Serological Evidence of Rickettsia, Orientia, and Coxiella in Domestic Animals from Bhutan: Preliminary Findings.

There is no information on rickettsial diseases in domestic animals in Bhutan. This study provides preliminary serological data on exposure of domestic animals to Rickettsia, Orientia, and Coxiella. Animal sera were collected opportunistically from Bhutan and tested in the Australian Rickettsial Reference Laboratory for IgG antibodies against spotted fever group (SFG) and typhus group (TG) Rickettsia, scrub typhus group (STG), and Q fever (QF). Of the 294 animals tested, 136 (46%) showed serological evidence of past exposure to one or more rickettsiae: 106 (36%), 62 (21%), 45 (15%), and 11 (4%) being positive against SFG Rickettsia, Orientia, TG Rickettsia, and Coxiella, respectively. Dogs appeared to exhibit the highest seropositivity against SFG (55%) and TG Rickettsia (45%), horses against STG (91%), while goats were mostly positive for Coxiella (9%). Dogs also appeared to have high risk of being exposed to SFG Rickettsia (odd ratios [OR] 5.71, 95% confidence interval [CI] 3.02-10.80, p < 0.001), TG Rickettsia (OR 48.74, 95% CI 11.29-210.32, p < 0.001), and STG (OR 6.80, 95% CI 3.32-13.95, p < 0.001), but not against QF (OR 1.95, 95% CI 0.42-8.95, p = 0.390). Differences in seropositivity rates between animal species may have been significant for SFG, TG, and STG, but not for QF. The differences in the seropositivity rates of the four infections between districts appeared to be significant for TG and STG, but not for SFG and QF. The seropositivity rates of domestic animals to the four rickettsial infections were consistent with similar studies on the human population in the same areas and appear to demonstrate a high prevalence of exposure to rickettsiae in Bhutan. These preliminary findings constitute baseline data for Bhutan. The findings of this study call for an increased human-livestock sector collaboration in rickettsial diseases research aimed at developing diagnostic and therapeutic guidelines and formulating preventive and control measures through a One Health approach.

Serologic Evidence of the Geographic Distribution of Bacterial Zoonotic Agents in Kenya, 2007.

Diseases of zoonotic origin contribute to the burden of febrile illnesses in developing countries. We evaluated serologic evidence of exposure to Bacillus anthracis, Brucella spp., spotted fever group rickettsioses (SFGR), and typhus group rickettsioses (TGR) from samples of persons aged 15-64 years collected during a nationwide human immunodeficiency virus (HIV) serosurvey conducted in 2007 in Kenya. The seropositivity observed for pathogens was B. anthracis 11.3%, Brucella spp. 3.0%, SFGR 23.3%, and TGR 0.6%. On univariate analysis, seropositivity for each pathogen was significantly associated with the following risk factors: B. anthracis with province of residence; Brucella spp. with sex, education level, and wealth; SFGR with age, education level, wealth, and province of residence; and TGR with province of residence. On multivariate analysis, seropositivity remained significantly associated with wealth and province for B. anthracis; with sex and age for Brucella spp; and with sex, education level, and province of residence for SFGR whereas TGR had no significance. High IgG seropositivity to these zoonotic pathogens (especially, B. anthracis and SFGR) suggests substantial exposure. These pathogens should be considered in the differential diagnosis of febrile illness in Kenya.

A survey for rickettsial agents on Rhipicephalus sanguineus (Ixodida, Ixodidae) ticks in Northeastern Brazil / Inquérito para agentes rickettsiais em carrapatos Rhipicephalus sanguineus (Ixodida, Ixodidae) no Nordeste do Brasil

In this study, rickettsial infection was searched in 108 canine blood samples and 22 Rhipicephalus sanguineus (Ixodida,Ixodidae) ticks collected on these dogs during 2011 and 2012 in Patos municipality, state of Paraíba, northeasternBrazil. Blood samples were tested through indirect immunofluorescence assay (IFA) by using antigens of six Rickettsiaspecies isolates from Brazil. All 108 dogs tested seronegative for R. rickettsii, R. parkeri, R. amblyommii, R. felis, R.rhipicephali, and R. bellii antigens, suggesting a non-endemic status of the studied region for spotted fever ricketsiosis.Among 22 R. sanguineus ticks, R. felis was detected in one (4.5%) specimen by PCR targeting a portion of the rickettsialgltA gene. The possible implications of this unusual PCR finding are discussed.

