Serosurvey of arthropod-borne diseases among shelter dogs in the Cumberland Gap Region of the United States.

BACKGROUND: The Cumberland Gap Region (CGR) of the United States is a natural corridor between the southeastern, northeastern, and midwestern regions of the country. CGR has also many species of ticks and mosquitos that serve as competent vectors for important animal and human pathogens. In this study, we tested dogs from six different animal shelters in the CGR for Rocky Mountain spotted fever (RMSF), anaplasmosis, Lyme disease, canine ehrlichiosis and canine heartworm disease. RESULTS: Sera from 157 shelter dogs were tested for antibodies to RMSF agent, Rickettsia rickettsii, using an indirect immunofluorescence assay. Sixty-six dogs (42.0%) were positive for either IgM or IgG, or both IgM and IgG antibodies to R. rickettsii. Moreover, the same set of sera (n = 157) plus an and additional sera (n = 75) from resident dogs at the same shelters were tested using the SNAP 4Dx Plus. Of 232 dogs tested, two (0.9%) were positive for antibodies to Anaplasma phagocytophilum/A. platys, nine (3.9%) were positive for antibodies to Borrelia burgdorferi, 23 (9.9%) for positive for antibodies to Ehrlichia canis/E. ewingii, and 13 (5.6%) were positive for Dirofilaria immitis antigen. Co-infection with two or more etiologic agents was detected in five animals. Three dogs had antibodies to both B. burgdorferi and E. canis/E. ewingii, and two dogs were positive for D. immitis antigen and antibodies to B. burgdorferi and E. canis/E. ewingii. CONCLUSIONS: Shelter dogs in the CGR are exposed to a number of important vector-borne pathogens. Further studies are required to ascertain the roles these animals play in maintenance and transmission of these pathogens.

Laboratory Blood-Based Testing for Non-Lyme Disease Tick-Borne Infections at a National Reference Laboratory.

OBJECTIVES: We evaluated trends in non-Lyme disease tick-borne disease (NLTBI) testing at a national reference laboratory. METHODS: Testing data performed at Quest Diagnostics during 2010 to 2016 were analyzed nationally and at the state level. RESULTS: Testing and positivity for most NLTBIs increased dramatically from 2010 through 2016 based on testing from a large reference laboratory. The number of positive cases, though not as stringent as criteria for public health reporting, generally exceeds that reported by the Centers for Disease Control and Prevention. The frequency of NLTBI in the US is seasonal but testing activity and positive test results are observed throughout all months of the year. Positive results for NLTBI testing mostly originated from a limited number of states, indicating the geographic concentration and distribution of NLTBIs reported in this study. CONCLUSIONS: This report provides an important complementary source of data to best understand trends in and spread of NLTBI.

Development of an electrochemical immunosensor for the diagnostic testing of spotted fever using synthetic peptides.

Spotted fever is a rare acute and multisystemic febrile infectious disease with a mortality rate of ≥50% without adequate antibiotic treatment, and in diagnosed and treated cases, of approximately 2.5%. Currently, the applied test to diagnose this disease is the indirect immunofluorescence reaction, however two samples of paired sera are necessary to confirm the diagnosis, since using only one sample may allow for confusion with cross reactions. OmpA is an outer membrane protein present in the R. rickettsia, the etiological agent of spotted fever, able to activate dendritic and macrophage cells. It also presents immunogenicity properties, and is considered a target for the development of diagnostic tests for spotted fever. In this context, an amperometric immunosensor was developed for the identification of sera antibodies (human IgG) from patients with spotted fever aimed at improving sensitivity and minimize sample volume. The development of the immunosensor was conducted using a synthetic peptide, derivative from the H6PGA4 R. rickettsia protein, homologous to OmpA. Amperometric responses were generated at -0.6 to 0.6V, at a scan rate of 0.025Vs-1 for 20 cycles, a limit of detection of approximately 10ngmL-1 for the synthetic peptides and 0.01µgmL-1 for the humam serum, a sensitivity of 2.59µA, adequate for the detection of spotted fever antibodies. The construction of this immunosensor, capable of identifying circulating antibodies in real time, can also be applied in the diagnosis of other infectious-parasitic diseases.

