Tick-borne Rickettsia Research Articles

Comparison of three oxytetracycline regimens for the treatment of persistent Anaplasma marginale infections in beef cattle

Anaplasmosis, caused by the tick-borne rickettsia, Anaplasma marginale, is an economically important disease of cattle in the United States and worldwide. Cattle that recover from acute infection become carriers in which low or microscopically undetectable A. marginale rickettsemia persists. Tetracycline antimicrobials are currently the only drug used in the US for treatment of acute anaplasmosis. There are currently no drugs specifically licensed for elimination of persistent infections. This study tested the efficacy of three oxytetracycline treatment regimens to clear A. marginale from cattle that were persistently infected. Forty Angus × Simmental steers, aged 6–12 months were experimentally infected with A. marginale. After the steers recovered from acute infection, seroconverted, and were confirmed infected using nested PCR followed by DNA hybridization, the carrier status of each animal was ascertained by sub-inoculation of blood into a separate, splenectomized Holstein calf. The steers were then blocked by bodyweight and randomly assigned as follows to four treatment groups: Treatment A, 300 mg/ml solution of oxytetracycline (Tetradure LA-300, Merial Canada Inc.) administered at 30 mg/kg, by intramuscular (IM) injection on day 0; Treatment B, the same 300 mg/ml solution of oxytetracycline administered at 30 mg/kg, IM on day 0 and again on day 5; Treatment C, a 200 mg/ml solution of oxytetracycline (Liquamycin LA-200, Pfizer Animal Health) administered at 22 mg/kg, intravenously (IV), q 24 h for 5 days (a treatment dose that corresponds with current Office International des Epizooties (OIE) recommendations for treatment prior to export). The fourth group consisted of untreated infected control cattle. All steers were still nested PCR and cELISA positive at 60 days after treatment. Infection was confirmed by subinoculation of blood into a splenectomized Holstein calf. These results demonstrated that the treatment regimens tested failed to clear A. marginale infections in carrier cattle.

Anaplasma marginale(Rickettsiales: Anaplasmataceae): recent advances in defining host–pathogen adaptations of a tick-borne rickettsia

The tick-borne intracellular pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae) develops persistent infections in cattle and tick hosts. While erythrocytes appear to be the only site of infection in cattle, A. marginale undergoes a complex developmental cycle in ticks and transmission occurs via salivary glands during feeding. Many geographic isolates occur that vary in genotype, antigenic composition, morphology and infectivity for ticks. In this chapter we review recent research on the host-vector-pathogen interactions of A. marginale. Major surface proteins (MSPs) play a crucial role in the interaction of A. marginale with host cells. The MSP1a protein, which is an adhesin for bovine erythrocytes and tick cells, is differentially regulated and affects infection and transmission of A. marginale by Dermacentor spp. ticks. MSP2 undergoes antigenic variation and selection in cattle and ticks, and contributes to the maintenance of persistent infections. Phylogenetic studies of A. marginale geographic isolates using msp4 and msp1alpha provide information about the biogeography and evolution of A. marginale: msp1alpha genotypes evolve under positive selection pressure. Isolates of A. marginale are maintained by independent transmission events and a mechanism of infection exclusion in cattle and ticks allows for only the infection of one isolate per animal. Prospects for development of control strategies by use of pathogen and tick-derived antigens are discussed. The A. marginale/vector/host studies described herein could serve as a model for research on other tick-borne rickettsiae.

03 鹿児島県の日本紅斑熱発生地における媒介マダニの調査

03 鹿児島県の日本紅斑熱発生地における媒介マダニの調査

Adhesion of outer membrane proteins containing tandem repeats of Anaplasma and Ehrlichia species (Rickettsiales: Anaplasmataceae) to tick cells

Infection of cells by tick-borne rickettsiae appears to be mediated by outer membrane proteins that allow pathogens to adhere to host cells. Major surface protein (MSP) 1a of Anaplasma marginale, the type species for the genus Anaplasma, was shown previously to be an adhesin for tick cells. The A. marginale MSP1a has a variable number of tandem 28 or 29 amino acid repeats located in the amino terminal region of the protein that contains an adhesion domain that is necessary and sufficient for infection of tick cells. The MSP1a studies demonstrated the importance of combining structural and functional characteristics for identification of adhesive proteins. In the present study other outer membrane proteins containing tandem repeats were selected from organisms of the family Anaplasmataceae and studied for their adhesive properties to tick cells. The adhesive properties and protein characteristics were then analyzed in order to provide a predictor of the adhesion function of proteins identified from genome sequences. Proteins selected included the A. marginale MSP1a, A. phagocytophilum 100 and 130 kDa, Ehrlichia chaffeensis 120 kDa, E. canis 140 kDa and E. ruminantium “mucin”, which were all cloned and expressed in Escherichia coli and then tested as adhesins for cultured IDE8 cells. Of the proteins studied, the A. marginale MSP1a and the E. ruminantium “mucin” were found to be adhesins for tick cells. Although all of these recombinant outer membrane proteins were glycosylated, the A. marginale MSP1a and E. ruminantium “mucin” adhesins shared a common feature of having a high Ser/Thr content in the tandem repeats. The results reported herein provide new information on the role of E. ruminantium “mucin” as an adhesin for tick cells and also suggest a role of glycans in adhesin molecules.