No presente trabalho, foi pesquisada infecção riquetsial em amostras de sangue de 108 cães e 22 carrapatos Rhipicephalussanguineus (Ixodida, Ixodidae) coletados destes animais durante 2011 e 2012, em Patos, Estado da Paraíba, Nordestedo Brasil. As amostras de sangue foram examinadas pela reação de imunofluorescência indireta (RIFI) utilizando-seantígenos de seis isolados de Rickettsia do Brasil. Todos os 108 cães foram soronegativos para os antígenos de R.rickettsii, R. parkeri, R. amblyommii, R. felis, R. rhipicephali e R. bellii, sugerindo que a região estudada não é endêmicapara riquetsioses do grupo da febre maculosa. Dos 22 carrapatos R. sanguineus, R. felis foi detectada em um (4,5%)espécime por PCR do gene riquetsial gltA. São discutidas as possíveis implicações desse achado incomum na PCR.

Real-time multiplex PCR assay for detection and differentiation of rickettsiae and orientiae.

The high incidence of rickettsial diseases in Southeast Asia necessitates rapid and accurate diagnostic tools for a broad range of rickettsial agents, including Orientia tsutsugamushi (scrub typhus) and Rickettsia typhi (murine typhus), but also spotted fever group infections, which are increasingly reported. We present an SYBR-Green-based, real-time multiplex PCR assay for rapid identification and differentiation of scrub typhus group, typhus group and spotted fever group rickettsiae using 47kDa, gltA and ompB gene targets. Detection limits for amplification of these genes in reference strains ranged from 24 copies/microl, 5 copies/microl and 1 copy/microl in multiplex and 2 copies/microl, 1 copy/microl and 1 copy/microl in single template format, respectively. Differentiation by melt-curve analysis led to distinct melt temperatures for each group-specific amplicon. The assay was subjected to 54 samples, of which all cell-culture and 75% of characterised clinical buffy coat samples were correctly identified. Real-time PCR has the advantage of reliably detecting and differentiating rickettsial and orientia cell-culture isolates in a single-template assay, compared with the more time-consuming and laborious immunofluorescence assay. However, further optimisation and validation on samples taken directly from patients to assess its clinical diagnostic utility is required.

Infección por Rickettsia slovaca tras la picadura de una garrapata / Rickettsia slovaca infection alter a tick bite

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[Microorganism "Montezuma" of the order Rickettsiales: the potential causative agent of tick-borne disease in the Far East of Russia]. / Mikroorganizm "Montezuma" iz poriadka Rickettsiales: potentsial'nyi vozbuditel' kleshchevogo transmissivnogo zabolevaniia na Dal'nem Vostoke Rossii.

A new microorganism, tentatively named "Montezuma" was detected in ticks and in specimens (blood, bioptic specimens of the primary affect) taken from patients with an acute fever disease, etiologically linked with the bites of Ixodes ticks in the Far East of the Russian Federation. After sequencing the products of the amplification of DNA isolated from ticks with wide-spectrum primers new primers were developed, highly specific to the unusual sequence thus obtained. The study revealed that ticks of the species Ixodes persulcatus (97%) and Haemophysalis concinnae (5%) contained DNA of this microorganism. The same DNA was detected in materials taken from the patients. The phylogenetic analysis of the gene showed that this organism formed an independent and well defined branch within the order Rickettsiales. The nearest homology (89%) was observed with recently detected endosymbiotes Acanthamoeba. The similarity with their relatives from the families Rickettsiaceae and Anaplasmataceae of the order Rickettsiales was within 81-86%, which made it possible to infer the existence of, probably, only a new genus, but also a family. The isolated DNA belonged, supposedly, to the new microoganism which caused a tick-borne disease in humans, transmitted through bites of Ixodes ticks, and was, supposedly, widely spread in the southern area of the Khabarovsk Territory.