Molecular and serological evidence of flea-associated typhus group and spotted fever group rickettsial infections in Madagascar.

BACKGROUND: Rickettsiae are obligate intracellular bacteria responsible for many febrile syndromes around the world, including in sub-Saharan Africa. Vectors of these pathogens include ticks, lice, mites and fleas. In order to assess exposure to flea-associated Rickettsia species in Madagascar, human and small mammal samples from an urban and a rural area, and their associated fleas were tested. RESULTS: Anti-typhus group (TGR)- and anti-spotted fever group rickettsiae (SFGR)-specific IgG were detected in 24 (39%) and 21 (34%) of 62 human serum samples, respectively, using indirect ELISAs, with six individuals seropositive for both. Only two (2%) Rattus rattus out of 86 small mammals presented antibodies against TGR. Out of 117 fleas collected from small mammals, Rickettsia typhi, a TGR, was detected in 26 Xenopsylla cheopis (24%) collected from rodents of an urban area (n = 107), while two of these urban X. cheopis (2%) were positive for Rickettsia felis, a SFGR. R. felis DNA was also detected in eight (31%) out of 26 Pulex irritans fleas. CONCLUSIONS: The general population in Madagascar are exposed to rickettsiae, and two flea-associated Rickettsia pathogens, R. typhi and R. felis, are present near or in homes. Although our results are from a single district, they demonstrate that rickettsiae should be considered as potential agents of undifferentiated fever in Madagascar.

Experimental infection of horses with Rickettsia rickettsii.

BACKGROUND: Rickettsia rickettsii is vectored by ticks, and some vertebrate hosts can be sources of infection to ticks during bacteremic periods. In Brazil, the main vector for R. rickettsii is the tick Amblyomma sculptum, a member of the A. cajennense complex. Horses, in turn, are one of the major hosts for A. sculptum. In this study, horses experimentally infected with R. rickettsii were assessed for clinical changes and their capability to transmit the infection to A. sculptum ticks. METHODS: Four horses were infected with R. rickettsii through either intraperitoneal injection or infestation with R. rickettsii-infected A. sculptum ticks. Simultaneously, the animals were infested with non-infected A. sculptum ticks. The horses were monitored for 30 days by clinical examination, hematological and biochemical tests, real-time PCR of blood for the detection of Rickettsia, and inoculation of blood in guinea pigs. IgG antibody titers were followed until the horses have shown seronegativity or until the end of the experiment. Uninfected ticks that fed on horses were subjected to real-time PCR and/or were fed on susceptible rabbits. RESULTS: The horses showed no clinical, hematological or blood biochemical alterations, and bacteremia was not detected by real-time PCR or by inoculation of horse blood into guinea pigs. Anti-R. rickettsii antibodies were detected in horses from 10 days to 2 years after infection. Uninfected ticks, after feeding on infected horses, showed 2.1 % positivity in real-time PCR, but failed to transmit the infection to rabbits at a next feeding stage. CONCLUSIONS: Rickettsia rickettsii-infected horses did not manifest illness and are not competent amplifier hosts of R. rickettsii for A. sculptum ticks.

High Seroprevalence for Rickettsia rickettsii in Equines Suggests Risk of Human Infection in Silent Areas for the Brazilian Spotted Fever.

Equines play a role in the epidemiology of Brazilian spotted fever (BSF) since they are a primary host for the tick Amblyomma sculptum. We studied the seroprevalence for three species of Rickettsia in equines in four endemic (with human cases) and in four non-endemic areas (no human cases) in the Piracicaba River Basin, São Paulo, Brazil. A serological survey of 504 equines was performed: around 63 animals were sampled in each area and tested through indirect immunofluorescence assay for R. rickettsii, R. parkeri, and R. bellii in 2012-2013. Blood samples were seropositive for 183 equines (36.3%) in which 73 (39.9%) were from non-endemic areas. In the studied sites equines were highly exposed to Rickettsia infection ranging from 6.1% to 54.7%, with Geometric Mean Titers greater in endemic area (p = 0.012). Results suggest that Rickettsia may be more widespread than the surveillance of BSF has detected. These results highlight the need to include data on the seroprevalence of sentinel animals to improve human diagnoses and surveillance in areas with no reported human cases.

