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.

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.

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.

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.

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.

Correlation of rickettsial titers, circulating endotoxin, and clinical features in Rocky Mountain spotted fever.

Blood rickettsial titers, skin biopsy results, and circulating endotoxin measurements were correlated with the clinical course of disease in patients with Rocky Mountain spotted fever (RMSF). Nine of 11 patients with documented RMSF had Rickettsia rickettsii isolated from plasma samples. Of the eight patients in whom rickettsial titers were measured, seven had 10(0.7) to 10(1.2) median tissue culture infective doses (TCID50) per milliliter; all seven had mild to moderately severe disease. One patient with fulminant, fatal untreated RMSF had 10(3) TCID50/mL of postmortem plasma. Two patients from whom rickettsiae were not isolated had positive direct immunofluorescent stains of skin biopsy material for R rickettsii. Circulating endotoxin was present in two patients, one with documented rickettsemia and one with a positive skin biopsy alone. Only low levels of circulating rickettsiae are present in patients with moderately severe disease. Measurement of plasma endotoxin is not useful in the early diagnosis of RMSF.

Rocky Mountain spotted fever presenting as thrombotic thrombocytopenic purpura.

A patient presented with findings compatible with thrombotic thrombocytopenic purpura. The diagnosis of Rocky Mountain spotted fever was also considered because the patient was a hunter in a tick-infested area. He was treated for both diagnoses. The patient recovered and a diagnosis of Rocky Mountain spotted fever was confirmed by serologic methods. Clinical symptoms and hematologic parameters of severe Rocky Mountain spotted fever may resemble thrombotic thrombocytopenic purpura, implying that there may be similarities in the pathophysiology of both disorders.

Predictors of prognosis and risk of acute renal failure in patients with Rocky Mountain spotted fever.

BACKGROUND: Acute renal failure has long been associated with severe Rocky Mountain spotted fever (RMSF). Despite many descriptions of the protean manifestations of this disease, relatively little is known concerning the risk factors for acute renal failure. Only a few studies have examined the outcome of patients infected with Rickettsia rickettsii who develop renal insufficiency, and these studies had methodological problems. OBJECTIVE: To study the incidence, risk factors, and outcomes of acute renal failure in a large group of hospitalized patients with definite or probable RMSF. METHODS: The clinical records of 114 patients with definite or probable RMSF were retrospectively reviewed to identify clinical and biochemical abnormalities at the time of admission that were associated with the development of acute renal failure and subsequent mortality. Renal failure was defined as a serum creatinine (Cr) above 2 mg/dL. Logistic regression was used to study the association between these variables and the outcomes during hospitalization: death and the development of acute renal failure. RESULTS: The mortality rate in this series was 14%; 19% of the patients developed acute renal failure. The development of acute renal failure increased the odds ratio (OR) of dying by a factor of 17 (P = 0.001). Factors at the time of hospitalization that were associated at a univariate level with subsequent mortality included elevated serum Cr, increased age, increased level of AST, increased level of bilirubin, decreased serum sodium and platelet count, the presence of neurological involvement, and being male. Both the presence of neurological involvement and an elevated serum Cr at presentation were independently associated with increased mortality by multivariate analysis. Three patients developed acute renal failure that required hemodialysis, and only 1 of these 3 patients survived; he was ultimately discharged with a normal serum Cr. Factors at presentation that were associated with the development of acute renal failure included increased bilirubin, increasing age, thrombocytopenia, and the presence of neurological involvement. Both age and decreased platelet count at presentation were independently associated with the development of acute renal failure by multivariate analysis. CONCLUSION: Acute renal failure was a frequent complication of RMSF in this series of patients from a tertiary referral medical center. The presence of acute renal failure was strongly associated with death. Clinical and biochemical variables are useful in predicting which patients will develop acute renal failure.

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.

Serologic characterization of Rocky Mountain spotted fever. Appearance of antibodies reactive with endothelial cells and phospholipids, and factors that alter protein C activation and prostacyclin secretion.

Rocky Mountain spotted fever (RMSF) takes its name from the characteristic rash that occurs as a consequence of vasculitis associated with rickettsial invasion of the endothelium. The authors examined sera from 14 patients with serologically confirmed RMSF for the presence of antibodies (IgG and IgM) reactive with human umbilical vein-derived endothelial cells and with the phospholipids cardiolipin (CL) and phosphatidyl serine (PS). Sera from 7 patients (50%) exhibited antiendothelial antibodies, and 10 (71%) patient sera reacted with CL and/or PS. Because such antibodies may interfere with or augment endothelial thrombosis-related activities, acute and convalescent sera were tested for their effects on endothelial PGI2 secretion and protein C activation. Acute sera from two patients and convalescent sera from four patients stimulated protein C activation. Additionally, sera from five acute and nine convalescent cases inhibited basal endothelial PGI2 secretion, but sera from two acute and three convalescent cases stimulated thrombin-dependent PGI2 secretion. These results demonstrated that, in a significant proportion of patients, RMSF was accompanied by the appearance of antibodies that bound to endothelial cells and to phospholipids; some of these antibodies may have altered anticoagulant endothelial functions.