Rickettsial diseases and their serological diagnosis.

Rickettsial diseases (typhus and spotted fever group rickettsioses, scrub typhus and Q fever) may pose a serious public health problem, namely when they are non-diagnosed or misdiagnosed. Although rickettsiae can be isolated from or detected in clinical specimens, serological tests still remain an indispensable tool in the diagnosis of rickettsial diseases. The complement fixation test widely used in the past is being replaced by other tests which make differentiation of immunoglobulin classes possible. Of these tests microimmunofluorescence is considered the test of choice followed by the latex agglutination, indirect hemagglutination, immunoperoxidase assay, and enzyme-linked immunosorbent assay. The last one is also suitable for seroepidemiological studies. Immunoblot analysis can be used to confirm the results of other tests. The use of the low-specific and low-sensitive Weil-Felix test should be reserved only for situations in which other serologic tests are not available.

Cowdria ruminantium antibodies in acaricide-treated and untreated cattle exposed to Amblyomma variegatum ticks in The Gambia.

An indirect enzyme-linked immunosorbent assay (ELISA), based on the major antigenic protein I fragment B (MAPI-B) of Cowdria ruminantium, was used to assess seroprevalence in cattle in The Gambia. Two groups of 20 N'Dama and 20 Gobra zebu cattle were monitored for 12 months with flumethrin treatment and for another 10 months without acaricidal treatment. Two groups of 20 N'Dama and 20 Gobra cattle served as untreated controls. During the period of acaricidal treatment, the cumulative proportions of positive serum samples were 25.6 +/- 5.6% (+/- confidence interval) and 34.7 +/- 6.8% in treated N'Dama and Gobra cattle respectively; the proportion of positive sera in untreated cattle was 52.2 +/- 6.9% in N'Damas and 61.4 +/- 7.3% in Gobras. Within breed, difference in antibody prevalence between treated and untreated cattle was significant (P < 0.001) but between breed differences were not significant. In the 10 months following suspension of acaricide application, there was an increase of proportion of positive serum samples in previously treated N'Dama and Gobra cattle. In both previously treated and untreated animals the peak of positive seroreactions occurred during and subsequent to the period of activity of Amblyomma variegatum adults. Cumulative seroprevalences in previously treated N'Dama and Gobra cattle were 32.6 +/- 6.9% and 44.7 +/- 8.5%, respectively; in untreated animals seroprevalence was 38.6 +/- 7.2% in N'Dama and 65.3 +/- 8.4% in Gobra cattle. Throughout the study period, within the N'Dama breed, the seropositive rate in previously treated cattle did not differ from that in untreated animals. Conversely, within the Gobra breed, the number of positive seroreactions was higher (P < 0.002) in untreated animals than in previously treated cattle. These results provide a support for designing A. variegatum and heartwater control strategies, if necessary, in The Gambia in relation to cattle breeds.

Species-specific BALB/c mouse antibodies to rickettsiae studied by western blotting.

BALB/c mice were inoculated intraperitoneally either once only, or up to four times at weekly intervals, with viable Rickettsia rickettsii, Rickettsia conorii or the Israeli spotted fever group rickettsia. Sera collected one week after the last inoculation were tested for the presence of antibodies reactive with the above organisms by indirect fluorescent antibody testing and Western blot. With repeated inoculations there was a general progressive rise in homologous and heterologous immunofluorescence titers although the increase after the first inoculation was always the greatest. For each rickettsia, the homologous titers were higher than the heterologous titers. Western blots showed that the reactive antibodies were against rickettsial high molecular mass species specific protein antigens and homologous species-specific antibody reactions were detectable earlier than heterologous cross-reacting antibody reactions. Antibodies in mice sera did not react with the group specific lipopolysaccharide-like antigens of the rickettsiae although such reactivity was strong in Western blots with sera from patients suffering from acute Rickettsia conorii infections. Our findings suggest that the intraperitoneal route of inoculation of BALB/c mice can be used for the differentiation of spotted fever group rickettsiae.

Analyses of Ehrlichia canis and a canine granulocytic Ehrlichia infection.