Clinical presentation, convalescence, and relapse of rocky mountain spotted fever in dogs experimentally infected via tick bite.

Rocky Mountain spotted fever (RMSF) is a tick-borne disease caused by R. rickettsii in North and South America. Domestic dogs are susceptible to infection and canine RMSF can be fatal without appropriate treatment. Although clinical signs of R. rickettsii infection in dogs have been described, published reports usually include descriptions of either advanced clinical cases or experimental infections caused by needle-inoculation of cultured pathogen rather than by tick bite. The natural progression of a tick-borne R. rickettsii infection has not been studied in sufficient detail. Here, we provide a detailed description of clinical, hematological, molecular, and serological dynamics of RMSF in domestic dogs from the day of experimental exposure to infected ticks through recovery. Presented data indicate that neither the height/duration of fever nor detection of rickettsial DNA in dogs' blood by PCR are good indicators for clinical prognosis. Only the apex and subsequent subsidence of neutrophilia seem to mark the beginning of recovery and allow predicting a favorable outcome in Rickettsia-infected dogs, even despite the continuing persistence of mucosal petechiae and skin rash. On the other hand the appropriate (doxycycline) antibiotic therapy of sufficient duration is crucial in prevention of RMSF relapses in dogs.

Rickettsial infections of dogs, horses and ticks in Juiz de Fora, southeastern Brazil, and isolation of Rickettsia rickettsii from Rhipicephalus sanguineus ticks.

The present study was performed in an area endemic for Brazilian spotted fever (BSF) in Juiz de Fora, state of Minas Gerais, Brazil, during the years 2007 and 2008, when fatal cases of BSF (caused by Rickettsia rickettsii) were reported. Adult ticks (Acari: Ixodidae) identified as Rhipicephalus sanguineus (Latreille) and Amblyomma cajennense (Fabricius) were collected from dogs and horses, respectively, and tested by polymerase chain reaction (PCR). Overall, 13.1% of the Rh. sanguineus ticks and none of the A. cajennense were found to be infected with R. rickettsii. Two isolates of R. rickettsii were successfully established in Vero cell culture from two Rh. sanguineus ticks. An indirect immunofluorescence assay (IFA) using R. rickettsii antigens detected blood serological reaction to R. rickettsii in 67.9% (53/78) of dogs and 41.0% (16/39) of horses living in the study area. Larval offspring from two Rh. sanguineus engorged females, naturally infected by R. rickettsii, were reared to adult stage in the laboratory. All active stages (larvae, nymphs, adults) remained 100% infected by R. rickettsii, which was efficiently transmitted to naïve rabbits. Overall, the results of the present study indicate a potential risk for transmission of R. rickettsii to humans by Rh. sanguineus, an occurrence yet to be documented in Brazil.

Serological survey of Rickettsia sp. in horses and dogs in a non-endemic area in Brazil.

Brazilian Spotted Fever (BSF) is a lethal rickettsiosis in humans caused by the bacteria Rickettsia rickettsii, and is endemic in some areas of Brazil. Horses and dogs are part of the disease's life cycle and they may also serve as sentinel animals in epidemiological studies. The first human BSF case in the State of Paraná was reported in 2005. The present study was conducted in the municipality of Almirante Tamandaré, where no previous case of BSF was reported. Serum samples were collected from 71 horses and 20 dogs from nine properties in the area. Ticks were also collected from these animals. All farmers completed a questionnaire about their knowledge of BSF and animal health management. Serum samples were analyzed by indirect immunofluorescent-antibody assay (IFA) using R. rickettsii and R. parkeri as antigens. Ticks were analyzed by PCR for Rickettsia sp., and all of them were PCR-negative. Six horses (8.45%) and 4 dogs (20%) were identified as seropositive. Farmers were not aware of the correlation between the presence of ticks and risk of BSF. Although a non-endemic area, Almirante Tamandaré is a vulnerable environment for BSF and effective tick control measures are required.