A comparison of the complement fixation, indirect fluorescent antibody, and microagglutination tests for the serological diagnosis of rickettsial diseases.

Three techniques for the serological diagnosis of Rocky Mountain spotted fever were compared by testing 417 sera from 178 patients who very probably did not have rickettsial infections and 88 sera from 41 patients who very probably had Rocky Mountain spotted fever (SF). The techniques were complement fixation (CF), indirect fluorescent antibody (IFA), and microagglutination (MA). To avoid possible degradation during unnecessary purification, the antigens were prepared by methods that were as simple as possible. In the CF tests of 417 sera from patients with nonrickettsial diseases there was only one titer of 8 and none at higher dilutions, whereas with the IFA and MA tests 4-8% of the sera reacted with SF antigens and 4-20% reacted with murine typhus (MT) antigens; the evidence indicated that these reactions were not caused by specific rickettsial antibody. With the SF sera, it could be seen that the IFA test was the most sensitive and the MA test was the least sensitive at each interval after infection. Moreover, the IFA results showed the least number of confusing cross-reactions with MT antigens and the MA test showed the most. The relative advantages of the three tests in serodiagnosis of rickettsial diseases are discussed.

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.

Studies on Macaca mulatta infected with Rocky Mountain spotted fever.

Acid-base alterations and changes in other selected serum constituents (free fatty acids, triglycerides, cholesterol, copper, cortisol, alpha1-acid glycoprotein, haptoglobin, and albumin) were measured during a study of Rocky Mountain spotted fever in 16 male rhesus macaques. Blood samples were taken from nonanesthetized macaques conditioned to repeated handling. Arterial pH increased and PCO2 decreased during the febrile period. Free fatty acids, triglycerides, copper, cortisol, alpha1-acid glycoprotein, and haptoglobin increased, whereas albumin decreased during the disease. Significant changes were not observed in arterial PO2. Cholesterol remained unchanged. The increase in arterial pH and decrease in PaCO2 indicated that respiratory alkalosis was present in macaques acutely affected with Rocky Mountain spotted fever.

Changes in blood serum constituents and hematologic values in Macaca mulatta with Rocky Mountain spotted fever.

Forty-seven male Macaca mulatta, 3 to 4 kg weight, were inoculated intravenously or subcutaneously with various doses of yolk sac-grown Rickettsia rickettsii. Thirty-four macaques became febrile and exhibited signs of infection ranging from transient illness with a few days of fever to severe illness with subsequent death. The rash appeared more frequently in the macaques inoculated subcutaneously. Febrile macaques that survived had leukocytosis, with concomitant neutrophilia. Febrile macaques that died had, in addition, marked terminal leukopenia and thrombocytopenia. Packed cell volume of all febrile macaques decreased. In almost all of the febrile macaques, there were increased serum urea nitrogen, glutamic-oxaloacetic transaminase, and lactate dehydrogenase and decreased total serum protein and amylase concentrations. A few febrile macaques had increased bilirubin values and decreased sodium, chloride, phosphorus, and alkaline phosphatase concentrations. Changes did not occur in serum glucose, potassium, calcium, and glutamic-pyruvic transaminase values. The experimental form of Rocky Mountain spotted fever in the macaque provides a subhuman primate model for studying the pathophysiology of this disease.

Vascular permeability and coagulation during Rickettsia rickettsii infection in dogs.

The vascular permeability of the ocular fundus, alterations in the coagulation system, and plasma concentrations of thromboxane B2 (TXB2) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) were studied in dogs following intradermal inoculation with 5 x 10(5) TCID50 of Rickettsia rickettsii. Twenty-four to 48 hours after the onset of fever and rickettsemia, multifocal areas of retinal vasculitis were evident, which corresponded to areas of altered vascular permeability demonstrated by fluorescein angiography. The number and intensity of retinal vessels with sodium fluorescein leakage peaked during the second week after inoculation, and retinal vascular permeability remained altered during the third week of infection, well past the phase of clinical and clinicopathologic recovery. Development of retinal vasculitic foci was associated with thrombocytopenia, increased concentrations of circulating fibrinogen, and slight prolongation of activated partial thromboplastin time. Increased concentrations of fibrin/fibrinogen degradation products were detected in 4 of 9 dogs. Despite the degree of vascular endothelial damage evident on fluorescein angiographic and histologic studies in these dogs, plasma TXB2 and 6-keto-PGF1 alpha concentrations were not increased.