Ehrlichia canis and canine granulocytic Ehrlichia sp. (CGE) infect canine monocytes and granulocytes, respectively. E. canis has been cultured in vitro and used to develop an immunofluorescence assay. CGE has not been cultured, and a serologic assay is not available. The sera of dogs infected with CGE were reported to react with E. canis by immunofluorescence. In this study, the temporal response of immunoglobulin G (IgG) was determined by an enzyme-linked immunosorbent assay (ELISA) with purified E. canis antigen in four dogs experimentally infected with E. canis, in two dogs experimentally infected with CGE, and in one dog infected with E. canis and subsequently infected with CGE. E. canis-infected dogs developed an IgG ELISA result of 1.5 or greater for the optical density signal/noise ratio by 2 months postinfection. CGE challenge of a dog with a previous E. canis infection induced an anamnestic increase in the IgG ELISA result; however, CGE infection alone did not induce a significant IgG ELISA response. Western immunoblot analysis showed that dogs infected with E. canis developed antibodies initially that reacted with low-molecular-mass proteins (30, 24, and 21 kDa) and subsequently with higher-molecular-mass proteins (160, 100, 78, 64, 47, and 40 kDa). In contrast, CGE-infected dogs showed reactions with the same higher-molecular-mass proteins of E. canis but, unlike E. canis-infected dogs, not with the low-molecular-mass proteins of E. canis. Of 10 serum samples collected in the field of Indonesia from dogs with tropical canine pancytopenia, all had an optical density signal minus noise value of 2.54 or greater in the IgG ELISA and reacted with E. canis antigen in a pattern similar to that of serum samples from dogs experimentally infected with E. canis in Western immunoblotting. This study suggests that the IgG ELISA and Western immunoblotting with purified E. canis as the antigen are useful in distinguishing between E. canis and CGE infections in dogs.

Lymphocyte subpopulations in peripheral blood of sheep experimentally infected with tick-borne fever.

The lymphocyte subpopulations in the peripheral blood of normal sheep and sheep experimentally infected with Cytoecetes phagocytophila, the causative agent of tick-borne fever, were analysed by flow cytometry, using a panel of monoclonal antibodies against specific lymphocyte epitopes. Experimental infection with tick-borne fever was characterised by a significant reduction in the total number of circulating lymphocytes six days after experimental infection (P less than 0.001). This lymphocytopenia was associated with a significant reduction in the number of B (LCAp220+) and T (CD5+) lymphocytes (P less than 0.001) but there was a significant increase in the number of cells which were neither T nor B (CD5-LCAp220-) cells (P less than 0.01). The reduction in the number of T lymphocytes was due to reduced numbers of circulating CD4+ (helper) T cells, CD8+ (cytotoxic/suppressor) T cells and those with the pan T cell marker (CD5+) but without CD4 or CD8 epitopes (CD4-CD8-). All lymphocytes returned to preinoculation levels 13 to 16 days after experimental infection.

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.

Expression of gamma interferon on circulating lymphocytes in viral infections.

Peripheral blood lymphocytes from a total of 111 patients and 40 healthy individuals were studied for gamma interferon (IFN-gamma) expression on their surfaces by indirect immunofluorescence assay and flow cytometry, with a new anti-IFN-gamma monoclonal antibody (IGMB-14) as a specific reagent. Of 64 patients with proven acute viral infections, 59 had a significantly higher percentage of lymphocytes expressing IFN-gamma on their membranes than healthy individuals did. On the other hand, only 3 (8.9%) of 34 patients with proven bacterial infections had an increased percentage of IFN-gamma-expressing lymphocytes. None of the eight patients with other infections and none of the five with systemic lupus erythematosus showed an increased percentage of IFN-gamma-positive lymphocytes. The percentage of IFN-gamma-expressing lymphocytes during a viral infection was found to be related to different stages of the disease. Finally, some applications of this rapid IFN-gamma assay method in viral diseases are discussed.

Disease features in horses with induced equine monocytic ehrlichiosis (Potomac horse fever).

