticks are the main vectors of the Brazilian spotted fever (29).

Little is known about the infection and the symptoms of rickettsia in other animals whether domestic (bovines) or wild.

Equines are the most studied domestic animals; studies indicate that when infected by R. rickettsii, they fail to display any clinical signs or hematological abnormalities. However, they do develop anti-rickettsia antibodies. Studies also conclude that equines are not good amplifiers of rickettsia, suggesting that they do not play our role as a natural amplifier of these microorganisms (30).

The wild animals studied include capybaras (Hydrochoerus hydrochaeris). It was demonstrated in Brazil that capybaras that underwent intraperitoneal inoculation of R. rickettsii displayed no symptoms of the disease, but did develop anti-rickettsia antibodies. They also demonstrated that capybaras developed high rickettsemia (12 days on average), capable of infecting ticks feeding on capybaras and guinea pigs inoculated with the blood of infected capybaras. In Colombia, capybaras have been studied as potential sentinels in rickettsiosis endemic areas (31).

Other studied animal species include small rodents such as Microtus pennsylvanicus, which has been implicated in the United States as one of the amplifiers for R. rickettsii. In Brazil, marsupials of the Didelphis aurita have also been implicated as competent amplifiers of R. rickettsii for Amblyomma cajennense ticks; however, other studies showed little or no amplification for R. felis, R. bellii, and R. parkeri species (32,33).

Future studies must address the role of domestic animals in good transmission dynamics of rickettsiosis, including the possibility of some animals to serve as reservoirs, amplifiers, or both (34).

Clinical manifestations in humans. Clinical symptoms of rickettsiosis are similar; however, their severity varies depending on the rickettsia involved in the infection. Currently, the Rocky Mountain spotted fever, caused by R. rickettsii is the most lethal rickettsiosis for humans in countries where it has been described. The main symptoms of rickettsiosis appear 6 to 14 days after the bite of the vector arthropod. Clinical manifestations include high fever, headache, muscle pain, nausea, vomiting, anorexia, abdominal pain, and diarrhea. Exanthema in the Rocky Mountain spotted fever is not usually apparent until the third day of fever, and begins with small irregular red macules that typically appear on the knees, elbows, and forearms. The exanthema can then evolve into wheals or petechiae. Complications include neurological manifestations, convulsions, and hemiplegia. Other manifestations of a severe disease include lung and kidney failure, myocarditis, the closest and gangrene in fingers, earlobes, and external genitalia (7).

DIAGNOSIS

Diagnosis of R.rickettsii infection in canines is generally done by detecting serum antibodies through indirect immunofluorescence, and a certain diagnosis requires sero-conversion or a fourfold elevation of antibody titers in paired samples. Alternatively, a sole titer above 1:1024 is also considered a diagnosis (35). However, this diagnostic method in canines is not usually used in Colombia and is reserved for research studies. Antibody detection with indirect immunofluorescence (IFI) is the gold standard and the best technique due to its sensitivity, specificity, and swiftness (8).

During the acute stage of rickettsiosis, immunohistochemical examination of biopsies taken from skin lesions is the most sensitive approximation to diagnosis. The drawbacks of this method are that it is not available in many laboratories and it cannot be applied until exanthema or an eschar appear, and that it does not differentiate the species involved in the infection.

Molecular techniques, such as the various methodologies of PCR and sequencing, allow for a quick and specific diagnosis by detecting rickettsia DNA in infected tissues, cultures, and ticks. The genes usually analyzed are those that code two external membrane proteins: rOmpA, rOmpB (7,36). The gltA gene is also used (Figure 5), which encode the citrate synthetase enzyme, present in all Rickettsias, the gene that codes the 17-kDa protein present in all SFG rickettsias, and gene D, found in most species. PCR should be the ideal diagnostic test; however it has some drawbacks such as the low sensitivity when the sample comes from animals that do not display highly rickettsemia, and the impossibility of obtaining isolation(8,37,38).

For diagnosis in humans, the most important epidemiological clinical finding is knowing about the exposure to ticks in endemic or high risk areas with vector population. At the early stage of the disease, symptoms are similar to many endemic tropical pathologies such as dengue, hantavirus cardiopulmonary syndrome, arenavirus hemorrhagic fever, yellow fever, leptospirosis, anaplasmosis, ehrlichiosis, and malaria. Thus the need for a differential diagnosis, and this is a complicated situation in the Colombian Tropic, given the endemic of some of these pathogens. Because of the similarities between the aforementioned endemic tropical pathologies, lab analysis are not very useful to diagnose rickettsiosis, and they usually show the same hematologic and biochemical abnormalities such as thrombocytopenia, and elevation of transaminase and VSG, hyponatremia and leukopenia (37, 39). The above-mentioned techniques are also used in human diagnosis.

Epidemiology, prevention, and control. Although rickettsias have a worldwide distribution, at least 13 species have been found in Central and South America, and most of the species classified as in the SFG (Figure 6) (40)

Studies in animals. Studies in domestic and wild animals determining rickettsia infections are scarce (31,34,41). However, the studies are key for a better understanding of the dynamics of rickettsiosis in a specific region; Table 2 shows the studies published in the country.

Studies in humans. In Colombia, the first cases of the Tobia fever, as the Rocky Mountain spotted fever caused by Rickettsia rickettsii is known, were reported in 1934 and 1936 in the municipality of Tobia, in Cundinamarca, Colombia (42). During a period of 65 years, the disease was unseen by health entities, and only until 2001 a study of seroprevalence of Rickettsia sp. in fieldworkers of the rural area of the municipality of Ciénaga de Oro (Córdoba, Colombia) a high and concerning 49% prevalence was found in the analyzed subjects. The high seroprevalence found in the area demonstrated the circulation of our rickettsia of the spotted fever group (43). Consequently, more studies were made in the country and isolated cases and outbreaks of the disease appeared in various parts of the country, summarized in table 2.

Treatment. Doxycycline is the drug of choice for canines and humans. There are also some other drugs with proven efficiency such as tetracycline, oxytetracycline, and chloramphenicol. The recommendation for canines is 10 mg/kg of doxycycline once a day for 28 days. In most of the cases, those in the acute stage of the disease responded to doxycycline treatment within 24 to 72 hours after the first injection (66). For humans, the dosage is 100 mg/12 H for 3 to 7 days depending on the seriousness of the case. For endemic and murine typhus, a single dose of 100 mg/12h is enough. Children may also receive doxycycline and azithromycin (8,67,68).

There is no vaccine and against rickettsiosis. Contact with ticks must be avoided, and tick removal immediately after detection, avoiding exposure to tick infested habitats such as forests, bushes, and grasslands, and the use of pyrethroids are the best strategies to prevent the disease (68,69).