Serological survey of Rickettsia sp. in horses and dogs in an non-endemic area in Brazil / Identificação sorológica de Rickettsia sp. em equinos e cães de área não endêmica no Brasil

Brazilian Spotted Fever (BSF) is a lethal rickettsiosis in humans caused by the bacteria Rickettsia rickettsii, and is endemic in some areas of Brazil. Horses and dogs are part of the disease's life cycle and they may also serve as sentinel animals in epidemiological studies. The first human BSF case in the State of Paraná was reported in 2005. The present study was conducted in the municipality of Almirante Tamandaré, where no previous case of BSF was reported. Serum samples were collected from 71 horses and 20 dogs from nine properties in the area. Ticks were also collected from these animals. All farmers completed a questionnaire about their knowledge of BSF and animal health management. Serum samples were analyzed by indirect immunofluorescent-antibody assay (IFA) using R. rickettsii and R. parkeri as antigens. Ticks were analyzed by PCR for Rickettsia sp., and all of them were PCR-negative. Six horses (8.45 percent) and 4 dogs (20 percent) were identified as seropositive. Farmers were not aware of the correlation between the presence of ticks and risk of BSF. Although a non-endemic area, Almirante Tamandaré is a vulnerable environment for BSF and effective tick control measures are required.

A Febre Maculosa Brasileira (FMB) é uma riquetsiose letal para humanos, causada pela bactéria Rickettsia rickettsii, e é endêmica em algumas regiões brasileiras. Equinos e cães podem participar do ciclo da doença e podem também servir como sentinelas em estudos epidemiológicos. O primeiro caso humano relatado no Estado do Paraná ocorreu em 2005. O presente estudo foi realizado no município de Almirante Tamandaré, região onde não há relatos de casos de FMB. Foram coletadas amostras de sangue de 71 cavalos e 20 cães em nove propriedades rurais na região. Carrapatos também foram colhidos dos animais. Todos os proprietários responderam a um questionário sobre o manejo sanitário dos animais e o conhecimento a respeito da FMB. As amostras de soro foram processadas pela técnica de Reação de Imunofluorescência Indireta (RIFI), utilizando-se os antígenos de R. rickettsii e R. parkeri. Os carrapatos foram analisados por PCR para Rickettsia sp. e todos foram negativos. Seis cavalos (8,45 por cento) e 4 cães (20 por cento) foram identificados como soropositivos. Todos os proprietários desconheciam a relação de carrapatos com a FMB. Embora considerada uma área não endêmica, Almirante Tamandaré é um ambiente vulnerável à FMB e um controle eficiente de carrapatos deve ser implementado.

Detection of Rickettsia rickettsii and Rickettsia sp. in blood clots in 24 patients from different municipalities of the State of Sao Paulo, Brazil.

The authors detected Rickettsia genus organisms using shell vial and polymerase chain reaction (PCR)/sequencing analysis in blood clots in patients suspected of having Brazilian spotted fever (BSF). DNA was detected using PCR with three sets of primers to access the gltA, ompA, and ompB genes. Sequence analysis was carried out using an automatic sequencer with Bioedit software. Seventy-five percent of the culture samples were positive and all samples amplified rickettsial gene fragments. To date, 46% of the samples have been sequenced.

Myocardial involvement in rocky mountain spotted fever: a case report and review.

Rocky Mountain Spotted Fever (RMSF), caused by Rickettia rickettsii, is a serious tickborne illness that is endemic in the southeastern United States. Although it is most commonly known as a cause of fever and rash, it can have systemic manifestations. The myocardium may rarely be involved, with symptoms that can mimic those of acute coronary syndromes. This report describes a case of serologically proven RMSF causing symptomatic myocarditis, manifested by chest pain, elevated cardiac enzyme levels, and decrease myocardial function. After treatment with antibiotics, the myocarditis resolved. Thus, although unusual, the clinician should be aware of myocardial disease in patients with appropriate exposure histories or other clinical signs of RMSF. Close monitoring and an aggressive approach are essential to reduce mortality rates.

Prevalence of antibodies to spotted fever group rickettsiae in humans and domestic animals in a Brazilian spotted fever-endemic area in the state of São Paulo, Brazil: serologic evidence for infection by Rickettsia rickettsii and another spotted fever group Rickettsia.