Fever after a tick bite: clinical manifestations and diagnosis of acute tick bite-associated infections in northeastern Switzerland

Different tick-borne infections can cause an acute febrile illness. The study objectives were to investigate the clinical manifestations and diagnosis of infections among patients who presented with fever after a tick-bite, and to detect newly described pathogens, including Ehrlichia, Babesia and Rickettsia helvetica, in North-Eastern Switzerland. : We studied 75 patients (41 male, 34 female, median age 38 years, among them 10 children) who had fever within 3 weeks after a tick-bite. Paired sera were tested for antibodies to Borrelia burgdorferi, tick-borne encephalitis virus, Anaplasma (Ehrlichia) phagocytophila, Babesia microti, B. divergens, and Rickettsia helvetica. In addition, microscopy and polymerase chain reaction was used to detect Ehrlichia. Clinical data were obtained at baseline and at 1 and 2 year follow-up. Tick-borne infections were confirmed or possible in 36 (48 %) patients: 7 (9 %) Erythema migrans, 6 (8 %) other specific manifestations of Lyme borreliosis, 6 (8 %) Lyme borreliosis presenting as non-specific febrile illness, 8 (11 %) tick-borne encephalitis, 7 (10 %) granulocytic ehrlichiosis, 1 B. microti infection in a traveler from the US and 6 (8 %) dual infections. In 8 (11 %) patients serological findings were suggesting possible acute or past R. helvetica infection. Among patients with fever after a tick-bite, Lyme borreliosis was most frequently found. There was no evidence for babesiosis among the resident population. Serologic data suggest that human granulocytic ehrlichiosis and R. helvetica infections may be endemic in Switzerland. Among 50 % of the patients no tick-borne infections could be diagnosed.

Development of an in vitro cloning method for Cowdria ruminantium.

Cowdria ruminantium is a tick-borne rickettsia which causes severe disease in ruminants. All studies with C. ruminantium reported so far were carried out with stocks consisting of infective blood collected from reacting animals or from the same stocks propagated in vitro. Cloned isolates are needed to conduct studies on immune response of the host, on genetic diversity of the parasite, and on mechanisms of attenuation and the development of vaccines. A method of cloning based on the particular chlamydia life cycle of Cowdria was developed. Instead of cloning extracellular elementary bodies, it appeared more convenient to clone endothelial cells infected by one morula resulting from the infection of the cell by one elementary body of Cowdria. Two hundred and sixteen clones were obtained by limiting dilution of infected cells. The method was experimentally validated by comparing randomly amplified polymorphic DNA fingerprints from individual clones obtained from endothelial cell cultures coinfected with two different stocks of C. ruminantium.

Inclusion appendages associated with the intraerythrocytic rickettsial parasiteAnaplasma marginale are composed of bundled actin filaments

Anaplasma marginale, a tick-borne rickettsia that infects erythrocytes of cattle, occurs within a parasitophorous vacuole or inclusion body. A tail-like inclusion appendage, composed of multiple filaments, occurs in association with the inclusion body membrane. The composition and function of the inclusion appendage have not been determined. In this study, theA. marginale inclusion appendage in bovine erythrocytes was found to be composed of actin filaments as determined by labeling with rhodamine-conjugated phalloidin. Electron microscopy studies revealed that theA. marginale inclusion appendages differed from F-actin tails reported previously in association with other pathogens in eukaryotic cells because these highly ordered structures were organized into regularly occurring striations, and the appendages were adhered directly to the parasitophorous vacuole membrane. In addition, actin appendages have not been described previously in erythrocytes. The potential role of the inclusion appendage associated withA. marginale in bovine erythrocytes and recently fed ticks is discussed.

Establishment of the tick (Acari:Ixodidae)-borne cattle pathogen Anaplasma marginale (Rickettsiales:Anaplasmataceae) in tick cell culture.