Fifty-five horses were inoculated IV and/or SC with materials containing Ehrlichia risticii, ie, infected whole blood, buffy coat cells, or cell culture, to study clinical and hematologic features of equine monocytic ehrlichiosis (Potomac horse fever). Major clinical and hematologic features of induced E risticii infection were biphasic increase in rectal temperature with peak increases of 38.9 C and 39.3 C on postinoculation days (PID) 5 and 12, respectively; depression; anorexia; decreased WBC count (maximal decrease of 47% on PID 12); and diarrhea from PID 14 to PID 18. Increased WBC count was an inconsistent feature, with a maximal increase of 51.5% on PID 20. During times of decreased and increased WBC counts, lymphocyte/neutrophil ratios remained fairly constant. However, not all horses had all clinical and hematologic features, and these features were present in different degrees among horses. Increased rectal temperature, depression, anorexia, and decreased WBC count were more consistent features, whereas diarrhea developed in 73% of the horses. Of 55 horses, 39 (71%) had all clinical and hematologic features of the disease (classic disease), whereas 16 (29%) horses did not have greater than or equal to 1 of these features (nonclassic disease). The E risticii titer in the blood (ehrlichemia) was maximum during the peak increase in rectal temperature. In 55 horses, mortality was 9%. Significant differences (P greater than 0.5) in clinical and hematologic features were not detected between horses that survived and those that died of E risticii infection.

Effect of equine ehrlichial colitis on the hemostatic system in ponies.

Hemostatic function was determined in 10 ponies at various times after inoculation with Ehrlichia risticii to determine whether equine ehrlichial colitis (EEC) caused changes in the hemostatic system and to determine the prognostic value of hemostatic function tests during EEC. Mean platelet count; plasma fibrinogen, fibronectin, factor VIII: coagulant, alpha 2-antiplasmin, and plasminogen values; and serum concentrations of fibrin/fibrinogen degradation products changed significantly (P less than 0.05) from base line (day 0, before inoculation) during 18 days after inoculation with E risticii. Four ponies that died or were euthanatized because of severe clinical signs of EEC had significantly (P less than 0.05) greater mean plasma fibrinogen concentrations plasma factor VIII:coagulant values, and activated partial thromboplastin times immediately before death than did the 6 surviving ponies. Factor V concentrations were significantly (P less than 0.05) lower on postinoculation days 10 and 20 in nonsurvivors. Seemingly, changes in hemostasis took place during EEC. Ponies that did not survive EEC had greater laboratory evidence of coagulopathy.

Anemia of inflammation in dogs infected with Ehrlichia platys.

Ten adult male dogs were inoculated with Ehrlichia platys, and blood samples were collected throughout the infection to evaluate the hematologic changes with respect to serum biochemical analytes. All dogs developed a mild, normocytic, normochromic anemia by postinoculation day 7, with significantly (P less than 0.05) decreased serum iron concentration and total iron-binding capacity. Stainable bone marrow iron appeared normal or increased throughout the infection. By postinoculation day 31, the PCV was not significantly different from the pretreatment value. All dogs became hypergammaglobulinemic, leukopenic, hypoalbuminemic, and hypocalcemic during the infection. These findings were compatible with the syndrome of anemia of inflammation.

Acute Ehrlichia platys infection in the dog.

Ten dogs were inoculated with Ehrlichia platys (E. platys) from an acutely infected dog. Two dogs were necropsied on each of days 7, 14, 21, 28, and 35 post-inoculation, and tissues were collected and either fixed in formalin or frozen for light microscopic examination of lesions or E. platys antigen localization in tissues. Serum antibody titers to E. platys and serum aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase activities were also determined. The significant light microscopic findings were lymph node follicular hyperplasia and crescent-shaped hemorrhages in the splenic periarteriolar lymphoid sheaths beginning day 7 post-inoculation. There was significant megakaryocyte hyperplasia of bone marrow on days 28 and 35 post-inoculation. Ehrlichia platys antigen was in macrophages at 14 days post-inoculation which corresponded to the initial decline in platelet numbers. Initial thrombocytopenia and splenic crescent-shaped hemorrhages were temporally related, however the degree of lesion development and prominence were not related to subsequent platelet numbers.