In serum samples obtained from all the healthy humans, horses, dogs, and donkeys present on three farms in the Pedreira Municipality, an endemic area for Brazilian spotted fever, an indirect immunofluorescence assay (IFA) detected antibodies against Rickettsia rickettsii in 17 (77.3%) horses, 5 (31.3%) dogs (titers ranging from 64 to 4,048), and none of 4 donkeys or 50 humans. Five canine and eight equine sera with high antibody titers to R. rickettsii were also tested by IFA against R. bellii, R. akari, and R. africae antigens. Sera from two horses and two dogs that showed similar high antibody titers against two rickettsial antigens were evaluated after cross-absorption. Sera from seven horses and two dogs contained antibodies specific for R. rickettsii, and one dog serum had antibodies against a Rickettsia species very closely related to R. africae. The latter may have been caused by infection with the recently identified COOPERI strain.

[Emerging and reemerging rickettsiosis in an endemic area of Minas Gerais State, Brazil]. / Rickettsioses emergentes e reemergentes numa região endêmica do Estado de Minas Gerais, Brasil.

This article describes a serological survey for rickettsiosis in the county of Novo Cruzeiro, Minas Gerais State, Brazil, in 1998, testing schoolchildren and dogs. Sera included 331 samples from schoolchildren from an endemic area and 142 samples from schoolchildren from a non-endemic area in the county. All children examined were healthy and had not reported clinical symptoms of Brazilian spotted fever prior to the serological survey. Some 35 children in the endemic area were reactive to Rickettsia rickettsiiby indirect fluorescent antibody (IFA) with a titer of 1:64, corresponding to 10.6%. Sera from 73 dogs were tested, showing seroreactivity (IFA 1:64) to Rickettsia rickettsi, Ehrlichia chaffeensis, and Ehrlichia canisin 3 (4.11%), 11 (15.07%), and 13 (17.81%), respectively. The results in schoolchildren and the presence of canine seroreactivity to Ehrlichiaspecies that are potentially pathogenic to humans suggests the risk of transmission of other Rickettsiaein the study area.

Hemostatic/fibrinolytic protein changes in C3H/HeN mice infected with Rickettsia conorii--a model for Rocky Mountain spotted fever.

Changes in plasma hemostatic and fibrinolytic proteins were determined during courses of a murine model of fatal and non-fatal Rocky Mountain spotted fever. C3H/HeN mice were infected with Rickettsia conorii and coagulation and histopathologic studies were performed at prescribed periods of time. A significant decrease in plasma factor VIII activity and rise in plasma factor V procoagulant activity correlated with a fatal infection. Factor VII levels were unchanged; factor XI levels dropped early in the course in the lethally infected animals, but returned to normal. Factor XII, high molecular weight kininogen, and prekallikrein levels were unchanged by the sublethal infection. Prekallikrein levels fell during the lethal infection. Antithrombin concentrations were decreased significantly in all animals, but plasma plasminogen levels did not change in either group of animals. Nonocclusive thrombi were microscopically observed rarely and only in animals surviving a sublethal infection. A fall in tissue plasminogen activator activity and a rise in plasminogen activator inhibitor activity highly correlated with a lethal outcome. Lethal infection with R. conorii is associated with primary endothelial cell injury resulting in decreased tissue plasminogen activator and increased plasminogen activator inhibitor.

Hemostatic changes in Rocky Mountain spotted fever and Mediterranean spotted fever.

Rocky Mountain spotted fever and Mediterranean spotted fever are rickettsial infections primarily of endothelial cells that normally have a potent anticoagulant function. As a result of endothelial cell infection and injury, the hemostatic system is perturbed and shows changes that vary widely from a minor reduction in the platelet count (frequently) to severe coagulopathies, such as deep venous thrombosis and disseminated intravascular coagulation (rarely). Changes favoring a hypercoagulable state include endothelial injury and release of procoagulant components, activation of the coagulation cascade with thrombin generation, platelet activation, increased antifibrinolytic factors, consumption of natural anticoagulants, and possibly high levels of coagulation-promoting cytokines. Yet, most studies have been performed on endothelial cell cultures that provide nonphysiologic, reductionistic, experimental conditions. The lack of flow, platelets, and WBCs makes these experiments far from simulating the response of endothelial cells in the human body. Coagulopathies and thrombotic events should be considered as potential complications of severe Rocky Mountain spotted fever and Mediterranean spotted fever.