Anaplasma marginale is a tick-borne rickettsia that causes bovine anaplasmosis worldwide. Despite its importance, A. marginale has thus far not been established in a continuous culture system. We have propagated A. marginale continuously for the 1st time in a tick cell line derived from the black-legged tick, Ixodes scapularis Say, using infected bovine blood as the inoculum. Erythrocytic stages invaded the tick cells and multiplied in membrane-lined vacuoles to form colonies typical of those observed in naturally infected ticks as demonstrated by light and electron microscopy. The rickettsiae have been passaged serially for 3 yr and have been cryopreserved in liquid nitrogen. Antigens present in A. marginale from tick cell culture were recognized by bovine immune serum against the blood stages of A. marginale. A. marginale grown in this tick cell line was infective for calves, and male ticks fed on the calves transmitted A. marginale to a susceptible calf. The ability to culture A. marginale removes a major impediment to the study of Anaplasma biology in vitro, and will enhance development of vaccines and diagnostic tests.

徳之島におけるマダニ類と紅斑熱群リケッチアの調査

徳之島におけるマダニ類と紅斑熱群リケッチアの調査

DNA typing of rickettsiae in naturally infected ticks using a polymerase chain reaction/restriction fragment length polymorphism system.

We used the polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) rickettsial typing system of Regnery and others to rapidly identify rickettsiae in naturally infected ticks. Unlike previously described methods, our PCR assays type rickettsiae directly from tick tissues without first isolating the organisms. We collected 226 adult Dermacentor andersoni ticks in the Bitterroot Mountains of western Montana and analyzed them for possible rickettsial infection by hemolymph test using the Gimenez stain. Thirteen (5.8%) of these ticks were positive by hemolymph test and selected for further analysis using the above PCR/RFLP typing system. The PCR assays performed using the first primer set (RpCS) resulted in amplification of fragments of the predicted size from nine of the 13 hemolymph test-positive tick samples. Only four of these nine tick samples were also positive in similar PCR assays performed with a second primer set (Rr190) that is presumed to be spotted fever group specific. The RFLP analyses of material amplified from these four ticks indicated they were infected with Rickettsia rickettsii (one sample) and R. rhipicephali (three samples). The PCR/RFLP analyses of the five PCR-positive tick samples that were positive only in assays performed with the RpCS primer set indicated that these ticks were infected with R. bellii. The remaining four of 13 hemolymph test-positive tick samples gave negative PCR results with both the RpCS and Rr190 primer sets. Infected hemocytes from these PCR-negative ticks contained organisms of distinctive bacillary morphology that appeared similar to those described previously as long forms, and it is possible that these organisms belong to a genus other than Rickettsia. We also examined established laboratory isolates of tick-borne rickettsiae from different regions of North America to determine whether this typing system produces consistent results. Multiple isolates of R. montana (nine isolates), R. bellii (five isolates), R. rickettsii (Hlp-like) (four isolates), and R. canada (two isolates) were tested and no significant variations in PCR/RFLP patterns were observed between members of the same serotypes. However, among the five isolates of R. rhipicephali tested, two slightly different RFLP patterns were noted. Our results suggest that this PCR/RFLP typing scheme has wide applicability for identifying rickettsiae directly from D. andersoni or D. variabilis tick tissues.

Antigenic diversity of Cowdria ruminantium isolates determined by cross-immunity

Antigenic diversity in five stocks of the tick-borne rickettsia Cowdria ruminandum, the causal agent of heartwater disease of ruminants, was studied by cross-immunity trials in goats and sheep. Complete absence of cross-protection was found only between the Kümm and Kwanyanga stocks, and in all other combinations there were various degrees of cross-immunity. Immunological strain differences were more pronounced in goats than in sheep.

Ehrlichiosis in Children

Tick-borne rickettsiae of the genus Ehrlichia have recently been recognized as a cause of human illness in the United States. In the years 1986-1988, 10 cases of ehrlichiosis were diagnosed in children in Oklahoma. Fever and headache were universal: myalgias, nausea, vomiting, and anorexia were also common. Rash was observed in six patients but was a prominent finding in only one. Leukopenia, lymphopenia, and thrombocytopenia were common laboratory abnormalities. Six patients were treated with tetracycline, three with chloramphenicol, and one was not treated with antibiotics: all recovered. The onset of illness in spring and early summer for most cases paralleled the time when Amblyomma americanum and Dermacentor variabilis are most active, suggesting that one or both ticks may be vectors of human ehrlichiosis in Oklahoma.