Hidden mortality attributable to Rocky Mountain spotted fever: immunohistochemical detection of fatal, serologically unconfirmed disease.

Rocky Mountain spotted fever (RMSF) is the most severe tickborne infection in the United States and is a nationally notifiable disease. Since 1981, the annual case-fatality ratio for RMSF has been determined from laboratory-confirmed cases reported to the Centers for Disease Control and Prevention (CDC). Herein, a description is given of patients with fatal, serologically unconfirmed RMSF for whom a diagnosis of RMSF was established by immunohistochemical (IHC) staining of tissues obtained at autopsy. During 1996-1997, acute-phase serum and tissue samples from patients with fatal disease compatible with RMSF were tested at the CDC. As determined by indirect immunofluorescence assay, no patient serum demonstrated IgG or IgM antibodies reactive with Rickettsia rickettsii at a diagnostic titer (i.e., >/=64); however, IHC staining confirmed diagnosis of RMSF in all patients. Polymerase chain reaction validated the IHC findings for 2 patients for whom appropriate samples were available for testing. These findings suggest that dependence on serologic assays and limited use of IHC staining for confirmation of fatal RMSF results in underestimates of mortality and of case-fatality ratios for this disease.

Platelet-associated immunoglobulin (antiplatelet antibody) in canine Rocky Mountain spotted fever and ehrlichiosis.

Antiplatelet antibodies were detected in the sera of dogs with naturally occurring and experimentally induced Rickettsia rickettsii and Ehrlichia canis infections. This is the first known report documenting elevated platelet-associated immunoglobulin (PAIg) titers in Rocky Mountain spotted fever (RMSF) infections. In the naturally occurring RMSF infections and ehrlichiosis, the antibodies persisted for weeks or months, even when the platelet counts had normalized. Results of this study indicate an immunological component for rickettsial thrombocytopenia. Therefore, current therapeutic recommendations, especially regarding avoiding the use of immunosuppressive drugs in patients with rickettsial diseases, need to be critically reviewed.

Dual infection with Ehrlichia chaffeensis and a spotted fever group rickettsia: a case report.

Well-documented cases of simultaneous human infection with more than one tick-borne pathogen are rare. To our knowledge only two dual infections have been reported: simultaneous human infection with the agent of human granulocytic ehrlichiosis and Borrelia burgdorferi and simultaneous human infection with B. burgdorferi and Babesia microti (1-2). Rocky Mountain spotted fever has long been known to be endemic in North Carolina; cases of human ehrlichial infection were recognized there soon after Ehrlichia chaffeensis was recognized as an important cause of tick-borne disease in the southeastern United States. Because both Rocky Mountain spotted fever and ehrlichiosis are prevalent in North Carolina, occasional cases of simultaneous human infection by rickettsial and ehrlichial agents would not be surprising; however, no such cases seem to have been reported.

Structure of the acid-labile galactosyl phosphate-containing O-antigen of the bacterium Proteus vulgaris OX19 (serogroup O1) used in the Weil-Felix test.

The structure of the O-specific polysaccharide chain of Proteus vulgaris OX19 lipopolysaccharide which determines the O1 specificity of Proteus and is used in the Weil-Felix test for diagnostics of rickettsiosis was established. On the basis of 1H- and 13 C-NMR spectroscopy, including two-dimensional correlation spectroscopy (COSY), H-detected 1H, 13C heteronuclear multiple-quantum coherence (HMQC), and rotating-frame nuclear Overhauser effect spectroscopy (ROESY), it was found that the polysaccharide consists of branched pentasaccharide repeating units containing D-galactose, 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose, and 2-acetamido-2,6-dideoxy-D-glucose (QuiNAc, two residues), which are connected to each other via a phosphate group (P): [formula: see text]. The polysaccharide is acid-labile, the glycosyl phosphate linkage being cleaved at pH 4.5 (70 degrees C) to give a phosphorylated pentasaccharide with a galactose residue at the reducing end. Structural analysis of the oligosaccharide and a product of its dephosphorylation with 48% hydrofluoric acid using 1H- and 13C-NMR spectroscopy and electrospray ionization mass spectrometry confirmed the structure of the polysaccharide.