Molecular Genetics of Populations of Intracellular Bacteria: The Spotted Fever Group Rickettsiae

The population structure of tick-borne rickettsiae show the following characteristics: (1) the amount of genetic differentiation between strains within subpopulations is very small. (2) The evolution of the subpopulations does not fit into models based on either host or geographic similarities, suggesting the need for more information on the frequency of vertical versus horizontal transmission of strains between hosts. (3) The species are highly clonal, with little evidence of genetic exchange between populations. (4) The dominant class of genetic change is single-nucleotide point mutation. No evidence for major rearrangements was observed. (5) Differentiation between species of the spotted fever group is equivalent to that seen between local strains of E. coli.

Ehrlichia canis Infection in a Child

Ehrlichia canis is a tick-borne rickettsia that causes disease in animals throughout the world. Its importance as a human pathogen, however, has only recently been documented. We report a child with serologically proven ehrlichiosis, to highlight differential diagnosis, to expand the spectrum of dermatologic manifestations, and to provide the first clinical evidence of therapeutic efficacy of chloramphenicol. CASE REPORT A 4-year-old boy from rural Missouri was admitted to the hospital on August 10, 1988, for evaluation of a fever of 2 weeks' duration. During the week before he became ill, his parents had removed numerous ticks from both the patient and the family's pet dog.

Rickettsia bellii sp. nov.: a Tick-Borne Rickettsia, Widely Distributed in the United States, That Is Distinct from the Spotted Fever and Typhus Biogroups

During studies to serotype tick-borne rickettsiae from various areas in the United States, particularly those areas associated with the vectors of Rickettsia rickettsii, we obtained 263 isolates of a rickettsia whose prototype is referred to as strain 369-C. Although rickettsiae of the strain 369-C serotype are distinct from rickettsiae of other serotypes, as determined by cross-micro-immunofluorescence tests of mouse antisera, they do possess one or more antigens that react with convalescent sera from Rocky Mountain spotted fever, epidemic typhus, and murine typhus patients. Because strain 369-C-like isolates have been recovered from eight species of argasid and ixodid ticks from widely separated areas of the United States, we set out to characterize these organisms more precisely, particularly with regard to their relationships to rickettsiae of the spotted fever and typhus groups. The characteristics which we used included morphology as determined by light microscopy and electron microscopy; cytopathogenicity in Vero cell cultures; pathogenicity for embryonated chicken eggs, laboratory mice, guinea pigs, and voles; protein composition as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; and deoxyribonucleic acid base composition. Our results indicated that strain 369-C-like rickettsiae are significantly different phenotypically from rickettsiae of both the spotted fever and typhus groups. Furthermore, the guanine-plus-cytosine content of the deoxyribonucleic acid of strain 369-C (30 mol%) was not different from the guanine-plus-cytosine content of rickettsiae of the typhus group, but it was lower than the guanine-plus-cytosine content recorded by other workers for rickettsiae of the spotted fever group. We propose that this species be named Rickettsia beliti sp. nov., with strain RML 369-C as the type strain; all serologically related rickettsiae should henceforth be referred to as the R. beliti serogroup.

Radiographic findings in Rocky Mountain spotted fever.

Rocky Mountain spotted fever is a febrile disease caused by a tick-borne rickettsia. Diffuse small vessel vasculitis may result in thrombosis of small vessels and capillary damage and can result in radiographic abnormalities. Cardiomegaly with pulmonary edema, interstitial infiltrates, patchy alveolar infiltrates, and pleural effusions may be detected in varying combinations on the chest radiograph. Soft-tissue necrosis may also be observed.

Comparative electrophoresis of spotted fever group rickettsial proteins

Electrophoretic analyses were performed to establish the polypeptide profiles of the following tick-borne typhus rickettsiae of the spotted fever group: Rickettsia rickettsii (Sheila Smith, Bitter Root, Iowa and R strains), R. sibirica, R. conorii, R. parkeri, R. australis and R. akari. Organisms were propagated in chick embryo fibroblast cells in roller bottles and purified by density centrifugation. After polyacrylamide gel electrophoresis, comparative electrophoretic profiles were examined by densitometric scanning. Of the more than 20 separate polypeptides detected, six of the most prominent were found in all rickettsiae examined except R. akari. No carbohydrate-containing moieties were detected in electrophoretic gels.

Rickettsioses (tick typhus, Q-fever, urban typhus) in Malaya.

Tick-borne rickettsiae occur primarily in the climatic climax forest formations which still cover much of peninsular Malaya. The jungle rats (Rattus bowersi, R. canus, R. cremoriventer, R. edwardsi, R. rajah, R. sabanus, R. surifer, R. muelleri, and R. whiteheadi) with their ectoparasitic Dermacentor, Ixodes and Haemaphysalis ticks are the reservoirs of tick typhus and Q-fever rickettsiae.